Virus enveloppés, vecteurs et immunothérapie

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Research

Viral entry and interconnection with lipid metabolism
Viral particles assembly
Host response against infection: sensing and evasion mechanisms
Eco-infectiology of HBV-like viruses: assessment of cross-species transmission
Innovative technologies: viral particle engineering and novel humanized mouse models
Translational developments: towards clinical and biotherapy applications

Summary

Viruses use, misuse and abuse their hosts to replicate, escape immune responses and invade new cells or organisms. By comparing virus genus/species, we aim at improving our understanding of how some human viral pathogens can establish persistent versus acute infections, how this is determined by interactions with host pathways, and how they are further transmitted and transgress species barriers. We focus, on the one hand, on questions regarding the biology of envelope surface glycoproteins of selected viral pathogens, more particularly on the mechanisms that allow their assembly on viral particles and the processes by which they mediate entry into cells through interactions with cell surface receptors and entry routes. We have also initiated projects aiming at unraveling the mechanisms of cross-species transmission and the (re)emergence of zoonotic infectious diseases transmitted by bats and ticks. On the other hand, we aimed at understanding some immune mechanisms that restrict infection or, alternatively, that are counteracted, subverted or perverted by pathogens. We expect that the understanding of such mechanisms will facilitate the development of antiviral strategies. Finally, we have invested solid efforts in innovative biotherapies against infectious diseases, by targeting different steps of virus replication. Such applications have required the development of viral engineering technologies and of viral vector platforms that we will assess in gene therapy, vaccinology, or immunotherapy studies.

A-Viral entry and interconnection with lipid metabolism

Lab members involved: B Bartosch, VL Dao-Thi, F Douam, M Dreux, N Freitas, C Granier, D Lavillette, J Mancip, G Maurin, G Neveu, J Perez-Vargas

HCV entry pathways and receptors
We have pursued our efforts aiming at characterizing the intimate steps of HCV entry into cells and its relation with lipid metabolism. During the first steps of infection, the virus enters hepatocytes, which is a multi-step and slow process. The initial capture of HCV particles by glycosaminoglycans (GAG) and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin and the tight junction proteins Claudin-1 and Occludin, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports from our team and others have indicated that HDL indirectly promotes HCV entry through interaction with SR-BI. Yet, in contrast to CD81, Claudin-1 and Occludin, the importance of SR-BI had not been addressed directly because of lack of cells in which functional complementation assays with mutant receptors could be performed. We have therefore developed SR-BI complementation assays to unambiguously demonstrate that SR-BI is an essential HCV entry factor. Then, by expressing different SR-BI mutants, we have revealed features of SR-BI intracellular and extracellular domains that influence HCV infectivity at the levels of i) attachment of viral particles, ii) stimulation of HCV entry induced by HDL/SR-BI interaction, and iii) SR-BI trafficking, most likely in relation with interaction with the other HCV entry factors and/or transport of HCV particles to specific cell micro-domains.
Altogether, these results highlighted specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection. Thus, as HCV particles assemble along the very-low-density lipoprotein (VLDL) pathway and are released from hepatocytes as entities varying in their degree of lipid and apolipoprotein (apo) association as well as buoyant densities, we have sought to characterize the cell entry pathway of these different HCV particle sub-populations. We have showed that HCV utilize SR-BI in a manifold manner during entry. First, SR-BI mediates primary attachment of HCV particles of intermediate density to cells. These initial interactions involve apolipoproteins, such as apoE, present on the surface of HCV particles, but not the HCV E2 surface glycoprotein, suggesting that lipoprotein components in the virion act as host-derived ligands for important capture molecules such as SR-BI. Second, we found that in contrast to this initial attachment, SR-BI mediates entry of HCV particles independent of their buoyant density. This function of SR-BI does not depend on E2/SR-BI interaction, but relies on the lipid transfer activity of SR-BI, probably by facilitating entry steps along with other HCV entry co-factors. Finally, our results underscored a third function of SR-BI governed by specific residues in the hypervariable region 1 of E2 leading to enhanced cell entry and depending on SR-BI ability to bind to E2.

Envelope glycoprotein functions in cell entry
Overall, the cell entry pathway of HCV appears to entail an initial phase that involves interactions between cell surface molecules (e.g., GAGs, SR-BI) with non-viral, cellular ligands incorporated on virus particles (e.g., ApoE, ApoB) and subsequent interactions between HCV E1E2 glycoproteins with alternative entry factors/functions (e.g., CD81, SR-BI). This second phase is likely to occur only once the virions have entered in a specific cellular micro-environment and that ultimately leads to membrane fusion. We characterized the role of both conserved and less conserved E1E2 domains required for a mandatory cooperation of either glycoprotein during HCV entry. Our working hypothesis was that a conserved protein function, involving different segments, involves the co-evolution of variable domains in order to maintain the most optimal direct or indirect interaction (“dialog”) between these domains. Through classical mutation analysis, we defined dialogs occurring both within E1 and between E1 amino-terminal and E2 carboxy-terminal domains that are involved in membrane fusion, and we also found, for the first time, that E1E2 complexes interact with the first extracellular loop of Claudin-1. Through an alternative approach involving a computational coevolution sequence analyses that we validated on Dengue virus E protein sequences, whose structure is known, we identified several co-evolution networks within or between E1 and E2. This offered for the first time an overall view on how E1 and E2 may be functionally organized, which we confirmed experimentally in cell entry and membrane fusion assays. Altogether, these results indicated that HCV harbors unusual fusion proteins and mechanisms.

