Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm.

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.

Two distinct conformers of PrPD type 1 of sporadic Creutzfeldt-Jakob disease with codon 129VV genotype faithfully propagate in vivo.

Current classifications of sporadic Creutzfeldt-Jakob disease (sCJD) identify five subtypes associated with different disease phenotypes. Most of these histopathological phenotypes (histotypes) co-distribute with distinct pairings of methionine (M)/valine (V) genotypes at codon 129 of the prion protein (PrP) gene and the type (1 or 2) of the disease-associated PrP (PrPD). Types 1 and 2 are defined by the molecular mass (~ 21 kDa and ~ 19 kDa, respectively) of the unglycosylated isoform of the proteinase K-resistant PrPD (resPrPD). We recently reported that the sCJDVV1 subtype (129VV homozygosity paired with PrPD type 1, T1) shows an electrophoretic profile where the resPrPD unglycosylated isoform is characterized by either one of two single bands of ~ 20 kDa (T120) and ~ 21 kDa (T121), or a doublet of ~ 21-20 kDa (T121-20). We also showed that T120 and T121 in sCJDVV have different conformational features but are associated with indistinguishable histotypes. The presence of three distinct molecular profiles of T1 is unique and raises the issue as to whether T120 and T121 represent distinct prion strains. To answer this question, brain homogenates from sCJDVV cases harboring each of the three resPrPD profiles, were inoculated to transgenic (Tg) mice expressing the human PrP-129M or PrP-129V genotypes. We found that T120 and T121 were faithfully replicated in Tg129V mice. Electrophoretic profile and incubation period of mice challenged with T121-20 resembled those of mice inoculated with T121 and T120, respectively. As in sCJDVV1, Tg129V mice challenged with T121 and T120 generated virtually undistinguishable histotypes. In Tg129M mice, T121 was not replicated while T120 and T121-20 generated a ~ 21-20  kDa doublet after lengthier incubation periods. On second passage, Tg129M mice incubation periods and regional PrP accumulation significantly differed in T120 and T121-20 challenged mice. Combined, these data indicate that T121 and T120 resPrPD represent distinct human prion strains associated with partially overlapping histotypes.

Exposure Risk of Chronic Wasting Disease in Humans.

The majority of human prion diseases are sporadic, but acquired disease can occur, as seen with variant Creutzfeldt-Jakob disease (vCJD) following consumption of bovine spongiform encephalopathy (BSE). With increasing rates of cervid chronic wasting disease (CWD), there is concern that a new form of human prion disease may arise. Currently, there is no evidence of transmission of CWD to humans, suggesting the presence of a strong species barrier; however, in vitro and in vivo studies on the zoonotic potential of CWD have yielded mixed results. The emergence of different CWD strains is also concerning, as different strains can have different abilities to cross species barriers. Given that venison consumption is common in areas where CWD rates are on the rise, increased rates of human exposure are inevitable. If CWD was to infect humans, it is unclear how it would present clinically; in vCJD, it was strain-typing of vCJD prions that proved the causal link to BSE. Therefore, the best way to screen for CWD in humans is to have thorough strain-typing of harvested cervids and human CJD cases so that we will be in a position to detect atypical strains or strain shifts within the human CJD population.

A novel mechanism of phenotypic heterogeneity in Creutzfeldt-Jakob disease.

One of remarkable features of sporadic Creutzfeldt-Jakob disease (sCJD) is the great phenotypic variability. Understanding the molecular basis of this variability has important implications for the development of therapeutic approaches. It is well established that, in many cases, phenotypic heterogeneity of sCJD is under control of two determinants: the genotype at the methionine (M)/valine (V) polymorphic codon 129 of the human prion protein gene and the type, 1 or 2, of the pathogenic and disease-related form of the prion protein, PrPD. However, this scenario fails to explain the existence of distinct heterozygous sCJDMV2 subtypes, where heterogeneity occurs without any variation of the 129 allotype and PrPD type. One of these subtypes, denoted sCJDMV2C, associated with PrPD type 2, is characterized by widespread spongiform degeneration of the cerebral cortex (C). The second variant, denoted sCJDMV2K, features prominent deposition of PrPD amyloid forming kuru type (K) plaques. Here we used a mass spectrometry based approach to test the hypothesis that phenotypic variability within the sCJDMV2 subtype is at least partly determined by the abundance of 129 M and 129 V polymorphic forms of proteinase K-resistant PrPD (resPrPD). Consistent with this hypothesis, our data demonstrated a strong correlation of the MV2C and MV2K phenotypes with the relative populations of protease-resistant forms of the pathogenic prion proteins, resPrPD-129 M and resPrPD-129 V, where resPrPD-129 M dominated in the sCJDMV2C variant and resPrPD-129 V in the sCJDMV2K variant. This finding suggests an important, previously unrecognized mechanism for phenotypic determination in human prion diseases.

