Anti-prion Protein Antibody 6D11 Restores Cellular Proteostasis of Prion Protein Through Disrupting Recycling Propagation of PrPSc and Targeting PrPSc for Lysosomal Degradation.

PrPSc is an infectious and disease-specific conformer of the prion protein, which accumulation in the CNS underlies the pathology of prion diseases. PrPSc replicates by binding to the cellular conformer of the prion protein (PrPC) expressed by host cells and rendering its secondary structure a likeness of itself. PrPC is a plasma membrane anchored protein, which constitutively recirculates between the cell surface and the endocytic compartment. Since PrPSc engages PrPC along this trafficking pathway, its replication process is often referred to as "recycling propagation." Certain monoclonal antibodies (mAbs) directed against prion protein can abrogate the presence of PrPSc from prion-infected cells. However, the precise mechanism(s) underlying their therapeutic propensities remains obscure. Using N2A murine neuroblastoma cell line stably infected with 22L mouse-adapted scrapie strain (N2A/22L), we investigated here the modus operandi of the 6D11 clone, which was raised against the PrPSc conformer and has been shown to permanently clear prion-infected cells from PrPSc presence. We determined that 6D11 mAb engages and sequesters PrPC and PrPSc at the cell surface. PrPC/6D11 and PrPSc/6D11 complexes are then endocytosed from the plasma membrane and are directed to lysosomes, therefore precluding recirculation of nascent PrPSc back to the cell surface. Targeting PrPSc by 6D11 mAb to the lysosomal compartment facilitates its proteolysis and eventually shifts the balance between PrPSc formation and degradation. Ongoing translation of PrPC allows maintaining the steady-state level of prion protein within the cells, which was not depleted under 6D11 mAb treatment. Our findings demonstrate that through disrupting recycling propagation of PrPSc and promoting its degradation, 6D11 mAb restores cellular proteostasis of prion protein.

Altered lymphocyte proliferation and innate immune function in scrapie 139A- and ME7-infected mice.

Lymphoid organs play an important role in prion disease development and progression. While the role of lymphoid organs and changes in immune-related genes have been extensively investigated in scrapie-infected animals, innate immunity has not. Previous studies examined lymphocyte function in scrapie-infected C3H/HeJ mice, which exhibit defects in lipopolysaccharide (LPS) response now known to result from a mutation in Toll-like receptor (TLR) 4. We examined immune function in scrapie-infected CD1 mice, which are LPS responders. Lymphocyte proliferation from CD1 mice infected with either 139A or ME7 scrapie was measured in response to concanavalin (Con) A or LPS at 1 and 3 months after infection. Following LPS exposure, mice infected 3 months with ME7, but not 139A, demonstrated significantly decreased lymphocyte proliferation compared to controls. After Con A exposure, lymphocyte proliferation in scrapie-infected mice did not differ from controls. Gender-specific comparison of lymphocyte proliferation showed significant decreases in mitogenic responses in females infected 3 months with either 139A or ME7, compared to controls. Males infected for 3 months with ME7, but not 139A, showed significantly decreased proliferation after lymphocyte exposure to LPS, but not Con A. Neither gender showed changes in lymphocyte proliferation after 1 month of scrapie infection. Innate immune activation of peritoneal macrophages was determined via production of nitric oxide (NO), IL-6, and TNF-α after exposure to TLR ligands. TNF-α and IL-6 production were reduced in macrophages from females infected with either scrapie strain for 3 months, while NO production after TLR agonist plus IFN-γ exposure was decreased in both females and males infected for 3 months with 139A, compared to ME7. These data demonstrated altered innate immunity, suggesting hormonal and/or other gender-specific regulation may contribute to gender differences in some immune functions. Our data demonstrate lymphocyte proliferation and innate immune functioning in scrapie-infected mice deteriorate with disease progression.

Inflammatory mediators are inhibited by a taurine metabolite in CpG oligodeoxynucleotide and IFN-r activated macrophage cell line.

