Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay

Summary

Background

Variant Creutzfeldt-Jakob disease (vCJD) has a pathogenesis distinct from other forms of human prion disease: disease-related prion protein (PrP^Sc) is readily detectable in lymphoreticular tissues. Quantitation of risk of secondary transmission, and targeting of risk reduction strategies, is limited by lack of knowledge about relative prion titres in these and other peripheral tissues, the unknown prevalence of preclinical vCJD, and a transmission barrier which limits the sensitivity of bioassay. We aimed to improve immunoblotting methods for high sensitivity detection of PrP^Sc to investigate the distribution of PrP^Sc in a range of vCJD tissues.

Methods

We obtained tissues at necropsy from four patients with neuropathologically confirmed vCJD and from individuals without neurological disease. Tissues were analysed by sodium phosphotungstic acid precipitation of PrP^Sc and western blotting using high sensitivity enhanced chemiluminescence.

Findings

We could reliably detect PrP^Sc in the equivalent of 50 nL 10% vCJD brain homogenate, with a maximum limit of detection equivalent to 5 nl. PrP^Sc could be detected in tissue homogenates when present at concentrations 10⁴–10⁵ fold lower than those reported in brain. Tonsil, spleen, and lymph node were uniformly positive for PrP^Sc at concentrations in the range of 0·1–15% of those found in brain: the highest concentrations were consistently seen in tonsil. PrP^Sc was readily detected in the retina and proximal optic nerve of vCJD eye at levels of 2·5 and 25%, respectively of those found in brain. Other peripheral tissues studied were negative for PrP^Sc with the exception of low concentrations in rectum, adrenal gland, and thymus from a single patient with vCJD. vCJD appendix and blood (Buffy coat fraction) were negative for PrP^Sc at this level of assay sensitivity.

Interpretation

We have developed a highly sensitive immunoblot method for detection of PrP^Sc in vCJD tissues that can be used to provide an upper limit on PrP^Sc concentrations in peripheral tissues, including blood, to inform risk assessment models. Rectal and other gastrointestinal tissues should be further investigated to assess risk of iatrogenic transmission via biopsy instruments. Ophthalmic surgical instruments used in procedures involving optic nerve and the posterior segment of the eye, in particular the retina, might represent a potential risk for iatrogenic transmission of vCJD. Tonsil is the tissue of choice for diagnostic biopsy and for population screening of surgical tissues to assess prevalence of preclinical vCJD infection within the UK and other populations.

Introduction

Prion diseases include Creutzfeldt-Jakob disease (CJD) and kuru in human beings, and scrapie and bovine spongiform encephalopathy (BSE) in animals. They are transmissible within the same or to different species by inoculation with, or dietary exposure to, infected tissues. According to the protein-only hypothesis,¹ prions are composed principally or entirely of abnormal isoforms of a host-encoded glycoprotein, prion protein (PrP).² The disease-related isoform, PrP^Sc, is derived from its normal cellular precursor, PrP^C, by a post-translational process that involves a conformational change. PrP^Sc can be distinguished biochemically from PrP^C by its partial protease resistance and detergent insolubility. Multiple prion strains are recognised and these are associated with distinct PrP^Sc types, which can be distinguished by western blot analysis with distinct cleavage sites to proteinase K implying different PrP^Sc conformations3, 4, 5, 6, 7, 8 and by differences in glycoform ratios of protease-digested PrP^Sc.⁵

The appearance of a new human prion disease, variant CJD (vCJD), in the UK from 1995 onwards, and the experimental confirmation that this is caused by the same prion strain as that causing BSE in cattle,5, 9, 10 has raised the possibility that a major epidemic of vCJD will occur in the UK and other countries as a result of dietary or other exposure to BSE prions. ¹¹

The pathogenesis of prion diseases varies with different host species and prion strain combinations. It is clear that the pathogenesis of vCJD differs substantially from that of other forms of CJD. PrP^Sc is readily detectable in lymphoreticular tissues in vCJD and not in classical CJD.12, 13 A distinctive PrP^Sc type, designated type 4t, is consistently seen in tonsil from patients with vCJD and tonsil biopsy can be used for diagnosis of vCJD before death.¹³ To date, all patients with a positive tonsil biopsy sample have been confirmed as having vCJD either at necropsy, or have had a subsequent clinical course consistent with vCJD. All patients with a negative biopsy sample have been confirmed to have other diagnoses at necropsy, or the subsequent clinical course (significant improvement or recovery) excludes this diagnosis (reference 13 and unpublished). Since in natural sheep scrapie and in experimental murine models of scrapie, in which there is also a prominent involvement of lymphoreticular tissues, PrP^Sc can be detected fairly early in the incubation period before detectable neuroinvasion has occurred, it has been suggested that large scale screening of surgical tonsillectomy and appendicectomy tissues for PrP^Sc could provide early warning of a high degree of vCJD prion infection and several such studies are in progress.13, 14

