Find an efficient solution for neutralization of organophosphorus nerve agents in vivo, in order to protect or cure people is a concern of public health. Such organophosphorus-based compounds are used as chemical weapons or pest control agents. The need of a biocompatible mean for their neutralization takes into account both their potential use within wartime or terrorist actions, and acute or chronic intoxications by insecticides. This project aims at unifying the efforts of two research groups in order to develop catalytic biocompatible scavengers. Depending on their biocompatibility, these agents will be used either in vivo as prophylactic or curative means, or will be incorporated into creams or foams for skin, mucosae and wound decontamination. Organophosphorus compounds (OP) are among the most toxic compounds synthesized due to their irreversible inhibition of acetylcholinesterase. A catalytic bioscavenger present in the bloodstream can neutralize the neurotoxics before they reach their biological target at the synapse or neuromuscular junction. Injected bioscavenger will be an important pretreatment tool in case of severe intoxication risk, for example for military personnel going in a conflict area where neurotoxics are potentially used, or for civilian emergency personnel going to the site of a chemical terrorist attack. For intoxicated people, a massive dose of bioscavenger can be injected to rapidly eliminate the toxic from the body, thus leading to a faster recovery. Human butyrylcholinesterase (BChE) is an enzyme for which modifying its catalytic activity has been an ongoing subject of interest. Considerable interest has been shown in BChE because it hydrolyses a wide range of toxic esters, including heroin and cocaine, and because it scavenges organophosphorus pesticides and nerve agents. Redesign of its active site has improved its cocaine hydrolase activity 2000-fold, turning it into a very efficient cocaine-detoxifying tool. This BChE variant is effective at detoxifying cocain in vivo, and we propose a similar approach for neurotoxics in the present project. IRBA teams and others have explored the development of catalytic scavenger for OP molecules during the last decades. Nowadays, the BChE G117H variant and human paraoxonase (PON1) have a catalytic activity against OP, but this activity is not efficient enough for practical use. In 2008, Baker succeeded in modifying the activity of a protein by adding an appropriate chemical function with the help of molecular modeling and bioinformatics tools. The aim here is to use these tools to modify BChE in order to introduce a performant OP hydrolase activity. Since 2002, our institute develops a softwaresoftware called SuMo whichSuMo, which will be included in the general methodology published by Baker. The use of this software will unlock the limitation of the choice of scaffold protein. Each protein constructed and validated in silico will be synthesized by the IRBA team and tested for hydrolase activity on nerve agents and pesticide. The lead hit will constitute the active ingredient of new medical countermeasure against the intoxication by organophosphorus neurotoxics.
Emergence of viral pathogens is a serious public health problem. Some highly pathogenic RNA viruses provoke severe and sometimes fatal human diseases called viral hemorrhagic fevers (VHF). These viruses are of strategic relevance for the French nation. They can potentially emerge in some French territories and affect civilian populations. Furthermore, they are of particular interest for the French army since these pathogens (i) are considered as potential viral bioterrorism agents and (ii) might be present in regions where French troops are deployed. The therapeutics currently used against viruses involved vaccines and antiviral molecules, which have proven to be highly effective in the past. However, the current model of antiviral drug discovery and development is still focused on a given virus, very expensive, and takes a long time to achieve marketing authorization approval. New approaches are therefore needed to address the urgent threats posed by viruses that cause VHF. Indeed, these ‘neglected’ viral pathogens, without clearly identified commercial market cannot take advantage of specific developments at a global scale. This project will therefore aim to unlock these scientific barriers. In order to develop new therapeutics against VHF, the following strategy will be applied: -We will use broad-spectrum antiviral molecules. This strategy will also be potentially advantageous in case of new viral emergence phenomena. -We will use some antiviral molecules already approved in industrialized countries. These drugs will allow for rapid therapeutic proposals to be considered. -Association of two antiviral molecules will be tested. Indeed, a disadvantage of using broad-spectrum molecules is often the need of using high doses to obtain satisfactory antiviral activity. Thus, during this project, a panel of broad-spectrum antiviral molecules with different modes of action will be evaluated on a set of highly pathogenic viruses of the Arenaviridae, Filoviridae and Flaviviridae families and with members of Bunyavirales that cause VHF. Some of these viruses will be manipulated in biosafety level-4 laboratory. The project will take place in three successive stages: -In vitro analysis of each broad-spectrum antiviral compound alone. -In vitro analysis of the best compounds in association two by two in order to find synergetic effects. -In vivo analysis of the best compound associations. A smaller panel of three virus will be use at this stage. The objective of this project is to (i) study systematically in vitro and in vivo activity of broad-spectrum antiviral compounds used alone or in association to find synergetic effects and (ii) to provide at the end one or two associations of broad-spectrum antiviral compounds. In addition, preferably in association with an industrial partner, the development of these therapeutics could subsequently involve a study using non-human primates. This could be the subject of an application for Astrid Maturation funding. Finally, this project will provide a dedicated, structured platform with validated protocols able of characterizing in vitro and in vivo antiviral molecules against highly pathogenic viruses, which could be used in the future by industrial partners or public laboratories.
Exposure to ionizing radiation can have serious consequences for the health of exposed people and potentially impact many victims. There is now well-established scientific knowledge of the health effects induced by different dose levels and their associated kinetics of occurrence. Thus, once the first triage has been carried out to identify irradiated victims on the basis of clinical symptoms, it is necessary to refine the evaluation of the dose received in order to make a precise diagnosis / prognosis for the remaining asymptomatic victims. During insidious scenarios, When the assessement of exposure condition is difficult or impossible, the quantification, on samples taken from the victims, of radio-induced chemical or biological effects is more suitable for an individualized dose reconstruction compared to theoretical calculation or Monte-Carlo simulations in regards to theirs great uncertainties. The standard and reference technique for biological dosimetry is the quantification of chromosomal aberrations in circulating lymphocytes. Its robustness and sensitivity range (from 150 mGy to 5 Gy) make it highly suitable both for the short-term diagnosis / prognosis phase of asymptomatic victims as well as for their long-term follow-up. However, the automated solutions proposed for the aberration recognition suffer up to day from lack of precision, especially in low doses due to the failure to eliminate false positive. The INCREASED project aims to adapt the most powerful algorithms of modern artificial intelligence to the context of automatic detection of chromosomal aberrations in dosimetry. This project proposes to revisit the semi-automatic or automatic detection methods currently used for the recognition of dicentrics in GIEMSA imagery in the light of the most recent advances in artificial intelligence and deep learning. These modern methods, which demonstrated their indisputable superiority in other areas of computer vision, will be deployed on GIEMSA images for an exhaustive multi-class count not only of dicentric chromosomes but also of ring-centric aberrations, acentric fragments , and even tricentrics. Furthermore, the INCREASED initiative is also quite innovative in dosimetry based on FISH imaging. To date, this type of dosimetric reconstruction is based on manual counting of non-exhaustive and very simplified annotations of the different possible forms of translocations. This protocol is therefore as heavy as imprecise. The INCREASED project offers two major improvements in this context: firstly in terms of exhaustive annotations of the different forms of observable FISH translocations (3 colors) through an universal and rigorous scoring. Secondly, the use of the most modern artificial intelligence algorithms able to detect and interpret co-locations/co-neighborhoods as different kind of translocations. The project shows a very strong dual character due to its applications targeting soldiers in operations, civilians in an accident context (NRBC-E, medical… etc) as well as the monitoring follow-up of military personnel, medical or industrial potentially exposed during their activity. Beyond the dosimetry framework, the results and methodologies developed during the INCREASED project could be applied to many fields based on massive cytogenetic images analyses.