Taurine plays an important role in brain and retinal development, and has an antiinflammatory and antioxidant function. Taurine chloramine (Tau-Cl) is produced in polymorphonuclear leukocytes via the myeloperoxidase/halide system. We previously demonstrated that Tau-Cl inhibits the production of nitric oxide (NO) and TNF-α in human and murine macrophages activated with IFN-γ in combination with individual Toll-like receptor (TLR) ligands including those for TLR2 and/or TLR4. In the current study, we further explored the effects of Tau-Cl in RAW 264.7 cells stimulated with the TLR9 ligand CpG oligodeoxynucleotide (ODN). Specifically, we examined the effect of CpG ODN plus IFN-γ on the production of NO and TNF-α, and the effect of Tau-Cl on this process. Our findings show that CpG ODN plus IFN-γ-activated RAW 264.7 cells secrete high levels of NO and TNF-α, and that Tau-Cl (0.8 mM) inhibits this effect in a dose-dependent manner, more potently inhibiting the production of NO (99% inhibition) than that of TNF-α (48% inhibition). Nitric oxide synthase (iNOS) protein was also induced by CpG ODN plus IFN-γ, and was also inhibited by Tau-Cl. Furthermore, while CpG ODN plus IFN-γ induced TNF-α and iNOS mRNAs, Tau-Cl transiently suppressed this effect. Taurine itself had no effects on any of these processes. Our findings in a macrophage cell line demonstrate that Tau-Cl inhibits proinflammatory mediators resulting from TLR9 activation, and have implications for the utility of Tau-Cl in scenarios where such activation is deleterious such as in autoimmune conditions or infections in which overwhelming inflammation may occur. CpG ODNs and Tau-Cl both have potential for topical treatment of autoimmune conditions, including psoriasis, vitiligo, and alopecia areata. As CpG ODNs may, under some conditions, up-regulate Tregs, addition of Tau-Cl to CpG ODN topical formulations has potential for improving cancer immunotherapy.

Anti-PrPC monoclonal antibody infusion as a novel treatment for cognitive deficits in an Alzheimer's disease model mouse.

BACKGROUND: Alzheimer's Disease (AD) is the most common of the conformational neurodegenerative disorders characterized by the conversion of a normal biological protein into a ß-sheet-rich pathological isoform. In AD the normal soluble Aß (sAß) forms oligomers and fibrils which assemble into neuritic plaques. The most toxic form of Aß is thought to be oligomeric. A recent study reveals the cellular prion protein, PrPC, to be a receptor for Aß oligomers. Aß oligomers suppress LTP signal in murine hippocampal slices but activity remains when pretreated with the PrP monoclonal anti-PrP antibody, 6D11. We hypothesized that targeting of PrPC to prevent Aß oligomer-related cognitive deficits is a potentially novel therapeutic approach. APP/PS1 transgenic mice aged 8 months were intraperitoneally (i.p.) injected with 1 mg 6D11 for 5 days/week for 2 weeks. Two wild-type control groups were given either the same 6D11 injections or vehicle solution. Additional groups of APP/PS1 transgenic mice were given either i.p. injections of vehicle solution or the same dose of mouse IgG over the same period. The mice were then subjected to cognitive behavioral testing using a radial arm maze, over a period of 10 days. At the conclusion of behavioral testing, animals were sacrificed and brain tissue was analyzed biochemically or immunohistochemically for the levels of amyloid plaques, PrPC, synaptophysin, Aß40/42 and Aß oligomers. RESULTS: Behavioral testing showed a marked decrease in errors in 6D11 treated APP/PS1 Tg mice compared with the non-6D11 treated Tg groups (p < 0.0001). 6D11 treated APP/PS1 Tg mice behaved the same as wild-type controls indicating a recovery in cognitive learning, even after this short term 6D11 treatment. Brain tissue analysis from both treated and vehicle treated APP/PS1 groups indicate no significant differences in amyloid plaque burden, Aß40/42, PrPC or Aß oligomer levels. 6D11 treated APP/PS1 Tg mice had significantly greater synaptophysin immunoreactivity in the dentate gyrus molecular layer of the hippocampus compared to vehicle treated APP/PS1 Tg mice (p < 0.05). CONCLUSIONS: Even short term treatment with monoclonal antibodies such as 6D11 or other compounds which block the binding of Aß oligomers to PrPC can be used to treat cognitive deficits in aged AD transgenic mice.