The surface glycoproteins of enveloped viruses govern entry into cells by interacting with different cellular factors. They also mediate virus interactions with soluble host factors such as, for example, complement proteins, lipoproteins or antibodies. Over the recent past years, we have investigated the biology of surface glycoproteins derived from several different membrane-enveloped viruses, such as retroviruses, HCV, and measles virus. Our recent studies with HCV have identified critical domains in both glycoproteins that interrelate with each other to permit HCV cells entry. Our objectives are to understand the function and the regulation of these proteins at the level of i) their interaction with the external environment, notably membrane-bound or soluble entry factors as well as inhibitory factors such as serum components and neutralizing antibodies, and ii) the molecular processes governing the cellular entry of enveloped viruses and subsequent membrane fusion. Particularly, we are now focusing our efforts on HCV, hepatitis B virus (HBV) and Crimean–Congo hemorrhagic fever virus (CCHFV), an arbovirus member of the family Bunyaviridae, all of which harboring a complex of two (or more) glycoproteins. Our studies aim at deciphering the role of specific domains of these oligomers that are involved in “cross-talks” between either glycoprotein through their conformational changes during envelope assembly or during cell entry. We expect that these projects will reveal novel viral and cellular targets or pathways for which we will be interested to develop specific inhibitors, antibodies or immunogens (see below).

Intersection of lipid metabolism and HCV replication
Chronically HCV-infected patients are often affected by metabolic disorders including insulin resistance, hypobetalipoproteinemia and liver steatosis. Importantly, HCV propagation depends on and also shapes several aspects of lipid metabolism such as cholesterol uptake and efflux through different lipoprotein receptors during its entry into cells, lipid metabolism modulating HCV genome replication and lipid droplets (LD) acting as essential platforms for recruitment of viral components along with enzymes utilized to generate viral particles. Understanding these interconnections is key to define the infectivity parameters of HCV in vivo and the physio-pathological processes, and to elaborate novel therapeutics. Importantly, the HCV-lipid metabolism interplay may find its most remarkable feature with the formation of hybrid infectious particles that combine viral and very-low-density lipoprotein (VLDL) components and that vary in their degree of lipid and apolipoprotein association. Consistently, HCV enters cells by using different entry co-factors that are lipoprotein and cholesterol transfer receptors, such as the low density-lipoprotein receptor (LDL-R), the scavenger receptor class B type I (SR-BI), a receptor of high-density lipoprotein (HDL), and the Niemann-Pick C1-like 1 (NPC1L1) cholesterol uptake receptor.

We have recently established in our animal facility a humanized liver mouse model, i.e., the immuno-deficient FRG (Fah-/-/Rag2-/-/IL2Rγc-/-) mice (see below), which is used to study in vivo and ex vivo the regulation of HCV infection by lipid metabolism. Thus, using these mice reconstituted with human liver cells as well as in vitro and ex vivo models, our studies now aim at addressing key questions regarding: i) the role of lipid storage and transport organelles, LDs and VLDLs, and lipid metabolism cofactors in recruitment of viral components, in assembly steps, and in lipidation and egress of HCV particles, ii) how cholesterol uptake and efflux mediated by different lipoprotein receptors modulate virus entry into cells through their interactions with lipid-containing viral particles, iii) how HCV association to lipoprotein lipids and apolipoproteins impacts escape from immune effectors such as neutralizing antibodies. We also investigate the role of the cellular factors involved in assembly and entry using humanized mice reconstituted with human liver cells, to address their functional relevance in vivo.

