High titers of mucosal and systemic anti-PrP antibodies abrogate oral prion infection in mucosal-vaccinated mice.

Significant outbreaks of prion disease linked to oral exposure of the prion agent have occurred in animal and human populations. These disorders are associated with a conformational change of a normal protein, PrP(C) (C for cellular), to a toxic and infectious form, PrP(Sc) (Sc for scrapie). None of the prionoses currently have an effective treatment. Some forms of prion disease are thought to be spread by oral ingestion of PrP(Sc), such as chronic wasting disease and variant Creutzfeldt-Jakob disease. Attempts to obtain an active immunization in wild-type animals have been hampered by auto-tolerance to PrP and potential toxicity. Previously, we demonstrated that it is possible to overcome tolerance and obtain a specific anti-PrP antibody response by oral inoculation of the PrP protein expressed in an attenuated Salmonella vector. This past study showed that 30% of vaccinated animals were free of disease more than 350 days post-challenge. In the current study we have both optimized the vaccination protocol and divided the vaccinated mice into low and high immune responder groups prior to oral challenge with PrP(Sc) scrapie strain 139A. These methodological refinements led to a significantly improved therapeutic response. 100% of mice with a high mucosal anti-PrP titer immunoglobulin (Ig) A and a high systemic IgG titer, prior to challenge, remained without symptoms of PrP infection at 400 days (log-rank test P<0.0001 versus sham controls). The brains from these surviving clinically asymptomatic mice were free of PrP(Sc) infection by Western blot and histological examination. These promising findings suggest that effective mucosal vaccination is a feasible and useful method for overcoming tolerance to PrP and preventing prion infection via an oral route.

Pall leukotrap affinity prion-reduction filter removes exogenous infectious prions and endogenous infectivity from red cell concentrates.

BACKGROUND AND OBJECTIVES: Three recent probable cases of transmission of a variant of human Creutzfeldt-Jakob disease (vCJD) through blood transfusion suggest that the disease can be transmitted through transfusion of blood components from presymptomatic blood donors. In this study, we investigated the performance of a new filter for reducing the levels of infectious prions (PrP(Sc)) from red cell concentrates (RCC). MATERIALS AND METHODS: Endogenous Infectivity: A pool of 500 ml of whole blood was collected from 263K-strain scrapie-infected hamsters into an anticoagulant, processed into non-leucoreduced RCC (NL-RCC), and then passed through a prion-reduction filter. Pre- and postfiltration samples were tested for PrP(Sc) by Western blot and infectivity by inoculation of healthy hamsters. Results of the endogenous infectivity study after 200 days post-inoculation are discussed. Exogenous (Spiking) Study: Scrapie-infected hamster brain homogenates containing PrP(Sc) were added to human RCC and then filtered. Levels of PrP(Sc) were determined by Western blot assay. The effect of prior leucodepletion of 'spiked' RCC on PrP(Sc) removal by the prion-removal filter was also assessed. RESULTS: In the endogenous infectivity study, at 200-day observation time, the prefiltered RCC transmitted disease to six of the 187 hamsters, whereas the filtered RCC did not transmit disease to any of 413 animals, P = 0.001. The prion filter also significantly reduced the concentration of leucocytes in the RCC by about 4 logs, P < 0.05. In the exogenous (spiking) study, the level of PrP(res) was significantly reduced in RCC P < 0.05. Prior leucodepletion of the RCC with a leucoreduction filter did not significantly reduce the concentration of exogenously spiked PrP(Sc), P > 0.05. CONCLUSION: The use of this new prion-reduction filter should reduce the risk of vCJD transmission through transfusion of RCC, the most widely transfused blood component.

Infected splenic dendritic cells are sufficient for prion transmission to the CNS in mouse scrapie.

