Protein enriched pasta: structure and digestibility of its protein network

International audienceWheat (W) pasta was enriched in 6% gluten (G), 35% faba (F) or 5% egg (E) to increase its protein content (13% to 17%). The impact of the enrichment on the multiscale structure of the pasta and on in vitro protein digestibility was studied. Increasing the protein content (W- vs. G-pasta) strengthened pasta structure at molecular and macroscopic scales but reduced its protein digestibility by 3% by forming a higher covalently linked protein network. Greater changes in the macroscopic and molecular structure of the pasta were obtained by varying the nature of protein used for enrichment. Proteins in G- and E-pasta were highly covalently linked (28-32%) resulting in a strong pasta structure. Conversely, F-protein (98% SDS-soluble) altered the pasta structure by diluting gluten and formed a weak protein network (18% covalent link). As a result, protein digestibility in F-pasta was significantly higher (46%) than in E- (44%) and G-pasta (39%). The effect of low (55 [degree]C, LT) vs. very high temperature (90 [degree]C, VHT) drying on the protein network structure and digestibility was shown to cause greater molecular changes than pasta formulation. Whatever the pasta, a general strengthening of its structure, a 33% to 47% increase in covalently linked proteins and a higher [small beta]-sheet structure were observed. However, these structural differences were evened out after the pasta was cooked, resulting in identical protein digestibility in LT and VHT pasta. Even after VHT drying, F-pasta had the best amino acid profile with the highest protein digestibility, proof of its nutritional interest

How the structure, nutritional and sensory attributes of pasta made from legume flour is affected by the proportion of legume protein

In this study, wheat in pasta was partially or completely replaced by faba to increase its protein quantity and improve its quality. Increasing the ratio of faba:wheat protein from 0:100 to 100:0 (g/g) in pasta enlarged its protein network at the microscopic scale and linearly diluted the covalently linked gluten network of wheat pasta by weakly linked proteins. A concomitant linear increase in the cooking loss (up to 2.6 fold), a decrease in resilience (up to 1.4 fold) and an increase of the in-vitro protein digestion (up to 25%) were observed in pasta. The increase in drying temperature (90 °C vs. 55 °C) promoted the covalent aggregation of proteins in all pasta, but was more efficient in legume pasta, enhancing their resilience and reducing their cooking loss, without altering the degree of protein hydrolysis. This was partly explained by the reduction in trypsin inhibitory activity in all legume pasta dried at 90 °C. Interestingly, scores for sensory attributes such as liking attributed to pasta containing 80% faba-protein were close to scores given to a commercial whole wheat pasta. Pasta made exclusively from faba dried at 55 °C or 90 °C tended to be liked more than their commercial gluten-free counterparts

Protein structure versus Nutrional and sensory properties of legume enriched pasta

Wheat pasta is a convenient starchy food presenting interesting low glycemic index as a result of its compact structure made of a gluten network entrapping starch granules. However, its protein content remains low (12-13%), its amino acid profile unbalanced and it is unsuitable for gluten-intolerant people. The aim of this study was to produce pasta enriched in protein and in lysine by incorporation of Faba flour (0-100%) and to evaluate the impact of such enrichment on pasta structure and nutritional properties

Qualités culinaires, sensorielles et nutritionnelles de pâtes alimentaires sansgluten à base de légumineuses

