An outbreed mouse model of yellow fever for study of pathogenesis and development of vaccines and therapeutics

Yellow fever (YF) is a mosquito-borne viral disease that is endemic in several African and South American countries. YF virus (YFV) causes subclinical infections with mild and non-specific symptoms, to severe, potentially lethal illness with jaundice, hemorrhage, and renal failure. Despite the existence of safe and efficient vaccines, epidemics continue to occur, mostly in Africa where the burden of YF is estimated to represent 1.7 million annual cases and up to 80,000 deaths per year. Moreover, emergence of YF has been reported in new, previously unaffected areas, because of the introduction of the mosquito vectors into these lands. There is no effective therapy against YFV infection but licensed vaccines are available, which are derivatives of a live attenuated strain that was first developed in 1937. These vaccines are currently being used in vaccination program in endemic countries and for travelers visiting these regions. They provide a long-lasting immunity against all the known genotypes of YFV. Although very rare, there are reports of serious adverse effects associated with these vaccines. One major drawback of YF vaccines is their preparation that is based on culture on embryonated eggs, a fastidious and lengthy process that limits the capacity to produce high volumes of stocks needed to respond to recurring epidemics and to prepare for a potential major outbreak. An effective therapy and new types of vaccine that can support rapid scale up is needed for efficient management of YF in the future. The best available animal models to enable these endeavors are currently non-human primates (NHP) in which YF cause a disease similar to human infection of YF. However, the cost of NHP studies is a limit to preclinical studies, in particular in the most affected areas of the globe. There are a few mouse models of YF. However, these models consist of genetically-deficient rodents that are not best suited to replicate the disease and to accurately evaluate new vaccines or therapies. We have developed a mouse model of YFV infection based on the Swiss Webster outbred strain. We have tested several epidemics isolates and identified strains that, when administrated by the intraperitoneal route, caused an acute infection leading to death. Interestingly, these YFV strains are lethal only when prepared from mouse organs and not when cultured on cell lines. We used this model to test the efficacy of the 17D YFV vaccine strain in protecting mice against lethal challenge showing that the model can be used to evaluate new YF vaccines and therapies

Systematic analysis of the regulation of type three secreted effectors in Salmonella enterica serovar Typhimurium

BACKGROUND: The type III secretion system (TTSS) is an important virulence determinant of Gram-negative bacterial pathogens. It enables the injection of effector proteins into the cytosol of eukaryotic cells. These effectors ultimately manipulate the cellular functions of the infected organism. Salmonella enterica serovar Typhimurium encodes two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 that are required for the intestinal and systemic phases of the infection, respectively. However, recent studies suggest that the roles of these TTSSs are not restricted to these compartments. The regulation of TTSSs in Salmonella is very complex with several regulators operating to activate or to repress expression depending on the environmental conditions. RESULTS: We performed a systematic analysis of the regulation of type III effectors during growth in vitro. We have tested the ability of seven regulatory genes to regulate ten effector genes. Each regulator was expressed in the absence of the other six to avoid cascade effects. Our results confirm and extend the previously reported regulation of TTSS1 and TTSS2 effectors by InvF-SicA and SsrB respectively. CONCLUSION: The set of strains constructed for this study can be used to quickly and systematically study the regulation of newly identified effector genes of Salmonella enterica. The approach we have used can also be applied to study complex regulatory cascades in other bacterial species

A Self-Assembling Lanthanide Molecular Nanoparticle for Optical Imaging

Chromophores that incorporate f-block elements have considerable potential for use in bioimaging applications because of their advantageous photophysical properties compared to organic dye, which are currently widely used. We are developing new classes of lanthanide-based self-assembling molecular nanoparticles as reporters for imaging and as multi-functional nanoprobes or nanosensors for use with biological samples. One class of these materials, which we call lanthanide "nano-drums", are homogeneous 4d-4f clusters approximately 25 to 30 angstrom in diameter. These are capable of emitting from the visible to near-infrared wavelengths. Here, we present the synthesis, crystal structure, photophysical properties and comparative cytotoxicity data for a 32 metal Eu-Cd nano-drum [Eu8Cd24L12(OAc)(48)] (1). We also explored the imaging capabilities of this nano-drum using epifluorescence, TIRF, and two-photon microscopy platforms.Welch Foundation F-816, F-1018, F1515Ministry of High Education (MOHE), Malaysia under High Impact Research (HIR) - MOHE project UM.C/625/1/HIR/MoE/CHAN/13/6 H-50001-00-A000034NIH/NIAID 1U01AI078008-3Centre for Blast Injury Study at Imperial College LondonCPRIT R1003NIH-NCI CA68682National Institutes of HealthNational Science FoundationCancer Prevention Research Institute of TexasNational Science Foundation CHE-0741973Chemistr