Gerstmann-Sträussler-Scheinker disease revisited: accumulation of covalently-linked multimers of internal prion protein fragments.

Despite their phenotypic heterogeneity, most human prion diseases belong to two broadly defined groups: Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS). While the structural characteristics of the disease-related proteinase K-resistant prion protein (resPrPD) associated with the CJD group are fairly well established, many features of GSS-associated resPrPD are unclear. Electrophoretic profiles of resPrPD associated with GSS variants typically show 6-8 kDa bands corresponding to the internal PrP fragments as well as a variable number of higher molecular weight bands, the molecular nature of which has not been investigated. Here we have performed systematic studies of purified resPrPD species extracted from GSS cases with the A117V (GSSA117V) and F198S (GSSF198S) PrP gene mutations. The combined analysis based on epitope mapping, deglycosylation treatment and direct amino acid sequencing by mass spectrometry provided a conclusive evidence that high molecular weight resPrPD species seen in electrophoretic profiles represent covalently-linked multimers of the internal ~ 7 and ~ 8 kDa fragments. This finding reveals a mechanism of resPrPD aggregate formation that has not been previously established in prion diseases.

Dengue-specific subviral nanoparticles: design, creation and characterization.

BACKGROUND: Dengue is today the most significant of arboviral diseases. Novel tools are necessary to effectively address the problem of dengue. Virus-like particles (VLP) offer a versatile nanoscale platform for developing tools with potential biomedical applications. From the perspective of a potentially useful dengue-specific tool, the dengue virus envelope protein domain III (EDIII), endowed with serotype-specificity, host receptor recognition and the capacity to elicit virus-neutralizing antibodies, is an attractive candidate. METHODS: We have developed a strategy to co-express and co-purify Hepatitis B virus surface (S) antigen in two forms: independently and as a fusion with EDIII. We characterized these physically and functionally. RESULTS: The two forms of the S antigen associate into VLPs. The ability of these to display EDIII in a functionally accessible manner is dependent upon the relative levels of the two forms of the S antigen. Mosaic VLPs containing the fused and un-fused components in 1:4 ratio displayed maximal functional competence. CONCLUSIONS: VLPs armed with EDIII may be potentially useful in diagnostic, therapeutic and prophylactic applications.

Evaluation of envelope domain III-based single chimeric tetravalent antigen and monovalent antigen mixtures for the detection of anti-dengue antibodies in human sera.

BACKGROUND: Flavivirus cross-reactive antibodies in human sera interfere with the definitive identification of dengue virus (DENV) infections especially in areas with multiple co-circulating flaviviruses. Use of DENV envelope domain-III (EDIII) can partially resolve the problem. This study has examined the effect of (i) incorporating the EDIIIs of four DENV serotypes into a single chimeric antigen, and (ii) immobilizing the antigen through specific interaction on the sensitivity and specificity of anti-DENV antibody detection. METHODS: A sera panel (n = 164) was assembled and characterized using commercial kits for infection by DENV and a host of other pathogens. Anti-DENV antibodies of both IgM and IgG classes in this panel were detected in indirect ELISAs using a mixture of monovalent EDIIIs, a chimeric EDIII-based tetravalent antigen, EDIII-T, and a biotinylated version of the latter as coating antigens. The sensitivity and specificity of these assays were compared to those obtained using the PanBio Dengue IgG/IgM ELISAs. RESULTS: The performance of dengue IgG and IgM indirect ELISAs, using either a physical mixture of four EDIIIs or the single chimeric EDIII-T antigen, were comparable. Coating of a biotinylated version of the tetravalent antigen on streptavidin plates enhanced sensitivity without compromising specificity. CONCLUSIONS: The incorporation of the EDIIIs of the four DENV serotypes into a single chimeric antigen did not adversely affect assay outcome in indirect ELISAs. Oriented, rather than random, immobilization of the tetravalent antigen enhanced sensitivity of detection of anti-DENV antibodies with retention of 100% specificity.