Anti-PrP Mab 6D11 suppresses PrP(Sc) replication in prion infected myeloid precursor line FDC-P1/22L and in the lymphoreticular system in vivo.

The pathogenesis of prion diseases is related to conformational transformation of cellular prion protein (PrP(C)) into a toxic, infectious, and self-replicating conformer termed PrP(Sc). Following extracerebral inoculation, the replication of PrP(Sc) is confined for months to years to the lymporeticular system (LRS) before the secondary CNS involvement results in occurrence of neurological symptoms. Therefore, replication of PrP(Sc), in the early stage of infection can be targeted by therapeutic approaches, which like passive immunization have limited blood-brain-barrier penetration. In this study, we show that 6D11 anti-PrP monoclonal antibody (Mab) prevents infection on a FDC-P1 myeloid precursor cell line stably infected with 22L mouse adapted scrapie strain. Passive immunization of extracerebrally infected CD-1 mice with Mab 6D11 resulted in effective suppression of PrP(Sc) replication in the LRS. Although, a rebound of PrP(Sc) presence occurred when the Mab 6D11 treatment was stopped, passively immunized mice showed a prolongation of the incubation period by 36.9% (pb0.0001) and a significant decrease in CNS pathology compared to control groups receiving vehicle or murine IgG. Our results indicate that antibody-based therapeutic strategies can be used, even on a short-term basis, to delay or prevent disease in subjects accidentally exposed to prions.

Induction of toll-like receptor 9 signaling as a method for ameliorating Alzheimer's disease-related pathology.

The pathogenesis of Alzheimer's disease (AD) is thought to be related to the accumulation of amyloid beta (Abeta) in amyloid deposits and toxic oligomeric species. Immunomodulation is emerging as an effective means of shifting the equilibrium from Abeta accumulation to clearance; however, excessive cell mediated inflammation and cerebral microhemorrhages are two forms of toxicity which can occur with this approach. Vaccination studies have so far mainly targeted the adaptive immune system. In the present study, we have stimulated the innate immune system via the Toll-like receptor 9 (TLR9) with cytosine-guanosine-containing DNA oligodeoxynucleotides in Tg2576 AD model transgenic mice. This treatment produced a 66% and 80% reduction in the cortical (p = 0.0001) and vascular (p = 0.0039) amyloid burden, respectively, compared with nontreated AD mice. This was in association with significant reductions in Abeta42, Abeta40, and Abeta oligomer levels. We also show that treated Tg mice performed similarly to wild-type mice on a radial arm maze. Our data suggest that stimulation of innate immunity via TLR9 is highly effective at reducing the parenchymal and vascular amyloid burden, along with Abeta oligomers, without apparent toxicity.

Accelerated prion disease pathogenesis in Toll-like receptor 4 signaling-mutant mice.

Prion diseases such as scrapie involve the accumulation of disease-specific prion protein, PrP(Sc), in the brain. Toll-like receptors (TLRs) are a family of proteins that recognize microbial constituents and are central players in host innate immune responses. The TLR9 agonist unmethylated CpG DNA was shown to prolong the scrapie incubation period in mice, suggesting that innate immune activation interferes with prion disease progression. Thus, it was predicted that ablation of TLR signaling would result in accelerated pathogenesis. C3H/HeJ (Tlr4(Lps-d)) mice, which possess a mutation in the TLR4 intracellular domain preventing TLR4 signaling, and strain-matched wild-type control (C3H/HeOuJ) mice were infected intracerebrally or intraperitoneally with various doses of scrapie inoculum. Incubation periods were significantly shortened in C3H/HeJ compared with C3H/HeOuJ mice, regardless of the route of infection or dose administered. At the clinical phase of disease, brain PrP(Sc) levels in the two strains of mice showed no significant differences by Western blotting. In addition, compared with macrophages from C3H/HeOuJ mice, those from C3H/HeJ mice were unresponsive to fibrillogenic PrP peptides (PrP residues 106 to 126 [PrP(106-126)] and PrP(118-135)) and the TLR4 agonist lipopolysaccharide but not to the TLR2 agonist zymosan, as measured by cytokine production. These data confirm that innate immune activation via TLR signaling interferes with scrapie infection. Furthermore, the results also suggest that the scrapie pathogen, or a component(s) thereof, is capable of stimulating an innate immune response that is active in the central nervous system, since C3H/HeJ mice, which lack the response, exhibit shortened incubation periods following both intraperitoneal and intracerebral infections.

