[show abstract][hide abstract] ABSTRACT: Yersinia pestis, the plague bacillus, has a rodent-flea-rodent life cycle but can also persist in the environment for various periods of time. There is now a convenient and effective test (F1-dipstick) for the rapid identification of Y. pestis from human patient or rodent samples, but this test cannot be applied to environmental or flea materials because the F1 capsule is mostly produced at 37°C. The plasminogen activator (PLA), a key virulence factor encoded by a Y. pestis-specific plasmid, is synthesized both at 20°C and 37°C, making it a good candidate antigen for environmental detection of Y. pestis by immunological methods. A recombinant PLA protein from Y. pestis synthesized by an Escherichia coli strain was used to produce monoclonal antibodies (mAbs). PLA-specific mAbs devoid of cross-reactions with other homologous proteins were further cloned. A pair of mAbs was selected based on its specificity, sensitivity, comprehensiveness, and ability to react with Y. pestis strains grown at different temperatures. These antibodies were used to develop a highly sensitive one-step PLA-enzyme immunoassay (PLA-EIA) and an immunostrip (PLA-dipstick), usable as a rapid test under field conditions. These two PLA-immunometric tests could be valuable, in addition to the F1-disptick, to confirm human plague diagnosis in non-endemic areas (WHO standard case definition). They have the supplementary advantage of allowing a rapid and easy detection of Y. pestis in environmental and flea samples, and would therefore be of great value for surveillance and epidemiological investigations of plague foci. Finally, they will be able to detect natural or genetically engineered F1-negative Y. pestis strains in human patients and environmental samples.
PLoS ONE 01/2013; 8(1):e54947. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The identification in the UK of 4 v-CJD infected patients thought to be due to the use of transfused Red Blood Cell units prepared from blood of donors incubating v-CJD raised major concerns in transfusion medicine. The demonstration of leucocyte associated infectivity using various animal models of TSE infection led to the implementation of systematic leuco-depletion (LD) of Red Blood cells concentrates (RBCs) in a number of countries. In the same models, plasma also demonstrated a significant level of infectivity which raised questions on the impact of LD on the v-CJD transmission risk. The recent development of filters combining LD and the capture of non-leucocyte associated prion infectivity meant a comparison of the benefits of LD alone versus LD/prion-reduction filters (LD/PR) on blood-borne TSE transmission could be made. Due to the similarity of blood/plasma volumes to human transfusion medicine an experimental TSE sheep model was used to characterize the abilities of whole blood, RBCs, plasma and buffy-coat to transmit the disease through the transfusion route. The impact of a standard RBCs LD filter and of two different RBCs LD/PR prototype filters on the disease transmission was then measured. Homologous recipients transfused with whole-blood, buffy-coat and RBCs developed the disease with 100% efficiency. Conversely, plasma, when intravenously administered resulted in an inconstant infection of the recipients and no disease transmission was observed in sheep that received cryo-precipitated fraction or supernatant obtained from infectious plasma. Despite their high efficacy, LD and LD/PR filtration of the Red Blood Cells concentrate did not provide absolute protection from infection. These results support the view that leuco-depletion strongly mitigates the v-CJD blood borne transmission risk and provide information about the relative benefits of prion reduction filters.
PLoS ONE 01/2012; 7(7):e42019. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.
Journal of Virology 12/2011; 86(4):2056-66. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC(50) of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD(50)). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention.
PLoS ONE 01/2011; 6(5):e20166. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrP(Sc) negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed.
