Structural basis for recognition of the central conserved region of RSV G by neutralizing human antibodies.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly, and yet there remains no effective treatment or vaccine. The surface of the virion is decorated with the fusion glycoprotein (RSV F) and the attachment glycoprotein (RSV G), which binds to CX3CR1 on human airway epithelial cells to mediate viral attachment and subsequent infection. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region of G have been shown to neutralize both subtypes of RSV and to protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated two human antibodies directed against the central conserved region of RSV G and demonstrated that they neutralize RSV infection of human bronchial epithelial cell cultures in the absence of complement. Moreover, the antibodies protected cotton rats from severe RSV disease. Both antibodies bound with high affinity to a secreted form of RSV G as well as to a peptide corresponding to the unglycosylated central conserved region. High-resolution crystal structures of each antibody in complex with the G peptide revealed two distinct conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with the structure of fractalkine (CX3CL1) alone or in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. Collectively, these results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide the development of new vaccines and antibody-based therapies for RSV.

A broadly neutralizing human monoclonal antibody exhibits in vivo efficacy against both human metapneumovirus and respiratory syncytial virus.

BACKGROUND: Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infection, with significant morbidity and mortality. No licensed vaccines or therapeutic agents exist. Monoclonal antibodies (mAbs) are effective at preventing other infectious diseases and could be used against HMPV in high-risk hosts. METHODS: In vitro assays were performed to assess the neutralizing activity and affinity kinetics of human mAb 54G10. A new mouse model was developed to assess prophylactic and therapeutic efficacy in vivo. The epitope of 54G10 was identified by generating mAb-resistant mutants (MARMs). RESULTS: At low concentrations, 54G10 neutralized all 4 subgroups of HMPV in vitro and had subnanomolar affinity for the fusion protein. DBA/2 mice were permissive for all 4 HMPV subgroups, and 54G10 was effective both prophylactically and therapeutically against HMPV in vivo. Sequencing of HMPV MARMs identified the 54G10 epitope, which was similar to an antigenic site on respiratory syncytial virus (RSV). 54G10 also exhibited in vitro neutralizing activity and in vivo protective and therapeutic efficacy against RSV. CONCLUSIONS: Human mAb 54G10 has broad neutralizing activity against HMPV and could have prophylactic and therapeutic utility clinically. The conserved epitope could represent a structural vaccine target for HMPV and RSV.

Lack of prion infectivity in fixed heart tissue from patients with Creutzfeldt-Jakob disease or amyloid heart disease.

In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP(Sc)) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. Although the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP(Sc) in the brain, none of the inoculated heart samples resulted in disease or the accumulation of PrP(Sc). Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.

Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid.

OBJECTIVE: Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B-cell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic. METHODS: We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy- and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections. RESULTS: Whereas humanized control rAbs derived from anti-myelin hybridomas and anti-myelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immunoreactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells. INTERPRETATION: The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein.

Trafficking of prion proteins through a caveolae-mediated endosomal pathway.

To understand the posttranslational conversion of the cellular prion protein (PrPC) to its pathologic conformation, it is important to define the intracellular trafficking pathway of PrPC within the endomembrane system. We studied the localization and internalization of PrPC in CHO cells using cryoimmunogold electron microscopy. At steady state, PrPC was enriched in caveolae both at the TGN and plasma membrane and in interconnecting chains of endocytic caveolae. Protein A-gold particles bound specifically to PrPC on live cells. These complexes were delivered via caveolae to the pericentriolar region and via nonclassical, caveolae-containing early endocytic structures to late endosomes/lysosomes, thereby bypassing the internalization pathway mediated by clathrin-coated vesicles. Endocytosed PrPC-containing caveolae were not directed to the ER and Golgi complex. Uptake of caveolae and degradation of PrPC was slow and sensitive to filipin. This caveolae-dependent endocytic pathway was not observed for several other glycosylphosphatidyl inositol (GPI)-anchored proteins. We propose that this nonclassical endocytic pathway is likely to determine the subcellular location of PrPC conversion.

A change in the conformation of prions accompanies the emergence of a new prion strain.