5 selected publications:
1. Douam F, Lavillette D, Cosset FL. 2015. The mechanism of hcv entry into host cells. Prog Mol Biol Transl Sci. 29:63-107.
2. Douam F, Dao Thi VL, Maurin G, Fresquet J, Mompelat D, Zeisel MB, Baumert TF, Cosset* FL, Lavillette* D. 2014. Critical interaction between e1 and e2 glycoproteins determines binding and fusion properties of hepatitis c virus during cell entry. Hepatology. 59:776-88.
3. Dao Thi, V. L., C. Granier, M. B. Zeisel, M. Guerin, J. Mancip, O. Granio, F. Penin, D. Lavillette, R. Bartenschlager, T. F. Baumert, M. Dreux*, and F. L. Cosset*. 2012. Characterization of hepatitis C virus particle subpopulations reveals multiple usage of the scavenger receptor BI for entry steps. The Journal of biological chemistry 287:31242-31257. *Corresponding and co-last authors
4. Maurin, G., J. Fresquet, O. Granio, C. Wychowski, D. Lavillette*, and F. L. Cosset*. 2011. Identification of Interactions in the E1E2 Heterodimer of Hepatitis C Virus Important for Cell Entry. The Journal of biological chemistry 286:23865-23876. *Corresponding and co-last authors
5. Dreux M, VL Dao Thi, J Fresquet, M Guérin, Z Julia, G Verney, D Durantel, F Zoulim, D Lavillette, B Bartosch*, and F. L. Cosset*. 2009. Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains. PLoS Pathogens. 5(2):e1000310. *Co-last authors.

B-Viral particles assembly

Lab members involved: B Boson, S Calattini, S Denolly, M Dreux, N Fontaine, O Granio, K Katsarou, C Mialon, F Turlure

HCV encodes a polyprotein that is translated and that undergoes maturation by cleavage at the endoplasmic reticulum (ER). The assembly of the viral structural components, including core, the capsid protein, the E1/E2 envelope glycoproteins, and the vRNA is believed to occur at the ER, requiring a coordinated integration of cellular and viral pathways in which the HCV non-structural proteins play a major role. The cytosolic lipid droplets (LDs) induce concentration of core close to the ER-located assembly site and may provide a physical link with the vRNA replication site, also localized in specialized, ER-derived structures called the ‘membranous web’. By comparing HCVcc from the low-titer JFH-1 virus isolate and the high-titer Jc1 virus chimera, we have analyzed the subcellular localization pattern of core protein in HCV-infected cells with a particular focus on core colocalization with E2 at the ER or with specific markers of the LDs. We found that ER localization, but not LD localization of core is required for efficient HCV assembly, challenging the current view that LD provides a platform required for HCV assembly. We then demonstrated that the p7 and NS2 non-structural proteins are key viral determinants governing the cellular localization of HCV core to LDs vs. ER and are required for initiation of the early steps of virus assembly. Our results also underscored a requirement for compatibilities between the p7 trans-membranes and the NS2 amino-terminus that dictates core-E2 colocalization in the ER, leading to initiation of virion assembly.
Our current studies aim at dissecting the cellular and viral pathways that permits the intracellular trafficking of viral structural components, core, RNA and envelope glycoproteins to the assembly site in a timely manner through their interplay with required cellular factors, structures and factories. Particularly, we aim at understanding i) how HCV non-structural proteins modulate core protein clustering and formation of viral particles and ii) the dynamics of intracellular sites responsible for core aggregation in association with viral RNA and envelopment of viral particles. In this respect, we have used daclatasvir, a potent inhibitor of NS5A, to help characterizing the formation of assembly sites. Indeed, previous results of others indicated that daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. On the other hand, NS5A is involved in replication of the HCV genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. We found a relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs and inhibition of viral particle assembly. We found that in addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins, because viral particles and replication complex vesicles cannot form and/or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection.

To broaden this research theme to other members of the Flaviviridae family, we have set up the dengue virus (DENV) model, which was a new topic in our laboratory. We have gathered tools and designed protocols necessary for molecular studies addressing DENV assembly and entry. Like for HCV, we hypothesized that non-structural (NS) proteins might regulate the DENV particle assemby step by temporally and spatially coordinating genome synthesis at the replication site with the biogenesis of viral particles at the assembly site. Our results underscore determinants of the viral protein NS2A and the NS3 protease that have key regulatory functions for the production of viral particles, but not for RNA replication, suggesting that NS2A and NS3 play a pivotal role for the biogenesis of DEN particles independently for their known function in viral replication. The cross-talks between these non-structural proteins required for particle envelopment, as well as their function in the regulation of the relative production of infectious versus non-infectious viral particle are currently under investigation.

5 selected publications:
1. Boson B, Denolly S, Turlure F, Chamot C, Dreux M, Cosset FL. 2017. Daclatasvir prevents hepatitis c virus infectivity by blocking transfer of the viral genome to assembly sites. Gastroenterology. 152:895-907 e14.
2. Yan Y, He Y, Boson B, Wang X, Cosset FL, Zhong J. 2017. A point mutation in the n-terminal amphipathic helix alpha0 in ns3 promotes hepatitis c virus assembly by altering core localization to the endoplasmic reticulum and facilitating virus budding. J Virol. 91.
3. Calattini S, Fusil F, Mancip J, Dao Thi VL, Granier C, Gadot N, Scoazec JY, Zeisel MB, Baumert TF, Lavillette D, Dreux M, Cosset FL. 2015Functional and biochemical characterization of hepatitis c virus (hcv) particles produced in a humanized liver mouse model. J Biol Chem. 290:23173-87.
4. Boson, B., O. Granio, R. Bartenschlager, and F. L. Cosset. 2011. A concerted action of hepatitis C virus p7 and nonstructural protein 2 regulates core localization at the endoplasmic reticulum and virus assembly. PLoS pathogens 7:e1002144.
5. Bartenschlager*, R., F. L. Cosset*, and V. Lohmann. 2010. Hepatitis C virus replication cycle. Journal of hepatology 53:583-585.