Ce numéro est constitué d’articles issus des « 1ères rencontres francophones sur les légumineuses » (RFL1), organisées les 31 mai et 1er juin 2016 à Dijon.Durum wheat semolina is traditionally used to produce pasta by successive hydration, mixing, extrusionand drying steps. These steps give a compact structure to pasta, with a protein network entrappingstarch granules. This specific structure is responsible for the low glycemic index of pasta and for itsorganoleptic qualities. However, wheat pasta remains nutritionally poor in lysine, an essential aminoacid. In addition, wheat pasta consumption can cause metabolic disorders in gluten intolerant people.Some gluten-free pasta made of gluten-free cereal are available on the market, but remains low inprotein. Legumes as a rich source in protein, lysine, and slowly digested carbohydrates, would be aninteresting raw material to produce gluten-free pasta. Gluten-free spaghetti made exclusively fromlegume flours have been produced; their culinary, sensory and nutritional properties are characterizedand compared to those of a 100% wheat and a commercial gluten-free cereal pasta. This studydemonstrates the nutritional benefit of legume pasta notably for the gluten intolerant people.Les pâtes alimentaires sont traditionnellement fabriquées à partir de semoule de blé dur en France.Leur procédé de fabrication, la pastification, consiste à hydrater, malaxer, extruder et sécher la pâte, luiconférant une structure finale compacte faite d’un réseau protéique enserrant les granules d’amidon.Cette structure spécifique est à l’origine de leur faible indice glycémique et de leurs qualitésorganoleptiques. D’un point de vue nutritionnel, les pâtes au blé demeurent néanmoins pauvres encertains acides aminés indispensables comme la lysine. Leur consommation engendrerait égalementdes désordres métaboliques chez les personnes intolérantes au gluten. Certaines pâtes fabriquées àpartir de céréales sans gluten sont aujourd’hui commercialisées, mais elles demeurent pauvres enprotéines. Les légumineuses, grâce à leur richesse en protéines et en lysine, et leur faible indiceglycémique, représenteraient une alternative pour la production de spaghetti sans gluten. Desspaghettis sans gluten ont ainsi été fabriqués exclusivement à partir de farines de légumineuses ; leurspropriétés culinaires, sensorielles et nutritionnelles ont été caractérisées et comparées à celles de pâtes100% blé et de pâtes aux céréales sans gluten du commerce. Cette étude montre l’intérêt potentiel depâtes 100% légumineuses en nutrition humaine notamment pour les populations intolérantes au gluten

A Vaccine Platform against Arenaviruses Based on a Recombinant Hyperattenuated Mopeia Virus Expressing Heterologous Glycoproteins

International audienceSeveral Old World and New World arenaviruses are responsible for severe endemic and epidemic hemorrhagic fevers, whereas other members of the Arenaviridae family are nonpathogenic. To date, no approved vaccines, antivirals, or specific treatments are available, except for Junín virus. However, protection of nonhuman primates against Lassa fever virus (LASV) is possible through the inoculation of the closely related but nonpathogenic Mopeia virus (MOPV) before challenge with LASV. We reasoned that this virus, modified by using reverse genetics, would represent the basis for the generation of a vaccine platform against LASV and other pathogenic arenaviruses. After showing evidence of exoribonuclease (ExoN) activity in NP of MOPV, we found that this activity was essential for multiplication in antigen-presenting cells. The introduction of multiple mutations in the ExoN site of MOPV NP generated a hyperattenuated strain (MOPVExoN6b) that is (i) genetically stable over passages, (ii) has increased immunogenic properties compared to those of MOPV, and (iii) still promotes a strong type I interferon (IFN) response. MOPVExoN6b was further modified to harbor the envelope glycoproteins of heterologous pathogenic arenaviruses, such as LASV or Lujo, Machupo, Guanarito, Chapare, or Sabia virus in order to broaden specific antigenicity while preserving the hyperattenuated characteristics of the parental strain. Our MOPV-based vaccine candidate for LASV, MOPEVACLASV, was used in a one-shot immunization assay in nonhuman primates and fully protected them from a lethal challenge with LASV. Thus, our hyperattenuated strain of MOPV constitutes a promising new live-attenuated vaccine platform to immunize against several, if not all, pathogenic arenaviruses.IMPORTANCE Arenaviruses are emerging pathogens transmitted to humans by rodents and responsible for endemic and epidemic hemorrhagic fevers of global concern. Nonspecific symptoms associated with the onset of infection make these viruses difficult to distinguish from other endemic pathogens. Moreover, the unavailability of rapid diagnosis in the field delays the identification of the virus and early care for treatment and favors spreading. The vaccination of exposed populations would be of great help to decrease morbidity and human-to-human transmission. Using reverse genetics, we generated a vaccine platform for pathogenic arenaviruses based on a modified and hyperattenuated strain of the nonpathogenic Mopeia virus and showed that the Lassa virus candidate fully protected nonhuman primates from a lethal challenge. These results showed that a rationally designed recombinant MOPV-based vaccine is safe, immunogenic, and efficacious in nonhuman primates

