Study of the mechanisms involved in neuropathogenic Flavivirus attenuation : the membrane protein as a target for viral attenuation

En raison de leur potentiel élevé de transmission, de leur vitesse de dissémination qui peut être fulgurante et des maladies parfois sévères qu’ils induisent, les Flavivirus sont considérablement préoccupants. Le virus West Nile (WNV) est actuellement l'un des arbovirus (arthropod-borne virus)le plus répandu dans le monde et sa forte capacité de (ré)émergence pose un problème de santé publique important. Ce virus est responsable de nombreux cas de maladies neurologiques sévères, parfois mortelles, notamment chez l’Homme et les chevaux. Aucun traitement ou vaccin humain anti-WNV n’est actuellement disponible sur le marché. Le(s) rôle(s) de la protéine de membrane (M) des Flavivirus est/sont encore peu caractérisé(s). Cette glycoprotéine est ancrée dans la membrane virale et joue un rôle structural important dans les processus de fusion et de maturation des particules virales. De plus, la protéine M a récemment été associée à la pathogénèse induite par les virus de l’encéphalite japonaise et du Zika (de Wispelaere et al., 2016; Yuanet al., 2017), suggérant que cette protéine contient des déterminants viraux essentiels à la virulence. Afin d’étudier les mécanismes sous-jacents, nous avons substitué le résidu isoleucine localisé en position 36de l’ectodomaine de la protéine M par une phénylalanine (M-I36F), et démontré que cette mutation provoque un encombrement allostérique qui affecte directement la structure de la protéine M.L’introduction d’une seconde mutation (substitution de l’alanine en position 43 par une glycine, résidu ne possédant pas de chaine latérale) dans la même protéine nous a permis de stabiliser la mutation MI36F. Si la mutation M-I36F seule ou associée à la mutation M-A43G n’affecte pas les étapes précoces (entrée,traduction, réplication) du cycle viral, elle est cependant à l’origine d’une modification de la morphologie des particules virales chez l’hôte mammifère. De plus, notre étude suggère que la sécrétion des particules de WNV néo-formées hors du réticulum endoplasmique, une étape encore peu caractérisée du cycle viral, dépend directement de leur morphologie. Enfin, cette étude nous a permis de mettre au point un modèle d’atténuation du WNV rationnellement dessiné, qui a été testé in vivo dans un modèle murin décrit d’encéphalite induite par le WNV. La protection complète induite par le virus double mutant, que nous observons lors d’une épreuve létale avec le virus sauvage, fait de ce virus mutant un potentiel candidat vaccin. Cette étude fait l’objet d’un dépôt de brevet.Because of their highly transmission potential, their speed of dissemination that can be dazzling, and the disease severity they might induced, flaviviruses are of great concern. West Nile virus (WNV) is currently one of the most common arboviruses (arthropod-borne virus) in the world and its high ability to (re-)emerge causes a global public health problem. This virus is responsible for many cases of severe neurological diseases, sometimes fatal, especially in humans and horses. No human anti-WNV treatment or vaccine is currently available. The role(s) of the Flavivirus membrane protein (M) is/are still poorly characterized. This glycoprotein is anchored in the viral membrane and plays an important structural role in the processes of fusion and maturation of viral particles. In addition, the M protein has recently been associated with Japanese Encephalitis and Zika-induced pathogenesis (de Wispelaere et al., 2016; Yuan et al., 2017), suggesting that this protein contains viral determinants essential for virulence. In order to study the underlying mechanisms, we substituted the isoleucine residue located in position 36 of the ectodomain of the M protein with a phenylalanine (M-I36F), and demonstrated that this mutation causes a steric hindrance that directly affects the structure of the M protein. The introduction of a second mutation (substitution of the alanine residue at position 43 with a glycine which has no side chain) in the same protein allowed stabilization of the M-I36F mutation. If the M-I36F mutation alone or associated with the M-A43G mutation does not affect the early stages (entry, translation, replication) of the viral cycle, it is however at the origin of a change in the morphology of viral particles in the mammalian host. In addition, our study strongly suggests that the secretion of newly formed WNV particles from the endoplasmic reticulum, a still poorly described stage of the viral cycle, directly depends on their morphology. Finally, this study allowed us to develop a rationally designed WNV attenuation model, which was tested in vivo in a previously described mouse model of WNV-induced encephalitis. The complete protection against a lethal challenge with the wild-type virus of mice previously infected with the double mutant virus makes this mutant virus a potential candidate vaccine. This study is being filed for a patent application