C-Host response against infection: sensing and evasion mechanisms

Lab members involved: M Dreux (co-PI), S Assil, E Décembre, V Grass, M Hillaire, B Webster

Note: this program is currently performed in the CIRI team of M. Dreux, created in 2016, and no longer explored in my team.

Viral nucleic acids, recognized within the infected cells, trigger an antiviral response characterized by the production of interferons (IFNs) and IFN-stimulated genes (ISGs) that suppress viral spread. Many viruses, including HCV and DENV have thus evolved potent mechanisms that preclude this response within infected cells. Nonetheless, HCV infection strongly triggers the expression of ISGs in the liver – the HCV replication site – of infected humans and chimpanzees, suggesting the existence of alternative pathogen-sensing mechanisms. Consistently, we demonstrated that HCV infected cells secrete viral RNA-containing exosomes that efficiently transfer immunostimulatory viral RNAs to immune cells, plasmacytoid dendritic cells (pDCs), which, in response, robustly produce IFN. We have identified key cellular factors required for pDC activation, including the ESCRT proteins that are required for multivesicular bodies (MVB) formation and exosome biogenesis. Collectively, our results revealed that pathogen-associated molecular patterns (PAMPs) can be carried by exosomes from infected cells in which viral RNA sensing is impaired to non-permissive cells that are specifically designed to produce IFN. Overall, we revealed a previously unsuspected mechanism of innate immunity in which exosomal export of viral RNA from infected cells serves as a host strategy to induce an innate response in cells whose innate signaling pathways are unopposed because they are not infected.

Like HCV, DENV-infected cells do not produce IFN themselves, but that they trigger a rapid IFN response by cocultured PMBCs. Our unpublished results demonstrate that among PBMCs, the pDCs are virtually the unique IFN producer cells in response to DENV-infected cells as well as to other flaviviruses such as West Nile virus. Moreover, we showed that pDC IFN response requires an active viral replication in infected cells that transmit DENV RNAs to IFN producing pDCs. However, our results imply that pDC activation triggered by DENV-infected cells is distinct from that induced by HCV-infected cells, which does not require HCV structural proteins. Indeed, we demonstrated that immature viral particles, which are produced in large numbers by inefficient cleavage of the prM glycoprotein, potently trigger IFN production by pDCs, and consistently, that infectious virus production is dispensable for pDC activation.

5 selected publications:
1. Decembre E, Assil S, Hillaire ML, Dejnirattisai W, Mongkolsapaya J, Screaton GR, Davidson AD, Dreux M. 2014. Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells. PLoS Pathog. 10:e1004434.
2. Cosset, F. L., and M. Dreux. 2013. HCV transmission by hepatic exosomes establishes a productive infection. Journal of hepatology 60:674-675.
3. Assil, S., E. Decembre, and M. Dreux. 2013. [Exosomes are carriers for immunostimulatory viral RNA]. Medecine sciences : M/S 29:104-106.
4. Dreux, M., U. Garaigorta, B. Boyd, E. Decembre, J. Chung, C. Whitten-Bauer, S. Wieland, and F. V. Chisari. 2012. Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity. Cell host & microbe 12:558-570.
5. Dreux, M., and F. V. Chisari. 2011. Impact of the autophagy machinery on hepatitis C virus infection. Viruses 3:1342-1357.

D-Eco-infectiology of HBV-like viruses: assessment of cross-species transmission

Lab members involved: F Amirache, J Perez-Vargas

Emerging infectious diseases represent a major threat for public health, biodiversity and global economies. In humans, the global burden of emerging and re-emerging infections already exceeds 300 million disability-adjusted life years (DALYs) (OMS 2011). Animals, and in particular wildlife, are the most likely sources of emerging infectious diseases threatening human populations, with >70% of emerging events being caused by zoonotic pathogens. Whereas information exists on human and commensal rendering control measures possible, little is known on non-human, non-agricultural systems.