Transmissible spongiform encephalopathies display long incubation periods at the beginning of which the titer of infectious agents (prions) increases in peripheral lymphoid organs. This "replication" leads to a progressive invasion of the CNS. Follicular dendritic cells appear to support prion replication in lymphoid follicles. However, the subsequent steps of neuroinvasion remain obscure. CD11c(+) dendritic cells, an unrelated cell type, are candidate vectors for prion propagation. We found a high infectivity titer in splenic dendritic cells from prion-infected mice, suggesting that dendritic cells carry infection. To test this hypothesis, we injected RAG-1(0/0) mice intravenously with live spleen cell subsets from scrapie-infected donors. Injection of infected dendritic cells induced scrapie without accumulation of prions in the spleen. These results suggest that CD11c(+) dendritic cells can propagate prions from the periphery to the CNS in the absence of any additional lymphoid element.

Development of a topical vaginal microbicide: lessons learned from human milk.

Vaccines are not presently available to prevent adherence and transmission of many common pathogens at mucosal surfaces. As a result, sexually transmitted diseases were one of the most commonly reported infections in the US in 1999. New methods are needed to reduce the spread of mucosal infections. Providing nonspecific protective factors, such as lipids and retinoids found in human milk to mucosal surfaces could reduce mucosal infection caused by viruses, e.g., herpes simplex virus-1 (HSV-1) and bacteria, e.g., Pseudomonas aeruginosa. Human milk lipids enzymatically modified to produce monoglycerides were antimicrobial and inactivated enveloped viruses, as well as gram-positive and gram-negative bacteria. Enveloped viruses were inactivated in seconds following contact with antimicrobial lipids, and P. aeruginosa infectivity was reduced by 99.9% after 2 hours. Transmission of pathogens at mucosal surfaces can also be prevented using retinoids that inhibit viral replication. In a human embryonic intestinal cell line the retinoic acid (RA) derivatives all-trans-RA and 9-cis-RA (10 microg/mL) decreased the production of HSV-1 and Echo-6 viruses by 1-2 log10 over a 48-hour period. In addition, all-trans-RA inhibited HSV-1 replication in Vero cells as effectively as interferon beta, reducing viral production by 2.5log10. These studies indicate that lipids and retinoids could be part of a topical microbicide to prevent mucosal infections.

Retinoic acid reduces the yield of herpes simplex virus in Vero cells and alters the N-glycosylation of viral envelope proteins.

Treatment of Vero cells with all-trans-retinoic acid (RA) decreased the production of infectious herpes simplex virus (HSV) by 1000-10000-fold when compared with control cultures. Levels of total HSV envelope glycoproteins gB, gC and gD produced following RA treatment, were comparable with those found in control cultures. Following 24 h of RA treatment, lower molecular weight variants of gB, gC and gD were produced in addition to the typical molecular mass of each protein found in control samples. Between 24 and 48 h of RA treatment, the proportion of the lower molecular mass variants increased. When control and RA treated samples were incubated with peptide N-glycosidase F (PNGase F), which removes N-glycosylated sugars, the molecular weights of the respective gB, gC and gD proteins produced were comparable in both the groups, indicating that RA did not alter the primary sequence of viral proteins during protein synthesis or increase viral protein proteolysis. RA treatment increased [3H]mannose incorporation into glycoproteins in HSV infected cells but did not change [3H]glucosamine incorporation. We conclude that RA treatment does not reduce the synthesis of three major viral envelope glycoproteins but alters their N-glycosylation and postulate that the inhibitory effect of RA is related to its action on N-glycosylation.

Characteristics of scrapie isolates derived from hay mites.

Previous epidemiological evidence suggested that in some instances a vector and/or reservoir is involved in the occurrence and spread of transmissible spongiform encephalopathies (TSEs). In a preliminary study, hay mite preparations from five Icelandic farms with a history of scrapie were injected into mice, and some of these mice became sick after long incubation periods. To confirm that the disease was scrapie, subsequent passages in mice were performed. In addition, the characteristics of the disease process in these passages were assessed and the results compared to those findings with standard scrapie strains. As expected for scrapie, subsequent passages in the same host led to shortened incubation periods compared to those in primary isolate mice, and all mice had spongiform changes in brain. Results were similar for three of four isolates with regard to clinical manifestations, the incubation periods in mice of the three scrapie incubation-period genotypes (s7s7, s7p7, p7p7), and the PrPSc Western blot (WB) pattern. The characteristics of the fourth isolate were markedly different from the other three isolates with regard to these parameters. Comparison of the characteristics of standard mouse-adapted scrapie strains and the four isolates revealed differences; these differences were particularly pronounced for the fourth isolate.