[show abstract][hide abstract] ABSTRACT: Prion diseases are transmissible neurodegenerative disorders for which no therapeutic or prophylactic regimens exist. Passive immunization with appropriate antibodies directed against the cellular form of the prion protein (PrPC) can delay the onset of prion disease after peripheral infection, but mechanisms and parameters determining their in vivo efficacy remain unknown. In the present study, we characterized the main pharmacokinetic properties of anti-PrP antibodies in different mouse models expressing various levels of PrPC (Prnp(0/0), C57BL/6 and tga20 mice) in correlation with therapeutic effect. Plasma levels of free antibodies, total endogenous PrPC and PrPC-antibody complexes were monitored after a single intraperitoneal monoclonal antibody (mAb) injection. Efficacy in delaying PrPSc peripheral accumulation seemed to be associated with mAb capacity to form long-lasting complexes with endogenous PrPC in the plasma. In agreement with previous observations on cellular models of transmissible spongiform encephalopathy infection, we observed that injection of anti-PrP antibodies induced a large (up to 100-fold) increase in circulating PrPC. Finally, the most efficient antibody extended the lifespan of infected animals greatly. These results allowed us to define critical characteristics of anti-PrP mAbs associated with therapeutic efficacy and could constitute a useful reference for designing optimized passive immunotherapies for prion diseases.
Journal of General Virology 06/2010; 91(Pt 6):1635-45. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A) is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC) as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains). Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14) and insect cells (Sf9). After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM) and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species.
PLoS ONE 01/2010; 5(10):e13245. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrP(Sc) accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 microg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.
[show abstract][hide abstract] ABSTRACT: This paper describes a procedure for evaluating the presence and the stability of the proteinase K-resistant form of the prion protein (PrP(res)) in slaughterhouse wastewater.
Wastewater samples were spiked with either scrapie or bovine spongiform encephalopathy agents and PrP(res) was concentrated and detected by western blotting. The detection limit was estimated to be 2-4 microg of either scrapie or BSE-infected brain tissue in 15 ml of sewage. Wastewater samples from three abattoirs were analysed, two of which had processed BSE-infected animals. No PrP(res) was detected. The effect of sewage on the inoculum and the persistence of transmissible spongiform encephalopathy agents in wastewater were also considered.
The results of the assay suggest that wastewaters from abattoirs where one positive BSE case has been identified would contain titres lower than 0.6-26 x 10(-4) cattle oral ID(50) per litre resulting from specified risk material tissue contamination. Moreover, the effect of abattoir wastewaters is to reduce the persistence of PrP(res).
The assay may be a useful tool for risk assessment studies and for reducing the potential risk of contamination with BSE via sewage sludge fertilizer procedures.
Journal of Applied Microbiology 12/2008; 105(5):1649-57. · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: The bovine spongiform encephalopathy (BSE) agent has been transmitted to humans, leading to variant Creutzfeldt-Jakob disease. Sheep and goats can be experimentally infected by BSE and have been potentially exposed to natural BSE; however, whether BSE can be transmitted to small ruminants is not known. Based on the particular biochemical properties of the abnormal prion protein (PrPsc) associated with BSE, and particularly the increased degradation induced by proteinase K in the N terminal part of PrPsc, we have developed a rapid ELISA designed to distinguish BSE from other scrapie strains. This assay clearly discriminates experimental ovine BSE from other scrapie strains and was used to screen 260 transmissible spongiform encephalopathy (TSE)-infected small ruminant samples identified by the French active surveillance network (2002/2003). In this context, this test has helped to identify the first case of natural BSE in a goat and can be used to classify TSE isolates based on the proteinase K sensitivity of PrPsc.
[show abstract][hide abstract] ABSTRACT: Faeces from infected animals have been suggested as a potential source of contamination and transmission of prion diseases in the environment. This work describes the development of a procedure for the detection of PrP(res) in stools which is based on a detergent-based extraction and immunoprecipitation (IP). The procedure was evaluated by analyzing TSE-spiked sheep and mice faeces, and proved to be specific for PrP(res) with sensitivities of 5-10 microg of infected brain tissue. In order to analyze the shedding of prions, we studied stools from orally inoculated mice over 4-days post-inoculation and also stools from terminally sick scrapie-infected mice. PrP(res) was only detected in stools shortly after the oral ingestion of TSE agents. The procedure described could be a useful tool for studying the excretion of prions and for evaluating potential environmental contamination by prions.
[show abstract][hide abstract] ABSTRACT: Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K (PK) digested abnormal prion protein (PrP(res)) identified on Western blotting (type 1 or type 2). These biochemically distinct PrP(res) types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrP(res) in the brain, the basis of this classification system and its relationship to agent strain are under discussion. Different brain areas from 41 sCJD and 12 iatrogenic CJD (iCJD) cases were investigated, using Western blotting for PrP(res) and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrP(Sc)) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrP(res) were identified. Despite this, the other two biochemical assays found that PrP(Sc) from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrP(Sc) subgroups were identified that correlated with the current sCJD clinico-pathological classification. In iCJD, four similar biochemical clusters were observed, but these did not correlate to any particular PRNP 129 polymorphism or western blot PrP(res) pattern. The identification of four different PrP(Sc) biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrP(res) isoform provides an alternative biochemical definition of PrP(Sc) diversity and new insight in the perception of Human TSE agents variability.
[show abstract][hide abstract] ABSTRACT: Following the two "mad cow" crises of 1996 and 2000, there was an urgent need for rapid and sensitive diagnostic methods to identify animals infected with the bovine spongiform encephalopathy (BSE) agent. This stimulated research in the field of prion diagnosis and led to the establishment of numerous so-called "rapid tests" which have been in use in Europe since 2001 for monitoring at-risk populations (rendering plants) and animals slaughtered for human consumption (slaughterhouse). These rapid tests have played a critical role in the management of the mad cow crisis by allowing the removal of prion infected carcasses from the human food chain, and by allowing a precise epidemiological monitoring of the BSE epizootic. They are all based on the detection of the abnormal form of the prion protein (PrP(Sc) or PrP(res)) in brain tissues and consequently are only suitable for post-mortem diagnosis. Since it is now very clear that variant Creutzfeldt-Jakob disease (vCJD) can be transmitted by blood transfusion, the development of a blood test for the diagnosis of vCJD is a top priority. Although significant progress has been made in this direction, including the development of the protein misfolding cyclic amplification (PMCA) technology, at the time this paper was written, this objective had not yet been achieved. This is the most important challenge for the years to come in this field of prion research.
Veterinary Research 01/2008; 39(4):33. · 3.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: A key molecular event in prion diseases is the conversion of cellular prion protein (PrP(c)) into an abnormal misfolded conformer (PrP(sc)). The PrP(c) N-terminal domain plays a central role in PrP(c) functions and in prion propagation. Because mammalian PrP(c) is found as a full-length and N-terminally truncated form, we examined the presence and amount of PrP(c) C-terminal fragment in the brain of different species. We found important variations between primates and rodents. In addition, our data show that the PrP(c) fragment is present in detergent-resistant raft domains, a membrane domain of critical importance for PrP(c) functions and its conversion into PrP(sc).
[show abstract][hide abstract] ABSTRACT: To study the pathogenesis of bovine spongiform encephalopathy infection in small ruminants, two Lacaune sheep with the AA136RR154QQ171 and one with the AA136RR154RR171 genotype for the prion protein, were inoculated with a brain homogenate from a French cattle BSE case by peripheral routes. Sheep with the ARQ/ARQ genotype are considered as susceptible to prion diseases contrary to those with the ARR/ARR genotype. The accumulation of disease-associated prion protein (PrP(d)) was analysed by biochemical and immunohistochemical methods. No PrP(d) accumulation was detected in samples from the ARR/ARR sheep 2 years post inoculation. In the two ARQ/ARQ sheep that had scrapie-like clinical symptoms, PrP(d) was found in the central, sympathetic and enteric nervous systems and in lymphoid organs. Remarkably, PrP(d) was also detected in some muscle types as well as in all peripheral nerves that had not been reported previously thus revealing a widespread distribution of BSE-associated PrP(d) in sheep tissues.