To investigate the role of the pathogenic prion protein (PrP(Sc)) in controlling susceptibility to foreign prions, two Syrian hamster (SHa) prion strains, Sc237 and DY, were transmitted to transgenic mice expressing chimeric SHa/mouse PrP genes, Tg(MH2M). First passage of SHa(Sc237) prions exhibited prolonged incubation times, diagnostic of a species barrier. PrP(Sc) of the new MH2M(Sc237) strain possessed different structural properties from those of SHa(Sc237), as demonstrated by relative conformational stability measurements. This change was accompanied by a disease phenotype different from the SHa(Sc237) strain. Conversely, transmission of SHa(DY) prions to Tg(MH2M) mice showed no species barrier, and the MH2M(DY) strain retained the conformational and disease-specific properties of SHa(DY). These results suggest a causal relationship between species barriers, changes in PrP(Sc) conformation, and the emergence of new prion strains.

Non-infectious aggregates of the prion protein react with several PrPSc-directed antibodies.

The key event in the pathogenesis of prion diseases is the conformational conversion of the normal prion protein (PrP) (PrP(C)) into an infectious, aggregated isoform (PrP(Sc)) that has a high content of beta-sheet. Historically, a great deal of effort has been devoted to developing antibodies that specifically recognize PrP(Sc) but not PrP(C), as such antibodies would have enormous diagnostic and experimental value. A mouse monoclonal IgM antibody (designated 15B3) and three PrP motif-grafted monoclonal antibodies (referred to as IgG 19-33, 89-112, and 136-158) have been previously reported to react specifically with infectious PrP(Sc) but not PrP(C). In this study, we extend the characterization of these four antibodies by testing their ability to immunoprecipitate and immunostain infectious and non-infectious aggregates of wild-type, mutant, and recombinant PrP. We find that 15B3 as well as the motif-grafted antibodies recognize multiple types of aggregated PrP, both infectious and non-infectious, including forms found in brain, in transfected cells, and induced in vitro from purified recombinant protein. These antibodies are exquisitely selective for aggregated PrP, and do not react with soluble PrP even when present in vast excess. Our results suggest that 15B3 and the motif-grafted antibodies recognize structural features common to both infectious and non-infectious aggregates of PrP. Our study extends the utility of these antibodies for diagnostic and experimental purposes, and it provides new insight into the structural changes that accompany PrP oligomerization and prion propagation.

Antibiotic-induced acceleration of type 1 diabetes alters maturation of innate intestinal immunity.

The early-life intestinal microbiota plays a key role in shaping host immune system development. We found that a single early-life antibiotic course (1PAT) accelerated type 1 diabetes (T1D) development in male NOD mice. The single course had deep and persistent effects on the intestinal microbiome, leading to altered cecal, hepatic, and serum metabolites. The exposure elicited sex-specific effects on chromatin states in the ileum and liver and perturbed ileal gene expression, altering normal maturational patterns. The global signature changes included specific genes controlling both innate and adaptive immunity. Microbiome analysis revealed four taxa each that potentially protect against or accelerate T1D onset, that were linked in a network model to specific differences in ileal gene expression. This simplified animal model reveals multiple potential pathways to understand pathogenesis by which early-life gut microbiome perturbations alter a global suite of intestinal responses, contributing to the accelerated and enhanced T1D development.

Transfer of scrapie prion infectivity by cell contact in culture.

BACKGROUND: When a cell is infected with scrapie prions, newly synthesized molecules of the prion protein PrP(C) are expressed at the cell surface and may subsequently be converted to the abnormal form PrP(Sc). In an experimental scrapie infection of an animal, the initial innoculum of PrP(Sc) is cleared relatively rapidly, and the subsequent propagation of the infection depends on the ability of infected cells to convert uninfected target cells to stable production of PrP(Sc). The mechanism of such cell-based infection is not understood. RESULTS: We have established a system in dissociated cell culture in which scrapie-infected mouse SMB cells are able to stably convert genetically marked target cells by coculture. After coculture and rigorous removal of SMB cells, the target cells express PrP(Sc) and also incorporate [35S]methionine into PrP(Sc). The extent of conversion was sensitive to the ratio of the two cell types, and conversion by live SMB required 2500-fold less PrP(Sc) than conversion by a cell-free prion preparation. The conversion activity of SMB cells is not detectable in conditioned medium and apparently depends on close proximity or contact, as evidenced by culturing the SMB and target cells on neighboring but separate surfaces. SMB cells were killed by fixation in aldehydes, followed by washing, and were found to retain significant activity at conversion of target cells. CONCLUSIONS: Cell-mediated infection of target cells in this culture system is effective and requires significantly less PrP(Sc) than infection by a prion preparation. Several lines of evidence indicate that it depends on cell contact, in particular, the activity of aldehyde-fixed infected cells.

Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation.

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations.

Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice.

There is increasing concern over the extent to which bovine spongiform encephalopathy (BSE) prions have been transmitted to humans, as a result of the rising number of variant Creutzfeldt-Jakob disease (vCJD) cases. Toward preventing new transmissions, diagnostic tests for prions in livestock have been developed using the conformation-dependent immunoassay (CDI), which simultaneously measures specific antibody binding to denatured and native forms of the prion protein (PrP). We employed high-affinity recombinant antibody fragments (recFab) reacting with residues 95-105 of bovine (Bo) PrP for detection and another recFab that recognizes residues 132-156 for capture in the CDI. We report that the CDI is capable of measuring the disease-causing PrP isoform (PrP(Sc)) in bovine brainstems with a sensitivity similar to that of end-point titrations in transgenic (Tg) mice expressing BoPrP. Prion titers were approximately 10(7) ID(50) units per gram of bovine brainstem when measured in Tg(BoPrP) mice, a figure approximately 10 times greater than that determined by bioassay in cattle and approximately 10,000x greater than in wild-type mice. We also report substantial differences in BoPrP(Sc) levels in different areas of the obex region, where neuropathology has been consistently observed in cattle with BSE. The CDI was able to discriminate between PrP(Sc) from BSE-infected cattle and Tg(BoPrP) mice as well as from chronic wasting disease (CWD)-infected deer and elk. Our findings argue that applying the CDI to livestock should considerably reduce human exposure to animal prions.

Cytosolic prion protein in neurons.

Localizing the cellular prion protein (PrPC) in the brain is necessary for understanding the pathogenesis of prion diseases. However, the precise ultrastructural localization of PrPC still remains enigmatic. We performed the first quantitative study of the ultrastructural localization of PrPC in the mouse hippocampus using high-resolution cryoimmunogold electron microscopy. PrPC follows the standard biosynthetic trafficking pathway with a preferential localization in late endosomal compartments and on the plasma membrane of neurons and neuronal processes. PrPC is found with the same frequency within the synaptic specialization and perisynaptically, but is almost completely excluded from synaptic vesicles. Unexpectedly, PrP is also found in the cytosol in subpopulations of neurons in the hippocampus, neocortex, and thalamus but not the cerebellum. Cytosolic PrP may have altered susceptibility to aggregation, suggesting that these neurons might play a significant role in the pathogenesis of prion diseases, in particular those mammals harboring mutant PrP genes.

Conformation of PrP(C) on the cell surface as probed by antibodies.

We have investigated the conformation of Syrian hamster PrP(C) on the surface of transfected CHO cells by performing cross-competition experiments between a set of nine monoclonal antibody fragments (Fab) directed to defined epitopes throughout the protein. No competition was observed between antibodies recognizing epitopes located within the unstructured N-terminal portion of PrP(C) and those recognizing epitopes located within the ordered C-terminal half of the molecule. However, competition was observed between antibodies recognizing overlapping epitopes and between antibodies recognizing epitopes lying adjacent to one another in the PrP sequence. Titrating the reactivity of each Fab against cell-surface PrP(C) revealed a clear heterogeneity in the accessibility of different specific epitopes. Fab D18, recognizing sequence incorporating the first alpha-helix of PrP(C), bound the largest fraction of the cell-surface PrP population. In contrast, Fab E123, binding an epitope at the extreme N terminus of PrP, and Fab 13A5, binding an epitope in the central region of PrP, were able to recognize fewer than half the number of PrP(C) molecules bound by Fab D18. The pattern of antibody reactivity we observed may, in part, result from N-terminal truncation of a proportion of PrP(C) molecules found at the cell surface. However, truncation cannot account for the marked disparity between exposure of the Fab D18 and 13A5 epitopes, which lie adjacent in the PrP sequence. The relative inaccessibility of the 13A5 epitope likely reflects either PrP(C)-PrP(C) interaction, interaction between PrP(C) and other constituents on the cell membrane, or the existence of PrP(C) subspecies with distinct conformations.