Ciblage de protéines hétérologues chez les bactéries lactiques (vers l'utilisation de Lactococcus lactis comme véhicule de molécules utiles dans le tube digestif de l'hôte)

Les bactéries lactiques (BL) sont les microorganismes les plus utilisés dans l'industrie alimentaire. Elles sont ainsi ingérées en grande quantité au travers des aliments et certaines espèces de BL font partie de la flore naturelle de l'homme ou des animaux. Du fait de ces intérêts, les BL sont très étudiées et les connaissances accumulées sur leur biologie permettent aujourd'hui d'envisager des applications nouvelles dans lesquelles ces bactéries seront utilisées comme vecteurs pour délivrer dans l'organisme des molécules biologiquement actives. Suivant l'utilisation souhaitée, ces molécules devront être produites dans des compartiments cellulaires différents d'où la nécessité de maîtriser le ciblage de protéines hétérologues chez les BL. Mes travaux de thèse ont permis la mise au point d'un système qui permet l'expression et le ciblage de protéines hétérologues dans le cytoplasme, la paroi bactérienne ou le milieu extracellulaire chez différentes espèces de BL.. Pour optimiser l'ancrage des protéines à la paroi des BL, nous avons entrepris la caractérisation de la sortase chez Lactococus lactis, la BL modèle. La sortase est une enzyme qui fixe les protéines, de façon covalente et selon un mécanisme conservé chez les bactéries à Gram positif. Nous avons identifié deux gènes de sortase sur le génome de Lactococcus lactis et démontré l'activité d'ancrage à la paroi du produit de l'un d'entre eux. Nous avons utilisé notre système de ciblage pour exprimer des antigènes du virus de la maladie de Gumboro chez Lactococcus lactis. La maladie de Gumboro est une affection immunodépressive du poulet qui cause des pertes importantes dans les élevages avicoles. Les souches de Lactococcus lactis exprimant les antigènes viraux ont été utilisées dans des tests de vaccination orale chez le poulet. Une réponse immunitaire significative a été obtenue avec une souche exprimant une forme ancrée à la paroi de l'antigène majeur du virus de Gumboro.Lactic acid bacteria (LAB) are widely used in the alimentary industry and are thus ingested in great quantities through food products. Furthermore, certain LAB species are members of the natural flora of man and animals. Because of these interests, LAB have been actively studied and the knowledge accumulated on their biology allow today to envisage new applications in which these bacteria will be used as vectors for the delivery of biologically active molecules into the body. According to the wished use, these molecules should be produced in different cellular compartments, which stress the need of controlling the targeting of heterologous proteins in LAB My thesis works allowed the settling of a system that permits the expression and the targeting of heterologous proteins in the cytoplasm, the cell wall or the extracellular medium of various LAB species. To optimize the anchoring of proteins in the cell wall, we tried to characterize the sortase of Lactococus lactis, the model LAB. Sortase is an enzyme that covalently anchors proteins, in a mechanism universal in Gram-positive bacteria. We identified two sortase genes on Lactococcus lactis genome and demonstrated the cell-wall-anchoring activity the product of the one of them. We used our protein targeting system to express antigens from the Gumboro disease virus in Lactococcus lactis. Gumboro disease is a chicken immunodepressive affection that causes important losses in poultry breeding. Lactococcus lactis strains expressing the viral antigens were used to immunize chickens and cells expressing cell-wall-anchored antigen induced significant immune response.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

The <it>Salmonella </it>Pathogenicity Island (SPI) 1 contributes more than SPI2 to the colonization of the chicken by <it>Salmonella enterica </it>serovar Typhimurium

Abstract Background Salmonella enterica serovar Typhimurium (Typhimurium) is an important pathogen that infects a broad range of hosts. In humans, Typhimurium causes a gastroenteritis characterized by vomiting, diarrhea, and abdominal pains. Typhimurium infection occurs mainly through the ingestion of contaminated food including poultry, pork, eggs, and milk. Chickens that are asymptomatic carriers of Typhimurium constitute a potential reservoir for infection. The type three secretion systems encoded by Salmonella pathogenicity islands (SPI) 1 and 2 are major virulence factors of Salmonella. However, only a few studies have investigated their role during the infection of chickens. Results We have taken a mixed infection approach to study the contribution of SPI1 and SPI2 to the colonization of the chicken by Typhimurium. We found that SPI1 contributes to colonization of both the cecum and spleen in the chicken. In contrast, SPI2 contributes to colonization of the spleen but not the cecum and, in the absence of SPI1, inhibits cecal colonization. Additionally, we show that the contribution of SPI1 in the spleen is greater than that of SPI2. These results are different from those observed during the infection of the mouse by Typhimurium where SPI2 is the major player during systemic colonization. Conclusion The co-infection model we used provides a sensitive assay that confirms the role of SPI1 and clarifies the role of SPI2 in the colonization of the chicken by Typhimurium.</p

Streptococcus mitis induces conversion of Helicobacter pylori to coccoid cells during co-culture in vitro.