Pathogenesis of recent Lassa virus isolates from lineages II and VII in cynomolgus monkeys

We thank P. Regnard, P.H. Moreau, and L. Fellmann (SILABE, Strasbourg) for medical care given to the monkeys. We thank S. Mundweiller, S. Godard, E. Moissonnier, D. Thomas, S. Mely, B. Labrosse, D. Pannetier, and C. Leculier (P4 INSERM–Jean Merieux, US003, INSERM) for assistance in conducting the BSL-4 experiments. We thank M-A Dillies (HUB, Institut Pasteur) for helpful discussions. We thank G. Fourcaud and B. Lafoux (UBIVE, CIRI, Institut Pasteur) for technical help. We also thank L. Branco (Zalgen Labs) for providing recombinant proteins. We are grateful to the Coalition for Epidemic Preparedness and Innovations (R. Hatchett, G. Thiry, and M. Saville) and C. Gerke (Department of Innovation Development, Institut Pasteur) for invaluable support.International audienceThe area of Lassa virus (LASV) circulation is expanding, with the emergence of highly pathogenic new LASV lineages. Benin recently became an endemic country for LASV and has seen the emergence of a new LASV lineage (VII). The first two outbreaks in 2014 and 2016 showed a relatively high mortality rate compared to other outbreaks. We infected cynomolgus monkeys with two strains belonging to lineage II and lineage VII that were isolated from deceased patients during the 2016 outbreak in Benin. The lineage VII strain (L7) caused uniform mortality. Death was associated with uncontrolled viral replication, unbalanced inflammatory responses characterized by increased concentrations of pro- and anti-inflammatory mediators, and the absence of efficient immune responses, resembling the pathogenesis associated with the prototypic Josiah strain in monkeys. The lineage II strain (L2) showed apparently lower virulence than its counterpart, with a prolonged time to death and a lower mortality rate. Prolonged survival was associated with better control of viral replication, a moderate inflammatory response, and efficient T-cell responses. Transcriptomic analyses also highlighted important differences in the immune responses associated with the outcome. Both strains caused strong inflammation in several organs. Notably, meningitis and encephalitis were observed in the cerebral cortex and cerebellum in all monkeys, independently of the outcome. Due to their apparently high pathogenicity, emerging strains from lineage VII should be considered in preclinical vaccine testing. Lineage II would also be beneficial in pathogenesis studies to study the entire spectrum of Lassa fever severity

Systemic viral spreading and defective host responses are associated with fatal Lassa fever in macaques

International audienceLassa virus (LASV) is endemic in West Africa and induces a viral hemorrhagic fever (VHF) with up to 30% lethality among clinical cases. The mechanisms involved in control of Lassa fever or, in contrast, the ensuing catastrophic illness and death are poorly understood. We used the cynomolgus monkey model to reproduce the human disease with asymptomatic to mild or fatal disease. After initial replication at the inoculation site, LASV reached the secondary lymphoid organs. LASV did not spread further in nonfatal disease and was rapidly controlled by balanced innate and T-cell responses. Systemic viral dissemination occurred during severe disease. Massive replication, a cytokine/chemokine storm, defective T-cell responses, and multiorgan failure were observed. Clinical, biological, immunological, and transcriptomic parameters resembled those observed during septic-shock syndrome, suggesting that similar pathogenesis is induced during Lassa fever. The outcome appears to be determined early, as differentially expressed genes in PBMCs were associated with fatal and non-fatal Lassa fever outcome very early after infection. These results provide a full characterization and important insights into Lassa fever pathogenesis and could help to develop early diagnostic tools