Bats are increasingly reported as important reservoirs for zoonotic viruses having significant impact on human health (such as Marburg virus, Nipah virus, Hendra virus, Ebola virus, Rabies virus, and coronaviruses). Apart from these newly emerging pathogens, shared human and animal viruses, including ancestor of measles, mumps, para-influenza and hepatitis B and C viruses may have originated in bats. To date, more than 200 viruses have been detected in bats, representing 27 virus families, which is the highest known diversity among mammals. The number of viruses that bats carry can vary broadly between species, e.g. frugivorous and gregarious species hosting more viruses than species with other diet and life style. As life style is strongly linked to phylogeny the reasons of these variations are not clear and may depend on specific genetic background/immunological functioning and/or may reflect the level of parasitic pressure; i.e. high in the inter-tropical areas and low in temperate regions. In many cases, the viruses naturally infecting bats do not appear to impact their health, suggesting selection of particular inherited traits resulting from the long co-evolution between bats and viruses. The hypotheses that explain such a viral richness and the potential absence of sign of disease in most cases are related to the particular life history traits and lifestyles of bats. This includes their relatively long life spans (exceptional relative to their body size; some living up to 30 years), the distances they cover by flight when feeding, dispersing or migrating, and their often social roosting behaviour (several thousands, up to millions of individuals per site), their sympatry (defined as the number of species with overlapping range) that may favour inter-species transmission. There is obviously some variation between species in all of these traits, but one thing all bats have in common is their ability to fly. Flight is very energetically expensive. Elevated metabolic rates and body temperatures accompanying flight might have resulted in bats developing excellent immune systems, which may allow them to fight off disease.

Hepatitis B is a major health problem affecting 350 million people worldwide, which represents a substantial economical and health burden with approximately 0.6-1 million deaths occurring each year as a result of HBV infection. HBV belongs to the Orthohepadnavirus genus of the Hepadnaviridae family, for which it is the only human representative. Some HBV-like viruses have been discovered in other mammals (woodchuck, ground squirrel and non-human primates) but to date, no cross species transmission events have been confirmed. Strikingly, since the advent of extensive sequencing, three novel HBV-like viruses’ sequences have been found in different bat species in Central America and Africa. This introduces a new element to be considered in the natural history and origin of this virus, which remains unresolved, giving rise to the hypothesis that bats may be a natural reservoir for HBV and sources of zoonotic transmission. However, only 54 species of bats – representing less than 5% of the total number of existing bat species – have been screened so far, suggesting that there is potentially a larger diversity of viruses yet to be discovered, which may change the current picture of HBV evolution.
This project aims at i) identifying new HBV-like viruses in bats living in different habitat conditions from South America, Africa and Europe, ii) characterizing the phylogenetic relationships with the other reported HBV viruses and iii) describing patterns of viral circulation both intra- and inter-species, and iv) characterizing the molecular cell entry determinants of HBV-like viruses compared to the human HBV from a mechanistic perspective. We focus on HBV-like viruses found in bats to understand whether and how the HBV-like virus envelope glycoproteins play a role in the potential crossing of the interspecies barrier. Thus, in collaboration with Dominique Pontier and the LabEx Ecofect, we are sampling bats from different places, including Gabon, Gyuana, Tunisia and France, and we are identifying HBV-like sequences present in the over 5,000 bat samples representing ca. 100 species already collected. Bioinformatics analyzes will be carried out on HBV and HBV-like viruses sequences to identify coevolution clusters in core and surface proteins of these viruses, which could mark molecular determinants involved in cell entry processes of HBV and HBV-like viruses. This will allow experimental characterization of the molecular cell entry determinants of HBV-like compared to the human HBV thanks to construction of HDVHBV and HDVHBV-like pseudo-viral particles.
Finally, all data generated from the ecological and molecular parts will be integrated in a mathematical model to test hypotheses on ecological and evolutionary factors potentially shaping virus host specificity and cross-species transmission. Taken together, the expected results should bring innovative insights into the zoonotic potential of bat hepadnaviruses. The present proposal will generate a wide range of empirical and theoretical results, as well as methodological developments, which could be valuable for a large range of host-pathogen systems.

E-Innovative technologies: viral particle engineering and novel humanized mouse models

Lab members involved: F Amirache, C Frecha, F Fusil, A Girard, C Levy, D Nègre, A Ollivier, J Szecsi, E Verhoeyen

Viral engineering: control of vector tropism and design of virus-like particle
Retroviral vectors derived from murine oncoretroviruses and lentiviruses provide powerful and flexible systems to mimic the surface of heterologous viruses because they can display heterologous viral envelope glycoproteins at their surface. Selective vector tropism was achieved in our lab by taking advantage of the natural tropisms of glycoproteins from other membrane-enveloped viruses. This is important for gene therapy applications since the capacity to transduce resting immune target cells and/or to overcome innate or adaptive immune responses against vectors as well as human complement-mediated inactivation would strongly promote their use in vivo, by direct inoculation of vector particles.
A major recent breakthrough of this program was the invention of novel lentiviral vectors that allowed efficient gene transfer into resting immune cells. Indeed, a major limitation of current lentiviral vectors (LVs) such as the generally used VSVG-pseudotyped LVs is their inability to govern efficient gene transfer into quiescent cells such as primary T cells and B cells, which hampers their application for gene therapy and immunotherapy. We invented a novel LV incorporating measles virus (MV) H and F surface glycoproteins (MV-LVs). Importantly, a single cell exposure to these MV-LVs allowed efficient and stable gene transfer of quiescent T cells and B cells, which are not susceptible to classical VSVG-LVs. Rather unexpectedly, MV-LVs did not induce cell-cycle entry upon transduction in either quiescent cell type and conserved the naïve or memory phenotypes of transduced resting T cells and B cells. Of utmost importance, MV-LVs allowed efficient gene transfer in both healthy and patient plasma B cells, demonstrating that they are the novel tools to direct stable transduction of B-cell chronic lymphocyte leukemia cells, one of the most prominent B-cell malignancies. Additionally, high-level transduction of immature dendritic cells (DCs) was also obtained without induction of their maturation. Thus, these MV-LVs are revolutionary for basic research as well as for gene therapy applications, and have been shared with over 100 labs in Europe, US and Canada.
A second major breakthrough was the development of LVs allowing high-level gene transfer in vivo in hematopoietic stem cells (HSCs). We engineered LVs that display at their surface ‘early acting cytokine’ such as stem cell factor (SCF), which allowed ex vivo and in vivo targeted gene transfer into HSCs in the bone marrow (BM). These novel LVs should facilitate HSC-based gene therapy by targeting primitive cells in the BM. Most importantly, SCF-LVs might completely omit ex vivo handling and simplify gene therapy for many hematopoietic defects by direct in vivo inoculation.

Vector pseudotypes also proved to be of great value for deciphering cell entry of viruses such as HCV, Lassa virus, Nipah virus, or MV as well as for designing inhibition assays that can be handled in low containment laboratories (BSL2 rather than BSL4 for e.g. Lassa virus). Furthermore, they allow to more readily studying drastic mutations of surface glycoproteins that would otherwise be impossible to study in the context of parental viruses that need to replicate in order to produce (mutant) viral particles. We will pursue the engineering of retroviruses in order to undertake projects addressing the entry functions and neutralization mechanisms of surface glycoproteins of alternative enveloped viruses of interest such as e.g., CCHFV, HCV and HBV (see above) and to design specific inhibitors.
We will also use such technologies to develop viral-like particle (VLP), containing no infectious material or viral genome, that can display immunogenic epitopes from the parental viruses and that can be used in vaccine studies (see below).

Basic vector development – vector production platform
Retroviral and more particularly lentiviral vectors have become routinely used research tools since they possess the unique characteristic to stably introduce DNA sequences (cDNA, mutated genes, sh/miRNA) into most cell types including primary cells, which are difficult to transfect.
Our Team (EVIR) has created, and hosts since 2004, the Vectorology platform (SFR BioSciences (UMS3444/US8)), which has now an established clientele in the whole of France and is locally of great value for several research projects. This vector production facility has received the IBISA label through its association in 2009 with the AniRA platform, emphasizing its high quality performance. The main activities of the Vectorology platform are the bio-safe production of retroviral vectors based on MLV, HIV-1 and SIV in conditions that can be defined with its clients (for more detail on the vector production platform see http://www.ifr128.prd.fr/ifr128.htm). Its also offers assistance in vector design and is involved in the transgenesis platform of the animal facility of the campus. Meanwhile, a continuous optimization of retroviral vector tools is pursued by members of the team in terms, for example, of vector design (inducible, tissue-specific, multicistronic vectors, etc…) and of pseudotyping. The close relationship between the EVIR team and the Vectorology platform allows efficient transfer of EVIR vector technologies (e.g. new pseudotyped vectors) to the platform and facilitates their access to the scientific community.

Humanized mouse models
The development of innovative preventive and therapeutic strategies against infectious hepatotropic pathogens such as HCV, HBV and Plasmodium falciparum has been strongly hampered by the lack of convenient small animal models. To overcome these limitations, novel animal models susceptible to hepatotropic pathogens have been developed. These animals are immunodeficient and allow engraftment of human hepatocytes since they present a mouse hepatocyte-lethal phenotype, such as the FRG mice (Fah-/-/Rag2-/-/IL2Rγc-/-) that we have established in our animal facility and we are using to study the full-life cycle of these hepatotropic viruses.
Although the humanized liver mice allow certain aspects of the natural course of HCV and HBV infection to be analyzed, these models cannot be used to decipher the protective or pathophysiological effects of anti-viral immune responses on infected hepatocytes as well as the preclinical investigation of novel immunotherapies and vaccines, since a functional human immune system is not present. To understand the cross-talks between the immune system and the liver during viral hepatitis, we are establishing novel mouse models in which human immune cells co-develop with human hepatocytes in the same mouse model. These mice will provide a unique opportunity to study the determinants of the human immune response to HBV or HCV infection and to address several aspects of viral pathophysiology. They will also address an urgent need of the academic research community as well as the pharmaceutical industry to develop immunotherapies and vaccines against viral hepatitis.