Etude des mécanismes impliqués dans l'atténuation des Flavivirus neurotropes : la protéine de membrane comme cible de l'atténuation

Because of their highly transmission potential, their speed of dissemination that can be dazzling, and the disease severity they might induced, flaviviruses are of great concern. West Nile virus (WNV) is currently one of the most common arboviruses (arthropod-borne virus) in the world and its high ability to (re-)emerge causes a global public health problem. This virus is responsible for many cases of severe neurological diseases, sometimes fatal, especially in humans and horses. No human anti-WNV treatment or vaccine is currently available. The role(s) of the Flavivirus membrane protein (M) is/are still poorly characterized. This glycoprotein is anchored in the viral membrane and plays an important structural role in the processes of fusion and maturation of viral particles. In addition, the M protein has recently been associated with Japanese Encephalitis and Zika-induced pathogenesis (de Wispelaere et al., 2016; Yuan et al., 2017), suggesting that this protein contains viral determinants essential for virulence. In order to study the underlying mechanisms, we substituted the isoleucine residue located in position 36 of the ectodomain of the M protein with a phenylalanine (M-I36F), and demonstrated that this mutation causes a steric hindrance that directly affects the structure of the M protein. The introduction of a second mutation (substitution of the alanine residue at position 43 with a glycine which has no side chain) in the same protein allowed stabilization of the M-I36F mutation. If the M-I36F mutation alone or associated with the M-A43G mutation does not affect the early stages (entry, translation, replication) of the viral cycle, it is however at the origin of a change in the morphology of viral particles in the mammalian host. In addition, our study strongly suggests that the secretion of newly formed WNV particles from the endoplasmic reticulum, a still poorly described stage of the viral cycle, directly depends on their morphology. Finally, this study allowed us to develop a rationally designed WNV attenuation model, which was tested in vivo in a previously described mouse model of WNV-induced encephalitis. The complete protection against a lethal challenge with the wild-type virus of mice previously infected with the double mutant virus makes this mutant virus a potential candidate vaccine. This study is being filed for a patent application.En raison de leur potentiel élevé de transmission, de leur vitesse de dissémination qui peut être fulgurante et des maladies parfois sévères qu’ils induisent, les Flavivirus sont considérablement préoccupants. Le virus West Nile (WNV) est actuellement l'un des arbovirus (arthropod-borne virus)le plus répandu dans le monde et sa forte capacité de (ré)émergence pose un problème de santé publique important. Ce virus est responsable de nombreux cas de maladies neurologiques sévères, parfois mortelles, notamment chez l’Homme et les chevaux. Aucun traitement ou vaccin humain anti-WNV n’est actuellement disponible sur le marché. Le(s) rôle(s) de la protéine de membrane (M) des Flavivirus est/sont encore peu caractérisé(s). Cette glycoprotéine est ancrée dans la membrane virale et joue un rôle structural important dans les processus de fusion et de maturation des particules virales. De plus, la protéine M a récemment été associée à la pathogénèse induite par les virus de l’encéphalite japonaise et du Zika (de Wispelaere et al., 2016; Yuanet al., 2017), suggérant que cette protéine contient des déterminants viraux essentiels à la virulence. Afin d’étudier les mécanismes sous-jacents, nous avons substitué le résidu isoleucine localisé en position 36de l’ectodomaine de la protéine M par une phénylalanine (M-I36F), et démontré que cette mutation provoque un encombrement allostérique qui affecte directement la structure de la protéine M.L’introduction d’une seconde mutation (substitution de l’alanine en position 43 par une glycine, résidu ne possédant pas de chaine latérale) dans la même protéine nous a permis de stabiliser la mutation MI36F. Si la mutation M-I36F seule ou associée à la mutation M-A43G n’affecte pas les étapes précoces (entrée,traduction, réplication) du cycle viral, elle est cependant à l’origine d’une modification de la morphologie des particules virales chez l’hôte mammifère. De plus, notre étude suggère que la sécrétion des particules de WNV néo-formées hors du réticulum endoplasmique, une étape encore peu caractérisée du cycle viral, dépend directement de leur morphologie. Enfin, cette étude nous a permis de mettre au point un modèle d’atténuation du WNV rationnellement dessiné, qui a été testé in vivo dans un modèle murin décrit d’encéphalite induite par le WNV. La protection complète induite par le virus double mutant, que nous observons lors d’une épreuve létale avec le virus sauvage, fait de ce virus mutant un potentiel candidat vaccin. Cette étude fait l’objet d’un dépôt de brevet