Reversion of prion protein conformational changes by synthetic beta-sheet breaker peptides.

BACKGROUND: Transmissible spongiform encephalopathies are associated with a structural transition in the prion protein that results in the conversion of the physiological PrPc to pathological PrP(Sc). We investigated whether this conformational transition can be inhibited and reversed by peptides homologous to the PrP fragments implicated in the abnormal folding, which contain specific residues acting as beta-sheet blockers (beta-sheet breaker peptides). METHODS: We studied the effect of a 13-residue beta-sheet breaker peptide (iPrP13) on the reversion of the abnormal structure and properties of PrP(Sc) purified from the brains of mice with experimental scrapie and from human beings affected by sporadic and variant Creutzfeldt-Jakob disease. In a cellular model of familial prion disease, we studied the effect of the peptide in the production of the abnormal form of PrP in intact cells. The influence of the peptide on prion infectivity was studied in vivo by incubation time assays in mice with experimental scrapie. FINDINGS: The beta-sheet breaker peptide partly reversed in-vitro PrP(Sc) to a biochemical and structural state similar to that of PrPc. The effect of the peptide was also detected in intact cells. Treatment of prion infectious material with iPrP13 delayed the appearance of clinical symptoms and decreased infectivity by 90-95% in mice with experimental scrapie. INTERPRETATION: Beta-sheet breaker peptides reverse PrP conformational changes implicated in the pathogenesis of spongiform encephalopathies. These peptides or their derivatives provide a useful tool to study the role of PrP conformation and might represent a novel therapeutic approach for prion-related disorders.

Biochemical and conformational variability of human prion strains in sporadic Creutzfeldt-Jakob disease.

The pathogenesis of prion (PrP) diseases is thought to be related to conformational changes of a normal cellular protein, PrPC, into a protease resistant protein called PrPSc, which is infectious by itself. A difficulty with this 'protein only' hypothesis is the existence of numerous PrP strains, that require PrPSc to have multiple conformations. Sporadic Creutzfeldt-Jakob disease (CJD), which accounts for nearly 80% of human prionoses, was reported to include at least two 'strains' termed types 1 and 2 which differ by electrophoretic patterns of their proteinase K (PK)-resistant fragments (PrP27-30). We have analyzed the biochemical and structural properties of PrPSc and PrP27-30 isolates from six sporadic CJD patients. Fourier transform-infra-red spectroscopy, PrP27-30 glycosylation patterns and studies of PK sensitivity revealed a striking heterogeneity. Furthermore, one isolate yielded a PrP27-30 fragment with a lower mobility clearly different from previously described sporadic CJD types. Although the average beta-sheet content was higher among type 1 isolates, there was overlap between the two types. Our study suggests that human sporadic CJD-related prions display a significant heterogeneity.

Immune surveillance and antigen conformation determines humoral immune response to the prion protein immunogen.

Transmissible spongiform encephalopathies (TSE) are progressive degenerative disorders of the central nervous system. PrP(Sc) is a TSE-specific marker derived from the host-encoded glycoprotein, PrPc. The generation of antibodies to PrP plays an important role in the diagnosis of these diseases. In this study the role of the PrP immunogen and the species being immunized was examined in relation to specific epitopes. Various mammals (mice, hamsters, rabbits and PrP null mice) were immunized with formic acid-treated PrP(Sc) isolated from mice, hamsters and sheep. Both the species being immunized and the source of immunogen played an important role in the antibody response. Response to a limited number of linear epitopes was seen among the various immunized animals. One region in the C-terminal portion of PrP appeared highly immunogenic in all species. Comparison of immunoreactivity and the pepscan-defined linear epitope sites suggests both linear and conformational directed responses in many of the animals. Information on the forces directing immune responses to PrP will lead to a better understanding of host-PrP interactions. It will also assist in the development of new strategies for generating additional tools for immunodiagnosis.