Helicobacter pylori (H. pylori) is a major gastric pathogen that has been associated with humans for more than 60,000 years. H. pylori causes different gastric diseases including dyspepsia, ulcers and gastric cancers. Disease development depends on several factors including the infecting H. pylori strain, environmental and host factors. Another factor that might influence H. pylori colonization and diseases is the gastric microbiota that was overlooked for long because of the belief that human stomach was a hostile environment that cannot support microbial life. Once established, H. pylori mainly resides in the gastric mucosa and interacts with the resident bacteria. How these interactions impact on H. pylori-caused diseases has been poorly studied in human. In this study, we analyzed the interactions between H. pylori and two bacteria, Streptococcus mitis and Lactobacillus fermentum that are present in the stomach of both healthy and gastric disease human patients. We have found that S. mitis produced and released one or more diffusible factors that induce growth inhibition and coccoid conversion of H. pylori cells. In contrast, both H. pylori and L. fermentum secreted factors that promote survival of S. mitis during the stationary phase of growth. Using a metabolomics approach, we identified compounds that might be responsible for the conversion of H. pylori from spiral to coccoid cells. This study provide evidences that gastric bacteria influences H. pylori physiology and therefore possibly the diseases this bacterium causes

Recombinant Helicobacter pylori Vaccine Delivery Vehicle: A Promising Tool to Treat Infections and Combat Antimicrobial Resistance

Antimicrobial resistance (AMR) has become a global public health threat. Experts agree that unless proper actions are taken, the number of deaths due to AMR will increase. Many strategies are being pursued to tackle AMR, one of the most important being the development of efficient vaccines. Similar to other bacterial pathogens, AMR in Helicobacter pylori (Hp) is rising worldwide. Hp infects half of the human population and its prevalence ranges from &lt;10% in developed countries to up to 90% in low-income countries. Currently, there is no vaccine available for Hp. This review provides a brief summary of the use of antibiotic-based treatment for Hp infection and its related AMR problems together with a brief description of the status of vaccine development for Hp. It is mainly dedicated to genetic tools and strategies that can be used to develop an oral recombinant Hp vaccine delivery platform that is (i) completely attenuated, (ii) can survive, synthesize in situ and deliver antigens, DNA vaccines, and adjuvants to antigen-presenting cells at the gastric mucosa, and (iii) possibly activate desired compartments of the gut-associated mucosal immune system. Recombinant Hp vaccine delivery vehicles can be used for therapeutic or prophylactic vaccination for Hp and other microbial pathogens

Cytokine response during non-cerebral and cerebral malaria: evidence of a failure to control inflammation as a cause of death in African adults

Background. With 214 million cases and 438,000 deaths in 2015, malaria remains one of the deadliest infectious diseases in tropical countries. Several species of the protozoan Plasmodium cause malaria. However, almost all the fatalities are due to Plasmodium falciparum, a species responsible for the severest cases including cerebral malaria. Immune response to Plasmodium falciparum infection is mediated by the production of pro-inflammatory cytokines, chemokines and growth factors whose actions are crucial for the control of the parasites. Following this response, the induction of anti-inflammatory immune mediators downregulates the inflammation thus preventing its adverse effects such as damages to various organs and death. Methods. We performed a retrospective, nonprobability sampling study using clinical data and sera samples from patients, mainly adults, suffering of non-cerebral or cerebral malaria in Dakar, Sénégal. Healthy individuals residing in the same area were included as controls. We measured the serum levels of 29 biomarkers including growth factors, chemokines, inflammatory and anti-inflammatory cytokines. Results. We found an induction of both pro- and anti-inflammatory immune mediators during malaria. The levels of pro-inflammatory biomarkers were higher in the cerebral malaria than in the non-cerebral malaria patients. In contrast, the concentrations of anti-inflammatory cytokines were comparable in these two groups or lower in CM patients. Additionally, four pro-inflammatory biomarkers were significantly increased in the deceased of cerebral malaria compared to the survivors. Regarding organ damage, kidney failure was significantly associated with death in adults suffering of cerebral malaria. Conclusions. Our results suggest that a poorly controlled inflammatory response determines a bad outcome in African adults suffering of cerebral malaria

Ability of Lactococcus lactis To Export Viral Capsid Antigens: a Crucial Step for Development of Live Vaccines

Thefood grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci