Transmissibility of Atypical Scrapie in Ovine Transgenic Mice: Major Effects of Host Prion Protein Expression and Donor Prion Genotype

Atypical scrapie or Nor98 has been identified as a transmissible spongiform encephalopathy (TSE) that is clearly distinguishable from classical scrapie and BSE, notably regarding the biochemical features of the protease-resistant prion protein PrPres and the genetic factors involved in susceptibility to the disease. In this study we transmitted the disease from a series of 12 French atypical scrapie isolates in a transgenic mouse model (TgOvPrP4) overexpressing in the brain ∼0.25, 1.5 or 6× the levels of the PrPARQ ovine prion protein under the control of the neuron-specific enolase promoter. We used an approach based on serum PrPc measurements that appeared to reflect the different PrPc expression levels in the central nervous system. We found that transmission of atypical scrapie, much more than in classical scrapie or BSE, was strongly influenced by the PrPc expression levels of TgOvPrP4 inoculated mice. Whereas TgOvPrP4 mice overexpressing ∼6× the normal PrPc level died after a survival periods of 400 days, those with ∼1.5× the normal PrPc level died at around 700 days. The transmission of atypical scrapie in TgOvPrP4 mouse line was also strongly influenced by the prnp genotypes of the animal source of atypical scrapie. Isolates carrying the AF141RQ or AHQ alleles, associated with increased disease susceptibility in the natural host, showed a higher transmissibility in TgOvPrP4 mice. The biochemical analysis of PrPres in TgOvPrP4 mouse brains showed a fully conserved pattern, compared to that in the natural host, with three distinct PrPres products. Our results throw light on the transmission features of atypical scrapie and suggest that the risk of transmission is intrinsically lower than that of classical scrapie or BSE, especially in relation to the expression level of the prion protein

Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay

Staphylococcal food poisoning (SFP) is one of the most common foodborne diseases worldwide, resulting from the ingestion of staphylococcal enterotoxins (SEs), primarily SE type A (SEA), which is produced in food by enterotoxigenic strains of staphylococci, mainly S. aureus. Since newly identified SEs have been shown to have emetic properties and the genes encoding them have been found in food involved in poisoning outbreaks, it is necessary to have reliable tools to prove the presence of the toxins themselves, to clarify the role played by these non-classical SEs, and to precisely document SFP outbreaks. We have produced and characterized monoclonal antibodies directed specifically against SE type G, H or I (SEG, SEH or SEI respectively) or SEA. With these antibodies, we have developed, for each of these four targets, highly sensitive, specific, and reliable 3-h sandwich enzyme immunoassays that we evaluated for their suitability for SE detection in different matrices (bacterial cultures of S. aureus, contaminated food, human samples) for different purposes (strain characterization, food safety, biological threat detection, diagnosis). We also initiated and described for the first time the development of monoplex and quintuplex (SEA, SE type B (SEB), SEG, SEH, and SEI) lateral flow immunoassays for these new staphylococcal enterotoxins. The detection limits in buffer were under 10 pg/mL (0.4 pM) by enzyme immunoassays and at least 300 pg/mL (11 pM) by immunochromatography for all target toxins with no cross-reactivity observed. Spiking studies and/or bacterial supernatant analysis demonstrated the applicability of the developed methods, which could become reliable detection tools for the routine investigation of SEG, SEH, and SEI

SipD and IpaD induce a cross-protection against Shigella and Salmonella infections

International audienceSalmonella and Shigella species are food- and water-borne pathogens that are responsible for enteric infections in both humans and animals and are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of strains resistant to antibiotics require the development of broadly protective therapies. Those bacteria utilize a Type III Secretion System (T3SS), necessary for their pathogenicity. The structural proteins composing the T3SS are common to all virulent Salmonella and Shigella spp., particularly the needle-tip proteins SipD (Salmonella) and IpaD (Shigella). We investigated the immunogenicity and protective efficacy of SipD and IpaD administered by intranasal and intragastric routes, in a mouse model of Salmonella enterica serotype Typhimurium (S. Typhimurium) intestinal challenge. Robust IgG (in all immunization routes) and IgA (in intranasal and oral immunization routes) antibody responses were induced against both proteins. Mice immunized with SipD or IpaD were protected against lethal intestinal challenge with S. Typhimurium or Shigella flexneri (100 Lethal Dose 50%). We have shown that SipD and IpaD are able to induce a cross-protection in a murine model of infection by Salmonella and Shigella. We provide the first demonstration that Salmonella and Shigella T3SS SipD and IpaD are promising antigens for the development of a cross-protective Salmonella-Shigella vaccine. These results open the way to the development of cross-protective therapeutic molecules

La protéine prion : structure, dynamique et conversion

En 1982, S.B. Prusiner a émis l’hypothèse selon laquelle l’agent infectieux des encéphalopathies spongiformes était uniquement constitué d’une protéine, la protéine prion. Celle-ci, une protéine cellulaire normale, peut adopter une conformation pathologique qui, ensuite, convertit la forme normale en forme pathologique, la protéine infectieuse. Cette hypothèse pose de nombreux problèmes relevant de la biologie structurale des protéines. Nos connaissances sur la structure spatiale et le repliement de la protéine prion ont progressé de façon importante et nous essayons de concilier les résultats expérimentaux actuels avec l’hypothèse prion. Si la structure de la protéine recombinante ne révèle rien de particulier, son repliement dans des conditions acides permet d’atteindre une structure en feuillet ß similaire à celle de la protéine trouvée chez les animaux infectés, mais qui, malheureusement, n’est pas infectieuse. La participation de co-facteurs comme les chaperons est évoqué, mais n’a pas permis d’atteindre la structure infectieuse. L’analyse des résultats expérimentaux ne permet ni d’infirmer ni de confirmer l’hypothèse prion, mais des aspects importants restent encore à explorer

Multititration: The New Method for Implementing Ultrasensitive and Quantitative Multiplexed In-Field Immunoassays Despite Cross-Reactivity?

International audienceThe accurate in-field titration of multiple pathogens is essential to efficiently describe and monitor environmental or biological contamination, isolate, act, and treat adequately. This underscores the requirement of portable, fast, quantitative, and multiplexed detection technologies, which, however, have not been properly developed so far, notably because it has been hindered by the phenomenon of cross-reactivity. In this work, we proposed a new analytical method based on the imaging through a portable device of lanthanide-based nanoparticles (YVO 4 :Eu) for spatially multiplexed detection, relying on a multiparameter analysis, i.e., a simultaneous analysis of all of the luminescence signals through the comparison to a calibration surface built in the presence of multiple analytes of interest. We then demonstrated the possibility to simultaneously quantify by multiplexed lateral flow assay (xLFA) the three enterotoxins SEG, SEH, and SEI in unknown mixtures, over two concentration decades (from a dozen of pg•mL −1 to few ng•mL −1). Assays were performed in less than an hour (25 min of strip migration followed by 30 min of drying at room temperature), the time during which the presence of the operator was not required for more than 5 min, in order to dip the strip and have it imaged by the reader. The concepts of nominal concentration recovery, coefficient of variation (CV), limit of blank (LOB), and limit of detection (LOD) were discussed in detail in the context of multiplexed assays. With our new definitions, quantitative results demonstrated a high recovery of the nominal concentrations (115%), reliability (CV = 20%), and sensitivity (LOBs of 3, 27, and 6 pg•mL −1 for SEG, SEH, and SEI respectively, and LODs of 6, 48, and 11 pg•mL −1 for SEG, SEH, and SEI, respectively). Based on this method, we observed an increase in sensitivity of 100 compared to the other multiplexed LFA labeled with gold particles and we approached the sensitivity of the simplex enzyme-linked immunosorbent assay (ELISA) performed with the same capture and detection antibodies. To conclude, our results, which are applicable to virtually any kind of multiplexed test, pave the way to the next generation of in-field analytical immunoassays by providing fast, quantitative, and highly sensitive multiplexed detection of biomarkers or pathogens

Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells

International audiencePrion 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

Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay

International audienceStaphylococcal food poisoning (SFP) is one of the most common foodborne diseases worldwide, resulting from the ingestion of staphylococcal enterotoxins (SEs), primarily SE type A (SEA), which is produced in food by enterotoxigenic strains of staphylococci, mainly S. aureus. Since newly identified SEs have been shown to have emetic properties and the genes encoding them have been found in food involved in poisoning outbreaks, it is necessary to have reliable tools to prove the presence of the toxins themselves, to clarify the role played by these non-classical SEs, and to precisely document SFP outbreaks. We have produced and characterized monoclonal antibodies directed specifically against SE type G, H or I (SEG, SEH or SEI respectively) or SEA. With these antibodies, we have developed, for each of these four targets, highly sensitive, specific, and reliable 3-h sandwich enzyme immunoassays that we evaluated for their suitability for SE detection in different matrices (bacterial cultures of S. aureus, contaminated food, human samples) for different purposes (strain characterization, food safety, biological threat detection, diagnosis). We also initiated and described for the first time the development of monoplex and quintuplex (SEA, SE type B (SEB), SEG, SEH, and SEI) lateral flow immunoassays for these new staphylococcal enterotoxins. The detection limits in buffer were under 10 pg/mL (0.4 pM) by enzyme immunoassays and at least 300 pg/mL (11 pM) by immunochromatography for all target toxins with no cross-reactivity observed. Spiking studies and/or bacterial supernatant analysis demonstrated the applicability of the developed methods, which could become reliable detection tools for the routine investigation of SEG, SEH, and SEI

Experimental TSEs transmission results to TgOvPrP4 ovine transgenic mice.

(1)<p>Animals were inoculated intracerebrally with 2 mg brain tissue.</p>(2)<p>Genotype of sheep or goat (amino acids 136, 141, 154, and 171).</p><p>Experimental inoculums were divided into 2 different groups. Group 3: small ruminants intracerebrally inoculated with various TSEs. Group 4: experimental strains initially derived from wild-type mice. Detection of PrP^res/PrP^sc was performed by Western blot or IHC analyses.</p

Natural scrapie isolates transmission results to TgOvPrP4 ovine transgenic mice.

(1)<p>Animals were inoculated intracerebrally with 2 mg brain tissue.</p>(2)<p>Genotype of sheep or goat (amino acids 136, 141, 154, and 171).</p>(Goat)<p>Goat isolate.</p>(α)<p>Isolates detected in the flock α.</p>(β)<p>Isolates detected in the flock β.</p><p>Natural inoculums were divided into 4 different groups, all originating from active surveillance of TSEs in small ruminants. Group 1a: atypical scrapie cases with <i>prnp</i> alleles associated with increased susceptibility to atypical scrapie (AF₁₄₁RQ or AHQ). Group 1b: atypical scrapie cases without <i>prnp</i> alleles associated with increased susceptibility to atypical scrapie. Group 2: classical usual scrapie cases. Group 3: CH1641-like scrapie cases. Detection of PrP^res/PrP^sc was performed by Western blot or IHC analyses.</p

Effects of variable PrP^c expression levels in TgOvPrP4 mice on TSEs transmission.

<p>Survival periods of PrP^sc positive TgOvPrP4 mice are shown according the level of PrP^c expression (∼0.25, 1.5 or 6× the PrP^c level expressed in a sheep brain control) and to the inoculums; Group 1a: atypical scrapie isolates carrying the AF₁₄₁RQ or AHQ alleles, Group 1b: atypical scrapie isolates without the AF₁₄₁RQ or AHQ alleles, Group 2: usual classical scrapie isolates, Group 3: CH1641-like scrapie isolates, Group 4: experimental isolates (SSBP1, CH1641, BSE^OVINE), Group 5: mouse-adapted strains (BSE, C506M3, 87V). (A) Transmission results. (B) Kaplan-Meier survival curves of PrP^res/PrP^sc positive mice expressing ∼1.5 (blue line) or ∼6× (red line) the PrP^c level expressed in a sheep brain control for each of the 6 different groups. p-values represent log-rank differences in cumulative survival between mice expressing ∼1.5 and ∼6× the PrP^c level expressed in a sheep brain control.</p