Biondi ring tangles in the choroid plexus of Alzheimer's disease and normal aging brains: a quantitative study.

The choroid plexus (CP) performs the vital function of producing up to 90% (450-1000 ml/day) of cerebrospinal fluid (CSF) to nourish and to protect the brain in the CSF suspension. The CP also acts as a selective barrier between blood and CSF to regulate ions and other essential molecules. However, the accumulation of intracellular inclusions called Biondi ring tangles (BRTs) in CP cells of Alzheimer's disease (AD)/aging brains may affect these vital functions of the CP. Statistical analysis of quantitative data on the numbers of CP cells containing BRTs from 54 brains (29 AD and 25 normal control), age range 1-100 years, indicated a significant difference (p<0.00004) between AD and control brains, using analysis of covariance (ANCOVA) with age as covariate. This study compiled the first set of archives to reveal the distribution pattern of BRTs in the CP of AD brains at various ages. Electron microscopy of negatively stained isolated BRTs revealed that these tangles are made of tightly packed bundles of long filaments with diameter around 10 nm that are morphologically distinct from the more loosely packed/shorter bundles of 6-8 nm amyloid fibrils of neuritic plaques (NPs) and from the 24 nm paired helical filaments of neurofibrillary tangles (NFTs) in AD brain. These data suggest that BRTs may represent a significant and measurable biomarker for AD in addition to NPs and NFTs.

Cell-lysate conversion of prion protein into its protease-resistant isoform suggests the participation of a cellular chaperone.

A conformational transition between the normal cellular prion protein (PrPC) and the beta-sheet-rich pathological isoform (PrPSc) is a central event in the pathogenesis of spongiform encephalopathies. The prion infectious agent seems to contain mainly, if not exclusively, PrPSc, which has the ability to propagate its abnormal conformation by transforming the host PrPC into the pathological isoform. We have developed an in vitro system to induce the PrPC --> PrPSc conversion by incubating a cell-lysate containing mouse PrPC with partially purified mouse PrPSc. After 48 h of incubation with a 10-fold molar excess of PrPSc, the cellular protein acquired PK-resistance resembling a PrPSc-like state. Time course experiments suggest that the conversion follows a stepwise mechanism involving kinetic intermediates. The conversion was induced by PrPSc extracted from mice infected with two different prion strains, each propagating its characteristic Western blot profile. The latter results and the fact that all the cellular components are present in the conversion reaction suggest that PrPC-PrPSc interaction is highly specific and required for the conversion. No transformation was observed under the same conditions using purified proteins without cell-lysate. However, when PrPC-depleted cell-lysate was added to the purified proteins the conversion was recovered. These findings provide direct evidence for the participation of a chaperone-like activity involved in catalyzing the conversion of PrPC into PrPSc.

Astrocytosis and amyloid deposition in scrapie-infected hamsters.