Bacteriophages are currently used in different countries to disinfect instruments, rooms, and food products. They are applied to treat acute and chronic infections in humans, animals and plants, and sometimes for prophylaxis in patients. Bacteriophages are also studied to face highly pathogenic bacteria that could represent a bioterrorism threat. In France, phage therapy is not presently allowed but some compassionate assays have been performed. Others are ongoing, notably to treat infection by multidrug resistant (MDR) Pseudomonas aeruginosa, although it could be also interesting to use phages early during the evolution of an infection, similarly to what is done in Georgia, Poland and Russia, i.e. before failure of classical treatment is recognized. At least two potential applications could lead to a renewed usage of phage therapy: non-chemical decontamination of instruments or rooms, and fight against MDR germs. An elevated frequency of MDR strains belonging to international clones is observed in the case of P. aeruginosa and Acinetobacter baumannii, two species frequently isolated in burn patients, or during post-surgery infections, particularly in soldiers with war wounds. Members of these clones often carry plasmids or transposons bearing antibiotic resistance genes. In addition some clones show a high capacity to form biofilms. In this context, it is essential to possess a collection of phages active against a large spectrum of strains, and, for maximal security, to anticipate the risk caused by phages and bacteria co-evolution after infection, in order to eliminate all danger for humans, and to limit the emergence of resistant bacteria. Progress of genome analysis techniques (next generation sequencing and bioinformatics) allows to examine this old approach with a new perspective. The project intends to constitute, for three bacterial species of interest in military medicine, a collection of strains resisting classical therapy, then to be able to elaborate phage cocktails with a strong efficiency against these strains. The use of cocktails rather than unique phages should lower the risk of emergence of resistant strains. The phage genomes will be sequenced and annotated, and their interactions with bacterial strains will be studied to answer 4 key questions addressing security issues: (i) is there a risk that the selected phages insert their DNA into the host genome, introducing new toxic genes and contributing to the emergence of more dangerous bacteria? (ii) during the lytic cycle, is there a risk that phages evolve through gene exchanges, between them or with the bacterial host? (iii) how frequent will be the emergence of bacteria resistant to different phages and will their resistance to antibiotics be affected? Similarly, will the bacteria accumulating antibiotic resistance see their phage susceptibility changing? Are there bacteria naturally resisting to all phages? (iv) What is the risk of transferring antibiotic resistance from one strain to another through phage transduction? The problem of DNA transfer by phages is considered unimportant as long as virulent phages are used. This is not proven and it is known that generalized transduction is possible at a low but not insignificant frequency when selection by antibiotics is used.
The tolerance limits of the human head to impacts have been mainly studied in the field of automotive industry, in connection with road safety. Nonetheless, head impacts are also frequent and need to be further studied in the field of defense. In particular, the effects of head exposure to low-level blasts or LLB have been recently of interest in a huge number of clinical and preclinical studies. This “craze” is linked to awareness that soldiers and law enforcement members are frequently, if not daily, exposed to these types of blasts. The latter, in the context of their professional activities, are required to use different types of firearms or explosives, as it is the case for breachers, snipers or mortar men. Although the underlying mechanisms have not been elucidated yet and without any real epidemiological study in support, it seems that chronic, repetitive, exposure to LLB can significantly alter the central nervous system. If the hypothesis according to which repeated exposure to LLB is associated with brain injuries and chronic neurological deficits is verified (or true), it will therefore become necessary to define new safety rules / good practices and potentially develop new protections for individuals using detonation devices generating LLB. This is what the research project presented here is aiming for. We propose to fill the gap in knowledge regarding the occurrence of brain injury following repeated exposure to blast threats and identify underlying mechanisms. This will be done through experiments on an instrumented headform, on the one hand, and on an animal model of “breachers” that can recapitulate neurologic symptoms that may arise from repeated use of LLB-generating weapons, on the other hand. The results obtained will then be used as boundary conditions for a new finite element model (FEM) developed within the framework of this project. This FEM will include, in an original way, the development and implementation of a new constitutive law of brain matter including the anisotropic architecture of this tissue (axonal fibers). In addition, it will take into account the mechanical-biological aspects of previous traumatic events in the mechanical model, in order to propose, for the first time ever, brain tolerance limits to repeated head impacts.