Pathologic prion protein infects cells by lipid-raft dependent macropinocytosis.

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrP(C)) to the infectious scrapie form (PrP(Sc)). However, the mechanism that exogenous PrP(Sc) infects cells and where pathologic conversion of PrP(C) to the PrP(Sc) form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrP(C) to the pathologic PrP(Sc) form in N2a cells exposed to strain RML PrP(Sc) infected brain homogenates, suggesting that a critical determinant of PrP(C) conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrP(Sc) infects cells by lipid raft dependent, macropinocytosis.

Identifying key components of the PrPC-PrPSc replicative interface.

In prion disease, direct interaction between the cellular prion protein (PrP(C)) and its misfolded disease-associated conformer PrP(Sc) is a crucial, although poorly understood step promoting the formation of nascent PrP(Sc) and prion infectivity. Recently, we hypothesized that three regions of PrP (corresponding to amino acid residues 23-33, 98-110, and 136-158) interacting specifically and robustly with PrP(Sc), likely represent peptidic components of one flank of the prion replicative interface. In this study, we created epitope-tagged mouse PrP(C) molecules in which the PrP sequences 23-33, 98-110, and 136-158 were modified. These novel PrP molecules were individually expressed in the prion-infected neuroblastoma cell line (ScN2a) and the conversion of each mutated mouse PrP(C) substrate to PrP(Sc) compared with that of the epitope-tagged wild-type mouse PrP(C). Mutations within PrP 98-110, substituting all 4 wild-type lysine residues with alanine residues, prevented conversion to PrP(Sc). Furthermore, when residues within PrP 136-140 were collectively scrambled, changed to alanines, or amino acids at positions 136, 137, and 139 individually replaced by alanine, conversion to PrP(Sc) was similarly halted. However, other PrP molecules containing mutations within regions 23-33 and 101-104 were able to readily convert to PrP(Sc). These results suggest that PrP sequence comprising residues 98-110 and 136-140 not only participates in the specific binding interaction between PrP(C) and PrP(Sc), but also in the process leading to conversion of PrP(Sc)-sequestered PrP(C) into its disease-associated form.

Toward molecular dissection of PrPC-PrPSc interactions.

Direct interaction between endogenous cellular prion protein (PrP(C)) and misfolded, disease-associated (PrP(Sc)) conformers is a key event in prion propagation, which precedes templated conversion of PrP(C) into nascent PrP(Sc) and prion infectivity. Although almost none of the molecular details of this pivotal process are understood, the persistence of individual prion strains suggests that assembly of the prion replicative complex is mechanistically precise. To systematically map defined regions of PrP(C) sequence that bind tightly to PrP(Sc), we have generated a comprehensive panel of over 45 motif-grafted antibodies containing overlapping peptide grafts collectively spanning PrP residues 19-231. Grafted antibody binding experiments, performed under stringent conditions, clearly identified only three distinct and independent high affinity PrP(Sc) recognition motifs. The first of these binding motifs lies at the very N-terminal region of the mature PrP molecule within PrP-(23-33); the second motif lies within PrP-(98-110); and the third is contained within PrP-(136-158). Mutational analyses of these PrP(Sc)-binding regions revealed that reactivity of the 23-33 and 98-110 segments are largely dependent upon the presence of multiple positively charged amino acid residues. These studies yield new insight into critical peptidic components composing one side of the prion replicative interface.

Selective incorporation of polyanionic molecules into hamster prions.

The central pathogenic event of prion disease is the conformational conversion of a host protein, PrPC, into a pathogenic isoform, PrPSc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrPC molecules in an autocatalytic process requiring accessory polyanions (Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9741-9746). Here we investigated the molecular mechanism by which polyanionic molecules facilitate infectious prion formation in vitro. Ina PMCA reaction lacking PrPSc template seed, synthetic polyA RNA molecules induce hamster HaPrPC to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrPSc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrPC molecules subjected to PMCA selectively incorporate an approximately 1-2.5-kb subset of [32P]polyA RNA molecules from a heterogeneous mixture ranging in size from approximately 0.1 to >6 kb. Neuropathological analysis of scrapie-infected hamsters using the fluorescent dye acridine orange revealed that RNA molecules co-localize with large extracellular HaPrP aggregates. These findings suggest that polyanionic molecules such as RNA may become selectively incorporated into stable complexes with PrP molecules during the formation of native hamster prions.