A sensitive and quantitative assay for normal PrP in plasma.

Transmissible spongiform encephalopathies can be transmitted by blood transfusion. The risk of spreading the disease among the human population could be mitigated with the implementation of a blood screening assay. We developed a two-antibody assay for PrP detection in plasma using the ORIGEN technology with a protocol modification to improve the limit of detection and to increase the sample volume assayed. In the standard 200 microL format, the assay had a detection limit of 7-10 pg of recombinant PrP and 3 pg in 1 mL final volume implementation. PrP concentration measured in normal and scrapie-infected hamster brains was 7.5+/-0.9 and 57.3+/-9.6 microg/g, respectively. After a concentration step with an immuno-affinity resin, plasma PrP(c) was detected by Western blot and its concentration was measured at 3.5+/-0.8 ng/mL. From these data and assuming that blood has the same specific infectivity as brain, we estimated the concentration of abnormal PrP in hamster-infected plasma to be 32 f g/mL. The assay also detected abnormal brain PrP spiked into plasma although the limit of detection was affected. This is a novel and sensitive assay for the detection of PrP in plasma that could be developed into a platform for a plasma-based TSE test.

CpG oligodeoxynucleotide-enhanced humoral immune response and production of antibodies to prion protein PrPSc in mice immunized with 139A scrapie-associated fibrils.

Prion diseases are characterized by conversion of the cellular prion protein (PrP(C)) to a protease-resistant conformer, the srapie form of PrP (PrP(Sc)). Humoral immune responses to nondenatured forms of PrP(Sc) have never been fully characterized. We investigated whether production of antibodies to PrP(Sc) could occur in PrP null (Prnp(-/-)) mice and further, whether innate immune stimulation with the TLR9 agonist CpG oligodeoxynucleotide (ODN) 1826 could enhance this process. Whether such stimulation could raise anti-PrP(Sc) antibody levels in wild-type (Prnp(+/+)) mice was also investigated. Prnp(-/-) and Prnp(+/+) mice were immunized with nondenatured 139A scrapie-associated fibrils (SAF), with or without ODN 1826, and were tested for titers of PrP-specific antibodies. In Prnp(-/-) mice, inclusion of ODN 1826 in the immunization regime increased anti-PrP titers more than 13-fold after two immunizations and induced, among others, antibodies to an N-terminal epitope, which were only present in the immune repertoire of mice receiving ODN 1826. mAb 6D11, derived from such a mouse, reacts with the N-terminal epitope QWNK in native and denatured forms of PrP(Sc) and recombinant PrP and exhibits a K(d) in the 10(-)(11) M range. In Prnp(+/+) mice, ODN 1826 increased anti-PrP levels as much as 84% after a single immunization. Thus, ODN 1826 potentiates adaptive immune responses to PrP(Sc) in 139A SAF-immunized mice. These results represent the first characterization of humoral immune responses to nondenatured, infectious PrP(Sc) and suggest methods for optimizing the generation of mAbs to PrP(Sc), many of which could be used for diagnosis and treatment of prion diseases.

Clearance and prevention of prion infection in cell culture by anti-PrP antibodies.

Prion diseases are transmissible and invariably fatal neurodegenerative disorders associated with a conformational transformation of the cellular prion protein (PrP(C)) into a self-replicating and proteinase K (PK)-resistant conformer, scrapie PrP (PrP(Sc)). Humoral immunity may significantly prolong the incubation period and even prevent disease in murine models of prionoses. However, the mechanism(s) of action of anti-PrP monoclonal antibodies (Mabs) remain(s) obscure. The murine neuroblastoma N2a cell line, infected with the 22L mouse-adapted scrapie strain, was used to screen a large library of Mabs with similar binding affinities to PrP, to identify those antibodies which could clear established infection and/or prevent infection de novo. Three Mabs were found capable of complete and persistent clearing of already-infected N2a cells of PrP(Sc). These antibodies were 6D11 (generated to PK-resistant PrP(Sc) and detecting PrP residues 93-109), and 7H6 and 7A12, which were raised against recombinant PrP and react with neighbouring epitopes of PrP residues 130-140 and 143-155, respectively. Mabs were found to interact with PrP(Sc) formation both on the cell surface and after internalization in the cytosol. Treatment with Mabs was not associated with toxicity nor did it result in decreased expression of PrP(C). Both preincubation of N2a cells with Mabs prior to exposure to 22L inoculum and preincubation of the inoculum with Mabs prior to infecting N2a cells resulted in a significant reduction in PrP(Sc) levels. Information provided in these studies is important for the rational design of humoral immune therapy for prion infection in animals and eventually in humans.

Nonenzymatic glycation at the N terminus of pathogenic prion protein in transmissible spongiform encephalopathies.

Transmissible spongiform encephalopathies (TSEs) are transmissible neurodegenerative diseases characterized by the accumulation of an abnormally folded prion protein, termed PrPSc, and the development of pathological features of astrogliosis, vacuolation, neuronal cell loss, and in some cases amyloid plaques. Although considerable structural characterization of prion protein has been reported, neither the method of conversion of cellular prion protein, PrPC, into the pathogenic isoform nor the post-translational modification processes involved is known. We report that in animal and human TSEs, one or more lysines at residues 23, 24, and 27 of PrPSc are covalently modified with advanced glycosylation end products (AGEs), which may be carboxymethyl-lysine (CML), one of the structural varieties of AGEs. The arginine residue at position 37 may also be modified with AGE, but not the arginine residue at position 25. This result suggests that nonenzymatic glycation is one of the post-translational modifications of PrP(Sc). Furthermore, immunostaining studies indicate that, at least in clinically affected hamsters, astrocytes are the first site of this glycation process.

Anti-prion antibodies for prophylaxis following prion exposure in mice.

Prion disease is characterized by a conformational change of the normal form of the prion protein (PrP(C)) to the scrapie-associated form (PrP(Sc)). Since the emergence of new variant Creutzfeldt-Jakob disease a potentially large human population is at risk for developing prion disease. Currently, no effective treatment or form of post-exposure prophylaxis is available for prion disease. We recently showed that active immunization with recombinant PrP prolongs the incubation period of scrapie. Here we show that anti-PrP antibodies following prion exposure are effective at increasing the incubation period of the infection. Stimulation of the immune system is an important therapeutic target for the prion diseases, as well as for other neurodegenerative illnesses characterized by abnormal protein conformation.

Immunization delays the onset of prion disease in mice.

The outbreak of new variant Creutzfeldt-Jakob disease has raised the specter of a potentially large population being at risk to develop this prionosis. None of the prionoses currently have an effective treatment. Recently, vaccination has been shown to be effective in mouse models of another neurodegenerative condition, namely Alzheimer's disease. Here we report that vaccination with recombinant mouse prion protein delays the onset of prion disease in mice. Vaccination was performed both before peripheral prion exposure and after exposure. A delay in disease onset was seen in both groups, but was more prolonged in animals immunized before exposure. The increase in the incubation period closely correlated with the anti-prion protein antibody titer. This promising finding suggests that a similar approach may work in humans or other mammalian species at risk for prion disease.