Etude des mécanismes impliqués dans l'atténuation des Flavivirus neurotropes : la protéine de membrane comme cible de l'atténuation

Because of their highly transmission potential, their speed of dissemination that can be dazzling, and the disease severity they might induced, flaviviruses are of great concern. West Nile virus (WNV) is currently one of the most common arboviruses (arthropod-borne virus) in the world and its high ability to (re-)emerge causes a global public health problem. This virus is responsible for many cases of severe neurological diseases, sometimes fatal, especially in humans and horses. No human anti-WNV treatment or vaccine is currently available. The role(s) of the Flavivirus membrane protein (M) is/are still poorly characterized. This glycoprotein is anchored in the viral membrane and plays an important structural role in the processes of fusion and maturation of viral particles. In addition, the M protein has recently been associated with Japanese Encephalitis and Zika-induced pathogenesis (de Wispelaere et al., 2016; Yuan et al., 2017), suggesting that this protein contains viral determinants essential for virulence. In order to study the underlying mechanisms, we substituted the isoleucine residue located in position 36 of the ectodomain of the M protein with a phenylalanine (M-I36F), and demonstrated that this mutation causes a steric hindrance that directly affects the structure of the M protein. The introduction of a second mutation (substitution of the alanine residue at position 43 with a glycine which has no side chain) in the same protein allowed stabilization of the M-I36F mutation. If the M-I36F mutation alone or associated with the M-A43G mutation does not affect the early stages (entry, translation, replication) of the viral cycle, it is however at the origin of a change in the morphology of viral particles in the mammalian host. In addition, our study strongly suggests that the secretion of newly formed WNV particles from the endoplasmic reticulum, a still poorly described stage of the viral cycle, directly depends on their morphology. Finally, this study allowed us to develop a rationally designed WNV attenuation model, which was tested in vivo in a previously described mouse model of WNV-induced encephalitis. The complete protection against a lethal challenge with the wild-type virus of mice previously infected with the double mutant virus makes this mutant virus a potential candidate vaccine. This study is being filed for a patent application.En raison de leur potentiel élevé de transmission, de leur vitesse de dissémination qui peut être fulgurante et des maladies parfois sévères qu’ils induisent, les Flavivirus sont considérablement préoccupants. Le virus West Nile (WNV) est actuellement l'un des arbovirus (arthropod-borne virus)le plus répandu dans le monde et sa forte capacité de (ré)émergence pose un problème de santé publique important. Ce virus est responsable de nombreux cas de maladies neurologiques sévères, parfois mortelles, notamment chez l’Homme et les chevaux. Aucun traitement ou vaccin humain anti-WNV n’est actuellement disponible sur le marché. Le(s) rôle(s) de la protéine de membrane (M) des Flavivirus est/sont encore peu caractérisé(s). Cette glycoprotéine est ancrée dans la membrane virale et joue un rôle structural important dans les processus de fusion et de maturation des particules virales. De plus, la protéine M a récemment été associée à la pathogénèse induite par les virus de l’encéphalite japonaise et du Zika (de Wispelaere et al., 2016; Yuanet al., 2017), suggérant que cette protéine contient des déterminants viraux essentiels à la virulence. Afin d’étudier les mécanismes sous-jacents, nous avons substitué le résidu isoleucine localisé en position 36de l’ectodomaine de la protéine M par une phénylalanine (M-I36F), et démontré que cette mutation provoque un encombrement allostérique qui affecte directement la structure de la protéine M.L’introduction d’une seconde mutation (substitution de l’alanine en position 43 par une glycine, résidu ne possédant pas de chaine latérale) dans la même protéine nous a permis de stabiliser la mutation MI36F. Si la mutation M-I36F seule ou associée à la mutation M-A43G n’affecte pas les étapes précoces (entrée,traduction, réplication) du cycle viral, elle est cependant à l’origine d’une modification de la morphologie des particules virales chez l’hôte mammifère. De plus, notre étude suggère que la sécrétion des particules de WNV néo-formées hors du réticulum endoplasmique, une étape encore peu caractérisée du cycle viral, dépend directement de leur morphologie. Enfin, cette étude nous a permis de mettre au point un modèle d’atténuation du WNV rationnellement dessiné, qui a été testé in vivo dans un modèle murin décrit d’encéphalite induite par le WNV. La protection complète induite par le virus double mutant, que nous observons lors d’une épreuve létale avec le virus sauvage, fait de ce virus mutant un potentiel candidat vaccin. Cette étude fait l’objet d’un dépôt de brevet