High titers of transmissible spongiform encephalopathy infectivity associated with extremely low levels of PrPSc in vivo.

Diagnosis of transmissible spongiform encephalopathy (TSE) disease in humans and ruminants relies on the detection in post-mortem brain tissue of the protease-resistant form of the host glycoprotein PrP. The presence of this abnormal isoform (PrP(Sc)) in tissues is taken as indicative of the presence of TSE infectivity. Here we demonstrate conclusively that high titers of TSE infectivity can be present in brain tissue of animals that show clinical and vacuolar signs of TSE disease but contain low or undetectable levels of PrP(Sc). This work questions the correlation between PrP(Sc) level and the titer of infectivity and shows that tissues containing little or no proteinase K-resistant PrP can be infectious and harbor high titers of TSE infectivity. Reliance on protease-resistant PrP(Sc) as a sole measure of infectivity may therefore in some instances significantly underestimate biological properties of diagnostic samples, thereby undermining efforts to contain and eradicate TSEs.

A recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo.

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is a major cause of lower-respiratory-tract disease. hMPV is associated with more severe disease in infants and persons with underlying medical conditions. Animal studies have shown that the hMPV fusion (F) protein alone is capable of inducing protective immunity. Here, we report the use of phage display technology to generate a fully human monoclonal antibody fragment (Fab) with biological activity against hMPV. Phage antibody libraries prepared from human donor tissues were selected against recombinant hMPV F protein with multiple rounds of panning. Recombinant Fabs then were expressed in bacteria, and supernatants were screened by enzyme-linked immunosorbent assay and immunofluorescent assays. A number of Fabs that bound to hMPV F were isolated, and several of these exhibited neutralizing activity in vitro. Fab DS7 neutralized the parent strain of hMPV with a 60% plaque reduction activity of 1.1 mug/ml and bound to hMPV F with an affinity of 9.8 x10(-10) M, as measured by surface plasmon resonance. To test the in vivo activity of Fab DS7, groups of cotton rats were infected with hMPV and given Fab intranasally 3 days after infection. Nasal turbinates and lungs were harvested on day 4 postinfection and virus titers determined. Animals treated with Fab DS7 exhibited a >1,500-fold reduction in viral titer in the lungs, with a modest 4-fold reduction in the nasal tissues. There was a dose-response relationship between the dose of DS7 and virus titer. Human Fab DS7 may have prophylactic or therapeutic potential against severe hMPV infection.

Probing the conformation of the prion protein within a single amyloid fibril using a novel immunoconformational assay.

The coexistence of multiple strains or subtypes of the disease-related isoform of prion protein (PrP) in natural isolates, together with the observed conformational heterogeneity of PrP amyloid fibrils generated in vitro, indicates the importance of probing the conformation of single particles within heterogeneous samples. Using an array of PrP-specific antibodies, we report the development of a novel immunoconformational assay. Uniquely, application of this new technology allows the conformation of multimeric PrP within a single fibril or particle to be probed without pretreatment of the sample with proteinase K. Using amyloid fibrils prepared from full-length recombinant PrP, we demonstrated the utility of this assay to define (i) PrP regions that are surface-exposed or buried, (ii) the susceptibility of defined PrP regions to GdnHCl-induced denaturation, and (iii) the conformational heterogeneity of PrP fibrils as measured for either the entire fibrillar population or for individual fibrils. Specifically, PrP regions 159-174 and 224-230 were shown to be buried and were the most resistant to denaturation. The 132-156 segment of PrP was found to be cryptic under native conditions and solvent-exposed under partially denaturing conditions, whereas the region 95-105 was solvent-accessible regardless of the solvent conditions. Remarkably, a subfraction of fibrils showed immunoreactivity to PrPSc-specific antibodies designated as IgGs 89-112 and 136-158. The immunoreactivity of the conformational epitopes was reduced upon exposure to partially denaturing conditions. Unexpectedly, PrPSc -specific antibodies revealed conformational polymorphisms even within individual fibrils. Our studies provide valuable new insight into fibrillar substructure and offer a new tool for probing the conformation of single PrP fibrils.