5 selected publications:
1. Levy C, Fusil F, Amirache F, Costa C, Girard-Gagnepain A, Negre D, Bernadin O, Garaulet G, Rodriguez A, Nair N, Vandendriessche T, Chuah M, Cosset* FL, Verhoeyen* E. 2016. Baboon envelope pseudotyped lentiviral vectors efficiently transduce human b cells and allow active factor ix b cell secretion in vivo in nod/scidgammac-/- mice. J Thromb Haemost. 14:2478-2492. *Corresponding and co-last authors
2. Girard-Gagnepain A, Amirache F, Costa C, Levy C, Frecha C, Fusil F, Negre D, Lavillette D, Cosset FL, Verhoeyen E. 2014. Baboon envelope pseudotyped lvs outperform vsv-g-lvs for gene transfer into early-cytokine-stimulated and resting hscs. Blood. 124:1221-31.
3. Levy, C., F. Amirache, C. Costa, C. Frecha, C. P. Muller, H. Kweder, R. Buckland, E. Verhoeyen*, and F. L. Cosset*. 2012. Lentiviral vectors displaying modified measles virus gp overcome pre-existing immunity in in vivo-like transduction of human T and B cells. Mol Ther 20:1699-1712. *Corresponding and co-last authors
4. Frecha, C., C. Costa, C. Levy, D. Negre, S. J. Russell, A. Maisner, G. Salles, K. W. Peng, E. Verhoeyen*, and F. L. Cosset*. 2009. Efficient and stable transduction of resting B-lymphocytes and primary chronic lymphocyte leukemia cells using measles virus gp displaying lentiviral vectors. Blood 114:3173-3180. *Corresponding and co-last authors
5. Frecha, C., C. Costa, D. Negre, E. Gauthier, S. J. Russell, E. Verhoeyen*, and F. L. Cosset*. 2008. Stable transduction of quiescent T cells without induction of cycle progression by a novel lentiviral vector pseudotyped with measles virus glycoproteins. Blood 112:4843-4852. *Corresponding and co-last authors

F-Translational developments: towards clinical and biotherapy applications

Lab members involved: F Amirache, O Bernadin, C Costa, F Fusil, A Girard, D Lavillette, A Gutteriez, C Levy, J Mancip, D Nègre, J Szecsi, E Verhoeyen

The manipulation of viral genomes and the engineering of viral particles lead to fascinating and powerful perspectives in several areas of biomedical research. Thanks to their capacity to integrate in host cell DNA, retroviruses provide attractive tools for gene delivery. Furthermore, the flexibility by which different viral or cellular components can be assembled on/in viral particles allows deriving macromolecular platforms that display miscellaneous polypeptides of interest, an approach useful in the domains of vaccinology, gene therapy and compound screening.

Vectors for gene therapy and immunotherapy
The efficient transduction of T cells, B cells and dendritic cells (DCs) is an essential asset for immuno-therapy and gene therapy since expression of a gene of interest would improve genetic vaccination against cancer or autoimmune diseases. Importantly, as lentiviral vectors engineered with measles virus envelope glycoproteins (MV-LVs) allow robust transduction of resting T and B cells as well as immature dendritic cells (DCs), they are excellent tools to study functions of the latter cells and to render T-cell, B-cell and DC based-gene therapy and immunotherapy applications feasible.
Using these novel MV-LV tools, we are exploring two gene transfer-based therapeutic approaches into autologous B cells, which are cells that are efficient at protein secretion (e.g. antibodies). In a first approach, we focus on an anti-HCV B-cell based immunotherapy permitting B-cells to express antiviral antibodies. Reinfusion of autologous B cells transduced with specific antibody cDNAs would allow a quick and continuous secretion of HCV neutralizing antibodies in vivo. This approach will be tested in the context of the humanized mouse models described above, in which HCV can replicate and induce B cell responses after adoptive transfer or reconstitution. In a second approach, we are developing a gene therapy for treatment of hemophilia by allowing secretion of blood coagulation factors (Factors IX and VIII) from transduced autologous B cells. We expect that this approach does not require high-level transduction because of the bystander effect and because mature B cells are known to persist durably in humans, which will be evaluated in vivo by adaptive transfer of the modified cells in NSG mice.