A LIVE AND ATTENUATED FLAVIVIRUS COMPRISING A MUTATED M PROTEIN FIELD OF THE INVENTION 5 BACKGROUND OF THE INVENTION

The application relates to the attenuation of flaviviruses, such as West Nile Virus (WNV). The application notably provides a live and attenuated flavivirus, such as a WNV, comprising a mutated M protein. Said mutated M protein comprises or consists of a sequence, wherein the amino acids at position 36 in the ectodomain and position 43 in the transmembrane 10 domain 1 in said sequence are mutated. The application also provides additional embodiments deriving from said live and attenuated flavivirus, such as a WNV, such as nucleic acids, cDNA clones, immunogenic compositions as well as uses and methods

Bioluminescent Ross River Virus Allows Live Monitoring of Acute and Long-Term Alphaviral Infection by In Vivo Imaging

International audienceArboviruses like chikungunya and Ross River (RRV) are responsible for massive outbreaks of viral polyarthritis. There is no effective treatment or vaccine available against these viruses that induce prolonged and disabling arthritis. To explore the physiopathological mechanisms of alphaviral arthritis, we engineered a recombinant RRV expressing a NanoLuc reporter (RRV-NLuc), which exhibited high stability, near native replication kinetics and allowed real time monitoring of viral spread in an albino mouse strain. During the acute phase of the disease, we observed a high bioluminescent signal reflecting viral replication and dissemination in the infected mice. Using Bindarit, an anti-inflammatory drug that inhibits monocyte recruitment, we observed a reduction in viral dissemination demonstrating the important role of monocytes in the propagation of the virus and the adaptation of this model to the in vivo evaluation of treatment strategies. After resolution of the acute symptoms, we observed an increase in the bioluminescent signal in mice subjected to an immunosuppressive treatment 30 days post infection, thus showing active in vivo replication of remnant virus. We show here that this novel reporter virus is suitable to study the alphaviral disease up to the chronic phase, opening new perspectives for the evaluation of therapeutic interventions

A molecular determinant of West Nile virus secretion and morphology as a target for viral attenuation