[show abstract][hide abstract] ABSTRACT: Human brain cellular prion protein (PrP(c)) is cleaved within its highly conserved domain at amino acid 110/111/112. This cleavage generates a highly stable C-terminal fragment (C1). We examined the relative abundance of holo- and truncated PrP(c) in human cerebral cortex and we found important inter-individual variations in the proportion of C1. Neither age nor postmortem interval explain the large variability observed in C1 amount. Interestingly, our results show that high levels of C1 are associated with the presence of the active ADAM 10 suggesting this zinc metalloprotease as a candidate for the cleavage of PrP(c) in the human brain.
[show abstract][hide abstract] ABSTRACT: Based on the hypothetical proposal of Sulkowski [E. Sulkowski, FEBS Lett. 307 (2) (1992) 129] for the implication of transition metal ions in the structural changes/oligomerisation of normal cellular prion protein (PrPc) resulting in the pathological isoform (PrPsc), we focused our study on the octarepat domain of this protein which has been supposed to be the metal binding site. We have studied the copper binding to synthetic prion octarepeat peptides (PHGGGWGQ)n (n=1, 3, 6) using metal chelate and size-exclusion modes of chromatographies. This copper binding induces oligomerisation resulting in multiple aggregates. Moreover, heterogeneity of metal bound octarepeat oligomers by ESI-MS has been demonstrated. In addition, anti prion antibodies specific to the octarepeat region were used to discriminate between metal free and copper, nickel and zinc bound hexamer octarepeat peptide. Differential recognition of Cu(II) and Zn(II) bound complexes has been observed which signify differences in exposed epitopes of aggregated peptides.
Journal of Chromatography B 05/2005; 818(1):75-82. · 2.49 Impact Factor
[show abstract][hide abstract] ABSTRACT: Prion diseases are transmissible neurodegenerative disorders affecting humans and animals for which no therapeutic or prophylactic regimens exist. During the last three years several studies have shown that anti-PrP monoclonal antibodies (mAbs) can antagonize prion propagation in vitro and in vivo, but the mechanisms of inhibition are not known so far. To identify the most powerful mAbs and characterize more precisely the therapeutic effect of anti-PrP antibodies, we have screened 145 different mAbs produced in our laboratory for their capacity to cure cells constitutively expressing PrPSc. Our results confirm for a very large series of antibodies that mAbs recognizing cell-surface native PrPc can efficiently clean and definitively cure infected cells. Antibodies having a cleaning effect are directed against linear epitopes located in at least four different regions of PrP, suggesting an epitope-independent inhibition mechanism. The consequence of antibody binding is the sequestration of PrPc at the cell surface, an increase of PrPc levels recovered in cell culture medium, and an internalization of antibodies. Taken together these data suggest that the cleaning process is more likely due to a global effect on the PrP trafficking and/or transconformation process. Two antibodies, Sha31 and BAR236, show an IC50 of 0.6 nM, thus appearing 10-fold more efficient than previous antibodies described in the literature. Finally, five co-treatments were also tested, and only one of them, described previously (SAF34 + SAF61), lowered PrPSc levels in the cells synergistically.
Journal of Biological Chemistry 04/2005; 280(12):11247-58. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The central molecular event in transmissible spongiform encephalopathies, such as scrapie in sheep, is the accumulation in tissues of an abnormal isoform of the cellular prion protein. A previous investigation of 26 sheep showed that the accumulation of PrP(res) in brain correlated more with the prnp genotype than with the severity of the clinical disease. Here, the ability of a sandwich ELISA to detect PrP(res) distribution in the brain was demonstrated. Immunohistochemistry also strongly supported the hypothesis that the dorsal motor nucleus of the vagus nerve is the possible entry site in the brain for the scrapie agent. Remarkably, three asymptomatic (or possibly asymptomatic for scrapie) sheep carrying an allele known to be associated with clinical scrapie resistance (ARR), which were negative for the detection of PrP(res) by Western blotting and immunohistochemistry, were positive for the presence of PrP(res) by ELISA, raising the possibility of carriers resistant to the disease and possibly contributing to the persistence of scrapie in certain flocks.
Journal of General Virology 12/2004; 85(Pt 11):3483-6. · 3.13 Impact Factor