For life-long correction of monogenetic blood diseases, the target cells of choice are the hematopoietic stem cells (HSCs) as they can give rise to all blood cell lineages. In vivo gene delivery into HSCs would mean a great step forward in the field of gene therapy. We developed LVs co-displaying on the surface of vector particles a modified RD114 envelope glycoprotein and an HSC-targeted cytokine, stem cell factor (SCF). Such RDTR/SCF-LVs allow efficient targeted transduction through specific vector-cell ligation and via SCF binding to its receptor present on the HSC and their subsequent penetration by membrane fusion via the RD114 glycoprotein counterpart. Remarkably, in vivo injection of RDTR/SCF-LVs into the bone marrow cavity of human immune system mice resulted in highly selective transduction of HSCs. Therefore, such RDTR/SCF-LVs might completely omit ex vivo handling of HSCs and simplify gene therapy for hematopoietic disease in the future by direct in vivo vector inoculation. For translation towards the clinic of this in vivo approach, we are focusing our efforts on a monogenetic disease: Fanconi Anemia (FA). FA HSC numbers are reduced in patients and their ex vivo manipulation induce their fragility, which could be circumvented by using allowing in vivo gene correction of FA in HSCs. Since FA mouse disease models do not recapitulate FA disease and since RDTR/SCF-LVs transduce human cells, we will first establish a humanized mouse model for FA by FANCA-knockdown in human HSCs before their engraftment in immunodeficient mice. Then, RDTR/SCF-LVs will be evaluated for in vivo correction of FA in this model. Importantly, this strategy will pave the way towards in vivo correction of FA and other monogenetic disease, such as e.g. beta-thalassemia.

Viral-like particles for vaccinology studies
The unraveling of the mechanisms driving assembly of viral particles and governing replication of viral genomes has allowed the design of VLP (viral-like particles)-based vaccines derived from retroviral Gag proteins incorporating on their surface the glycoproteins from several enveloped viruses. They represent novel immunogens that combine the advantages of sub-unit vaccines, i.e., high bio-safety features, and those of inactivated or attenuated viruses, i.e., strong immunogenicity. Furthermore, several gene delivery vectors are available that allow sustained expression of VLP antigenic formulations for the necessary time frames and within the appropriate tissues and/or intracellular localizations, as needed to raise suitable immune responses. Finally, the assembly of VLPs does not require viral replication and propagation, as opposed to true viruses. This offers many possibilities for structural modifications of the displayed antigens through genetic manipulation, aiming to present epitopes that otherwise would be difficult to reveal in conventional attenuated/inactivated vaccines and that could trigger immune responses that may eventually be poorly or not induced naturally.
We have obtained proofs of concept for VLP-based vaccine candidates against HCV and respiratory viruses such as highly pathogenic avian influenza viruses (HPAIV) and human metapneumovirinae (HMPV). VLPs associating envelope glycoproteins derived from HCV and HPAIV induced potent neutralizing antibody responses in mice and macaques. Interestingly, VLPs engineered to display the HCV E1 glycoprotein elicited neutralizing antibody responses of broad specificity against major HCV genotypes. Likewise, VLPs incorporating appropriately engineered HPAIV and HMPV induced cross-reactive neutralizing antibodies. Vectorization of these novel immunogens induced strong and potent humoral responses that were protective against lethal challenge. We are seeking to develop VLP immunogens for fighting emerging hemorrhagic fever viruses such as DENV and Crimean–Congo hemorrhagic fever virus (CCHFV) in partnership with private companies.

5 selected publications:
1. Fusil F, Calattini S, Amirache F, Mancip J, Costa C, Robbins JB, Douam F, Lavillette D, Law M, Defrance T, Verhoeyen E, Cosset FL. 2015. A lentiviral vector allowing physiologically regulated membrane-anchored and secreted antibody expression depending on b-cell maturation status. Mol Ther. 23:1734-47.
2. Levy, C., L. Aerts, M. E. Hamelin, C. Granier, J. Szecsi, D. Lavillette, G. Boivin*, and F. L. Cosset*. 2013. Virus-like particle vaccine induces cross-protection against human metapneumovirus infections in mice. Vaccine. 31:2778-2785. *Corresponding authors
3. Frecha, C., C. Costa, D. Negre, F. Amirache, D. Trono, P. Rio, J. Bueren, E. Verhoeyen*, and F. L. Cosset*. 2012. A novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice. Blood 119:1139-1150. *Corresponding and co-last authors
4. Garrone, P., A. C. Fluckiger, P. E. Mangeot, E. Gauthier, P. Dupeyrot-Lacas, J. Mancip, A. Cangialosi, I. Du Chene, R. Legrand, I. Mangeot, D. Lavillette, B. Bellier, F. L. Cosset*, F. Tangy*, D. Klatzmann*, and C. Dalba*. 2011. A prime-boost strategy using virus-like particles pseudotyped for HCV proteins triggers broadly neutralizing antibodies in macaques. Science translational medicine 3:94ra71. *Co-last authors
5. Szecsi, J., G. Gabriel, G. Edfeldt, M. Michelet, H. D. Klenk, and F. L. Cosset. 2009. DNA Vaccination with a Single-Plasmid Construct Coding for Viruslike Particles Protects Mice against Infection with a Highly Pathogenic Avian Influenza A Virus. The Journal of infectious diseases 200:181-190.

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