A single-shot Lassa vaccine induces long-term immunity and protects cynomolgus monkeys against heterologous strains

Acknowledgments: We thank P. Regnard (Silabe, Strasbourg) for medical care given to the monkeys. We thank S. Mundweiller, S. Godard, E. Moissonnier, D. Thomas, S. Mély, B. Labrosse, D. Pannetier, and C. Léculier (P4 INSERM–Jean Mérieux, US003, INSERM) for assistance in conducting the BSL-4 experiments. We are grateful to G. Fourcaud and B. Lafoux (Institut Pasteur, CIRI, Lyon) for technical help with histological studies. We thank S. Becker for providing us with the Josiah strain and T. G. Ksiasek, P. E. Rollin, and P. Jahrling for the LASV monoclonal antibodies. We also thank L. Branco (Zalgen Labs) for providing recombinant proteins. We are grateful to THEMIS Bioscience GmbH, a wholly owned subsidiary of Merck & Co. Inc. (E. Tauber, A. Kort, K. Ramsauer, S. Schrauf, Y. Tomberger, and R. Tschismarov), to the Coalition for Epidemic Preparedness and Innovations (R. Hatchett, G. Thiry, and M. Saville), and to C. Gerke (Department of Innovation Development, Institut Pasteur) for invaluable supportInternational audienceA safe and protective Lassa virus vaccine is crucially needed in Western Africa to stem the recurrent outbreaks of Lassa virus infections in Nigeria and the emergence of Lassa virus in previously unaffected countries, such as Benin and Togo. Major challenges in developing a Lassa virus vaccine include the high diversity of circulating strains and their reemergence from 1 year to another. To address each of these challenges, we immunized cynomolgus monkeys with a measles virus vector expressing the Lassa virus glycoprotein and nucleoprotein of the prototypic Lassa virus strain Josiah (MeV-NP). To evaluate vaccine efficacy against heterologous strains of Lassa virus, we challenged the monkeys a month later with heterologous strains from lineage II or lineage VII, finding that the vaccine was protective against these strains. A second cohort of monkeys was challenged 1 year later with the homologous Josiah strain, finding that a single dose of MeV-NP was sufficient to protect all vaccinated monkeys. These studies demonstrate that MeV-NP can generate both long-lasting immune responses and responses that are able to protect against diverse strains of Lassa virus

Hemostasis defects underlying the hemorrhagic syndrome caused by Mammarenaviruses in a cynomolgus macaque model

Viral hemorrhagic fevers (HF) are a group of acute febrile diseases with high mortality rates. While hemostatic dysfunction appears to be a major determinant of the severity of the disease, it is still unclear what pathogenic mechanisms lead to it. In clinical studies, arenaviruses such as Lassa, Machupo and Guanarito viruses caused HF that vary in symptoms and biological alterations. In this study we aimed to characterize the hemostatic dysfunction induced by arenaviral HF to determine its implication in the severity of the disease and to elucidate the origin of this syndrome. We found that lethal infection with Machupo, Guanarito and Lassa viruses is associated with cutaneo-mucosal, cerebral, digestive and pulmonary hemorrhages. The affected animals developed a severe alteration of the coagulation system, which was concomitant with acute hepatitis, minor deficit of hepatic factor synthesis, presence of a plasmatic inhibitor of coagulation and dysfunction of the fibrinolytic system. Despite signs of increased vascular permeability, endothelial cell infection was not a determinant factor of the hemorrhagic syndrome. There were also alterations of the primary hemostasis during lethal infection, with moderate to severe thrombocytopenia and platelet dysfunction. Finally, we show that lethal infection is accompanied by a reduced hematopoietic potential of the bone marrow. This study provides an unprecedented characterization of the hemostasis defects induced by several highly pathogenic Arenaviruses