In scrapie infection, prion protein (PrPSc) is localized in areas where there is neurodegeneration and astrocytosis. It is thought that PrPSc is toxic to neurons and trophic for astrocytes. In our study, paraffin sections from scrapie infected (263K and 139H) and control hamsters were examined with histological and immunocytochemical staining. We found that PrPSc was present in the ependymal cells of both 263K- and 139H-infected hamsters. In 139H-infected hamsters, PrPSc was found in the cytoplasm of neurons in cerebral cortex and in hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. In contrast, neuronal cytoplasm and nuclei, were positive for PrPSc in most areas such as cortex, hippocampus, and thalamus in 263K-infected hamsters. Many aggregations of PrPSc could be seen in the cortex, hippocampus, substantia nigra and around the Pia mater, corpus callosum, fimbria, ventricles, and blood vessels in sections from 139H- and/or 263K-positive animals. Furthermore, PrPSc was also co-localized with glial fibrillary acidic protein (GFAP) in many reactive astrocytes (approximately 90%) in certain areas such as the hippocampus in 263K-infected hamsters, but not 139H-infected hamsters. The patterns of astrocytosis and PrPSc formation were different between 139H- and 263K-infected hamsters, which may be used for a diagnosis purpose. Our results suggest a hypothesis that multiple cell-types are capable of PrPSc production. Our results also confirm that reactive astrocytes can produce and/or accumulate PrPSc during some scrapie strain infections. The findings suggest a 'snowball effect', that is: astrocytosis might play an important role in amyloidosis, while amyloidosis may induce further astrocytosis at least in 263K-infected hamsters.

Astrocytosis and proliferating cell nuclear antigen expression in brains of scrapie-infected hamsters.

Scrapie is a neurodegenerative disease in sheep and goats. Neuropathological examination shows astrocytosis. One issue is whether the astrocytosis seen in scrapie is a function of an increase in reactivity of individual cells, or whether there is actual replication of astrocytes. We used double-label immunohistochemistry for proliferating cell nuclear antigen (PCNA) and for glial fibrillary acidic protein (GFAP) to determine the mitotic state of cells and to confirm their identity as astrocytes. Brain sections from hamsters (strain LVG/LAK) infected with 139H or 263K scrapie isolates were examined. GFAP immunostaining was increased in astrocytes in most regions of the brains of scrapie-infected hamsters. These qualitative observations were confirmed by computerized image analysis quantification. A proportion of the hypertrophic astrocytes (0.5-10.8%, depending on specific location) were PCNA immunoreactive. The PCNA-immunopositive astrocytes were most frequently found in cerebral cortex, corpus callosum, subependymal areas, fimbria, caudate, thalamus, hypothalamus, hippocampus, and dentate gyrus. Our results suggest that the astrocytosis seen in scrapie-infected animals is, at least in part, owing to actual replication of astrocytes in these animals. We hypothesize that the astrocytes may be an important locus for the disease process.

Immunodiagnosis of prion disease.

The immunodiagnosis of prion diseases is of critical importance due to the transmissibility of these conditions and their fatal prognosis. A panel of monoclonal and polyclonal antibodies have been generated for use in the study and diagnosis of these diseases. This manuscript describes the generation and characterization of these antibodies as well as their diagnostic application.

Inhibition of herpes simplex virus replication by retinoic acid.

The retinoic acid (RA) isomers all-trans-RA, 9-cis-RA and 13-cis-RA as well as other retinoids were tested for their ability to reduce the yield of herpes simplex virus-1 (HSV-1). RA isomers reduced HSV-1 replication whereas the other retinoids, retinol, retinal, beta-carotene and amide derivatives of RA were not inhibitory. All-trans-RA reduced the yield of HSV-1 by 100-fold at 5 micrograms/ml but 9-cis-RA and 13-cis-RA reduced viral replication by 10-fold. At a concentration of 10 micrograms/ml all-trans-RA and 9-cis-RA reduced virus yield by 1000-fold while 13-cis-RA decreased HSV-1 production by 100-fold. RA isomers at a concentration of 10 micrograms/ml were not cytotoxic for the Vero cells used in these studies. Immunofluorescence studies showed that all-trans-RA treated cell cultures exhibited small foci of virus specific immunostaining while untreated cultures displayed intense HSV-1 immunoreactivity in virtually the entire cell population. RA-dependent inhibition of HSV-1 replication required the presence of RA with the virus. HSV-1 replication proceeded when RA was removed from infected cells. Treatment of cell cultures with RA did not induce gene expression for type-1 interferon (IFN) or for the type-1 IFN inducible genes studied suggesting that RA inhibition of HSV-1 replication is not mediated by IFN. These studies have established the ability of RA to reduce the replication of HSV-1 in vitro.