That substantial numbers in the population may be incubating vCJD, allied with the evidence of extensive lymphoreticular involvement, raises concerns that iatrogenic transmission of vCJD could be a major issue. Although there is no epidemiological evidence that blood transfusion is a major risk factor for sporadic CJD,¹⁵ the evidence that B lymphocytes are crucial for prion neuroinvasion,¹⁶ the distinctive pathogenesis of vCJD,¹³ and the unknown but potentially high prevalence of preclinical vCJD infection, has led the UK and other governments to take risk reduction measures on a precautionary basis with respect to blood and blood products.¹⁷ Major concerns also relate to possible iatrogenic transmission via contaminated surgical instruments. Prions can resist normal sterilisation procedures and it is known that neurosurgical instruments can act as a vector for transmission of classical CJD.¹⁸ Epidemiological evidence also suggests that previous abdominal surgery might be a risk factor for classical CJD.¹⁹ Stainless steel wires bind prions efficiently on exposure to brain homogenates and readily transmit the disease when implanted into mice.²⁰ Risk assessments that might guide policy decisions on risk reduction, for instance, extending use of single-use instruments for certain procedures, are limited by lack of data on the tissue distribution of infectivity in vCJD.

Bioassay remains the most sensitive method for prion detection within the same species of laboratory animals. Indeed, the lack of a precise molecular definition of the infectious prion means that bioassay is the only method to formally demonstrate infectivity. However, bioassay across species can be severely limited by species barrier effects. Such transmission barriers effectively prevent bioassay of CJD prions in rodents, and transmissions of classical CJD have until quite recently largely involved primates. Such studies have shown some evidence for infectivity in a number of tissues.²¹ Transgenic mice that express human, but not mouse PrP lack a species barrier to transmission of CJD and are highly susceptible to all types of classical CJD.9, 22 Extensive studies of peripheral tissues have not been reported to date. Interestingly, however, vCJD prions do not transmit easily to such transgenic mice, and transmission of vCJD to both transgenic and conventional mice involves a transmission barrier,9, 10 which severely limits the ability to detect low titre infectivity. We have now attempted to transmit 12 vCJD cases to transgenic and conventional mice (reference 9 and unpublished) by intracerebral inoculation with brain homogenates. Two cases failed to transmit at all to transgenic mice and transmission to wildtype mice was inefficient with sustained and variable incubation periods, and less than 100% of inoculated mice succumbed to disease.

Extensive studies on the pathogenesis of BSE in experimentally infected cattle have been done by bioassay in conventional mice. The cattle to mouse barrier has been determined by comparative endpoint titration at around 1000, such that mice can only detect around 1000 cattle infectious units per inoculum.²³ Assay sensitivity is also limited by the volume of inoculum that can be injected per mouse (typically 30 μL via the most sensitive, intracerebral, route). Such mouse bioassays have shown infectivity almost only in nervous tissues (brain, spinal cord, dorsal root, and trigeminal ganglia). The only other tissues in which infectivity has been reported are the terminal ileum of calves at 6 months after exposure²³ and possibly in sternal bone marrow.²⁴ Surprisingly, no infectivity has been detected in the lymphoreticular system. Because of the limitations of this barrier, the UK Ministry of Agriculture, Fisheries, and Food are currently undertaking bioassays in calves, in which there should be no transmission barrier. Since PrP^Sc is readily detectable in tissues of the central nervous system in BSE, it is possible that mouse bioassay for BSE might not be much more sensitive than western blot detection of PrP^Sc to establish which tissues might pose an infection risk. Given the remarkable persistence of biological properties of the BSE prion strain on passage in various species,²⁵ including human beings,9, 10 we reasoned that current mouse bioassays for vCJD prions could be limited by a similar order such that extensive bioassays of peripheral tissues, which would in any case be extremely timeconsuming and require the use of many animals, may add little to our current knowledge.

Indeed, PrP^Sc is readily detectable in all vCJD brain homogenates we have studied using our standard western blot protocol,⁵ including those that have not transmitted to transgenic mice, which suggests that immunoassays for PrP^Sc can be more sensitive than present bioassays. Although there are encouraging developments in new transgenic models,²⁶ pending development of a transmission barrier-free model for bioassay of vCJD prions, we reasoned that useful data to inform vCJD risk assessments would best be gained from high sensitivity immunodetection of PrP^Sc.

To date there have been no reports showing PrP^Sc deposition by western blotting in vCJD peripheral tissues other than those of the lymphoreticular system12, 13 and potential degrees of infectivity in such tissues remain unquantified. In sporadic, familial, and iatrogenic CJD, transmission experiments in non-human primates have indicated that infectivity is irregularly present and at very much lower concentrations in tissues outside the central nervous system.²¹ As yet there has been no demonstration of the detection of PrP^Sc in peripheral tissues from classical CJD cases.

We have therefore attempted to improve methods for selective precipitation of PrP^Sc from tissue homogenates (thereby allowing concentration of PrP^Sc from much larger volumes of tissue than could be injected into mice) and western blot detection methods for PrP^Sc. Combination of these has allowed us to develop a highly sensitive detection method that we have applied to study the distribution of PrP^Sc in patients with vCJD.