International audienceWest Nile virus (WNV), a member of the Flavivirus genus and currently one of the most common arboviruses worldwide, is associated with severe neurological disease in humans. Its high potential to re-emerge and rapidly disseminate makes it a bona fide global public health problem. The surface membrane glycoprotein (M) has been associated with Flavivirus-induced pathogenesis. Here we identify a key amino acid residue at position 36 of the M protein whose mutation impacts WNV secretion and promotes viral attenuation. We also identified a compensatory site at position M-43 whose mutation stabilizes M-36 substitution both in vitro and in vivo. Moreover, we find that introduction of the two mutations together confers a full attenuation phenotype and protection against wild-type WNV lethal challenge, eliciting potent neutralizing antibody production in mice. Our study thus establishes the M protein as a new viral target for rational design of attenuated WNV strains.IMPORTANCE West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus. However, such an approach is not acceptable nowadays and the development of vaccine rationally designed is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence is a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses that are an increasing threat to global human health

Comparison of a human neuronal model proteome upon Japanese encephalitis or West Nile Virus infection and potential role of mosquito saliva in neuropathogenesis

International audienceNeurotropic flavivirus Japanese encephalitis virus (JEV) and West Nile virus (WNV) are amongst the leading causes of encephalitis. Using label-free quantitative proteomics, we identified proteins differentially expressed upon JEV (gp-3, RP9) or WNV (IS98) infection of human neuroblastoma cells. Data are available via ProteomeXchange with identifier PXD016805. Both viruses were associated with the up-regulation of immune response (IFIT1/3/5, ISG15, OAS, STAT1, IRF9) and the down-regulation of SSBP2 and PAM, involved in gene expression and in neuropeptide amidation respectively. Proteins associated to membranes, involved in extracellular matrix organization and collagen metabolism represented major clusters down-regulated by JEV and WNV. Moreover, transcription regulation and mRNA processing clusters were also heavily regulated by both viruses. The proteome of neuroblastoma cells infected by JEV or WNV was significantly modulated in the presence of mosquito saliva, but distinct patterns were associated to each virus. Mosquito saliva favored modulation of proteins associated with gene regulation in JEV infected neuroblastoma cells while modulation of proteins associated with protein maturation, signal transduction and ion transporters was found in WNV infected neuroblastoma cells

Tailoring silicon for dew water harvesting panels

International audienceDew water, mostly ignored until now, can provide clean freshwater resources, just by extracting the atmospheric vapor available in surrounding air. Inspired by silicon-based solar panels, the vapor can be harvested by a concept of water condensing panels. Efficient water harvesting requires not only a considerable yield but also a timely water removal from the surface since the very beginning of condensation to avoid the huge evaporation losses. This translates into strict surface properties, which are difficult to simultaneously realize. Herein, we study various functionalized silicon surfaces, including the so-called Black Silicon, which supports two droplet motion modes-out-of-plane jumping and in-plane sweeping, due to its unique surface morphology, synergistically leading to a pioneering combination of above two required characteristics. According to silicon material's scalability, the proposed silicon-based water panels would benefit from existing infrastructures toward dual functions of energy harvesting in daytime and water harvesting in nighttime

A human domain antibody and Lewisb glycoconjugate that inhibit binding of helicobacter pylori to Lewisb receptor and adhesion to human gastric epithelium

Increasing antibiotic resistance has prompted development of alternative approaches to antimicrobial therapy, including blocking microbial adhesion to host receptors. The BabA adhesin of Helicobacter pylori binds to fucosylated blood group antigens, such as the Lewisb antigens in human primate gastric mucosa. We have isolated a human domain antibody specific for BabA that inhibits binding of BabA to Lewisb and prevents adhesion of H. pylori to human gastric epithelium. In addition, Lewisb oligosaccharides covalently linked to poly‐d‐lysine inhibited BabA binding to Leb. The poly‐d‐lysine‐Leb hexasaccharide and an Leb human serum albumin conjugate not only inhibited adherence of H. pylori to gastric epithelium but also displaced adherent bacteria when added to human stomach sections. Combinations of Leb and sialyl Lex or domain antibody 25 and sialyl Lex acted synergistically. Domain antibody 25 inhibitor may have potential for prophylactic use and, in combination with Leb glycoconjugates, therapeutic use in treatment of drug‐resistant H. pylori infection