Abnormal prion protein, infectivity and neurofilament light-chain in blood of macaques with experimental variant Creutzfeldt-Jakob disease

Abstract

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative infections. Variant Creutzfeldt-Jakob disease (vCJD) and sporadic CJD (sCJD) are human TSEs that, in rare cases, have been transmitted by human-derived therapeutic products. There is a need for a blood test to detect infected donors, identify infected individuals in families with TSEs and monitor progression of disease in patients, especially during clinical trials. We prepared panels of blood from cynomolgus and rhesus macaques experimentally infected with vCJD, as a surrogate for human blood, to support assay development. We detected abnormal prion protein (PrP^TSE) in those blood samples using the protein misfolding cyclic amplification (PMCA) assay. PrP^TSE first appeared in the blood of pre-symptomatic cynomolgus macaques as early as 2 months post-inoculation (mpi). In contrast, PMCA detected PrP^TSE much later in the blood of two pre-symptomatic rhesus macaques, starting at 19 and 20 mpi, and in one rhesus macaque only when symptomatic, at 38 mpi. Once blood of either species of macaque became PMCA-positive, PrP^TSE persisted through terminal illness at relatively constant concentrations. Infectivity in buffy coat samples from terminally ill cynomolgus macaques as well as a sample collected 9 months before clinical onset of disease in one of the macaques was assayed in vCJD-susceptible transgenic mice. The infectivity titres varied from 2.7 to 4.3 infectious doses ml^-1. We also screened macaque blood using a four-member panel of biomarkers for neurodegenerative diseases to identify potential non-PrP^TSE pre-symptomatic diagnostic markers. Neurofilament light-chain protein (NfL) increased in blood before the onset of clinical vCJD. Cumulatively, these data confirmed that, while PrP^TSE is the first marker to appear in blood of vCJD-infected cynomolgus and rhesus macaques, NfL might offer a useful, though less specific, marker for forthcoming neurodegeneration. These studies support the use of macaque blood panels to investigate PrP^TSE and other biomarkers to predict onset of CJD in humans.

Keywords: assay; biomarkers; blood; infectivity; macaque; prion.

Fig. 1.

Western blot analysis of macaque brain homogenates and inocula. Inoculum ‘vCJD brain’ (lane 1) and brain homogenates of cynomolgus macaques C16999, CO7422 and CO7423 (lanes 2–4) inoculated with vCJD brain. Inoculum ‘vCJD brain pool’ (lane 5) and brain homogenates of rhesus macaques DER9 and DF20 (lanes 6 and 7) inoculated with vCJD brain pool (lane 5) and MKL (lane 8) inoculated with vCJD brain. Proteinase-K-undigested normal macaque brain served as a negative control (lane 9).

Fig. 2.

Western blot analysis of cynomolgus macaque blood samples assayed by PMCA. Representative results with cynomolgus C16999 blood samples collected at the indicated months post-inoculation (mpi) and tested by PMCA. The negative-control lane represents a PMCA reaction with blood from an uninfected macaque. The negative-PK lane represents normal vole brain homogenate control without proteinase-K digestion.

Fig. 3.

Western blot analyses of rhesus macaque blood samples assayed by PMCA. Blood samples from rhesus macaques DER9, DF20 and MKL collected at the indicated months post-inoculation (mpi) tested by PMCA. The negative control lane represents PMCA reaction with uninfected macaque blood. The negative-PK lane represents normal vole brain homogenate without proteinase-K digestion.

Fig. 4.

NfL levels in plasma of infected cynomolgus and rhesus macaques. NfL concentrations as measured by the Simoa assay in plasma collected at the indicated months post-inoculation (mpi) from three vCJD-infected cynomolgus macaques (a) and three infected rhesus macaques (b). The average NfL value for samples of uninfected control macaque plasma was 7.3±3 pg ml⁻¹.

Fig. 5.

Summary of blood marker studies. Each line represents one macaque. The numbers in parentheses indicate the month post-inoculation. Arrowheads indicate the first time PrP^TSE was detected by PMCA. Thick arrows indicate the first time NfL values rose above the cut-off. Thin arrows indicate the time when onset of clinical illness was first reported. NfL, neurofilament light; Euth, euthanasia; CO, clinical onset.