Madame Beatriz

Eugenia Escudero Pérez

M Viktor

Volchkov Devant

M Renaud

M Christophe Peyrefitte

M Daniel Pinschewer

M Winfried Weissenhorn

The role of shed GP in Ebola virus pathogenicity

Keywords:

J'aimerais aussi remercier les membres de mon jury pour m'avoir fait l'honneur d'évaluer mon travail et pour l'échange qui aura lieu le jour de ma défense: M. Peyrefitte, qui a très aimablement accepté de juger mon travail en tant que représentant de la DGA (Délégation Générale pour l'Armement), M. Walzer et M. Pinschewer et M.Weissenhorn qui ont accepté d'être les rapporteurs de ma thèse ainsi que le Professeur Mahieux, qui a accepté d'être le président de mon jury. Je tiens à remercier spécialement à Thierry Walzer pour sa totale et inconditionnelle disponibilité pour m'aider avec des doutes en immunologie et pour ses conseils professionnels. Je l´admire profondément pour son intelligence, son honnêteté, et pour sa capacité à motiver et inspirer les autres. J'aimerais aussi exprimer ma profonde gratitude aux membres de son équipe pour leur gentillesse, leur sympathie, leur disponibilité, leur soutien, leur intérêt et leur aide constante avec tout ce dont j'ai eu besoin. Je remercie en spécial à Antoine, Sophie, Fabrice et Sébastien. Un grand merci également à Armelle, avec qui j'ai passée des très bons moments ensemble et qui a réussi à m'arracher du laboratoire pour jouer au foot. Je remercie aussi à Thierry Defrance, Denis Gerlier et Mathias Faure pour leur aide désintéressée. Je les admire énormément non seulement parce qu´ils sont des grands scientifiques mais aussi parce qu´ils sont des personnes honnêtes, intelligentes et intègres. Je voudrais aussi exprimer ma plus sincère gratitude à Marc Jamin et de nouveau à Thierry Walzer pour m'avoir fait l'honneur de faire partie de mon comité de thèse il y a deux ans. Je les remercie fortement pour m'avoir aidé à prendre du recul sur mon projet de thèse et pour les enrichissantes discussions qui m'ont permis d'avoir un regard plus critique sur mes résultats et d´avancer dans mon projet. J'aimerais bien remercier aussi à Veronique Santyi pour sa gentillesse, sa disponibilité et pour m´avoir initiée au Biacore et l' AKTA, machines que sans son aide, je n'aurais pas réussi à maîtriser. Je remercie également le plateau technique de cytometrie, en particulier à Sébastian et Thibault pour leur aide. Je ne peux pas non plus oublier de remercier à Renaud Colisson qui a toujours essayé de m'aider, de me faire faire des pauses pendant les intenses journées de travail et qui m'a appris patientent (même les week-ends) les bases de la cytometrie. Un grand merci aussi à tout l'équipe P4, en spécial à Audrey, pour son amabilité et son professionnalisme, à Delphine, pour sa sympathie et les bons moments passés pendant la formation, à Stephanie et à Stephan ainsi qu´au poste central de biosécurité pour m'avoir permis de réaliser toutes mes expériences dans d'excellentes conditions. Je suis très reconnaissante a la Délégation Générale pour l'Armement (DGA), La Caixa en Espagne et l'INSERM pour m´avoir permis de réaliser ce travail.

Je remercie aussi à ma petite étudiante, Camille Gibourt, avec qui, malgré le fait que le temps passé ensemble fut bref, j'ai beaucoup profité de cette échange qui m´a permis de ratifier ma passion pour l'enseignement et la recherche.

Un grand merci à Mathieu Mateo, qui à été mon premier maître de stage et qui m'a surtout appris dès le début l´engagement au quotidien envers la recherche.

de profiter de son large expérience dans beaucoup de domaines. Je présente aussi mes remerciements à Valentina pour avoir été ma deuxième maître de stage et m'avoir introduit au intéressant monde des GPs du virus Ebola; je la remercie également pour sa gentillesse et sa douceur qui imprègnent l´esprit du laboratoire. Même s'il n'est plus là, je tiens à remercier spécialement à Kirill Nemirov, qui est arrivé au laboratoire au même temps que moi et qui a été un énorme soutien et un très bon ami: il est une des meilleures personnes que je n'ai jamais connues. Je remercie aussi à Nicolas pour les très bons moments passés ensemble, pour sa disponibilité, ses encouragements et sa précieuse connaissance du mystérieux monde de la statistique. J'aimerais remercier à Audrey Baule, qui m'a beaucoup appris sur l'immunologie et qui a été une amie et un appui en tout moment. Je la remercie aussi pour toujours me faire confiance, pour sa compréhension et pour me pousser à élargir mes ambitions professionnelles. Sa présence au laboratoire m'a beaucoup manqué.

Finalement, le plus grand remerciement est pour Philip Lawrence, qui a été mon phare dans le laboratoire depuis son arrivée. Il m`a poussé à mettre en valeur mes résultats, il m'a corrigé, écouté, recorrigé, il a toujours respecté mes opinions, il m'a défendu et il a cru en moi. Au delà des remerciements pour avoir été ma bouée professionnelle, je le remercie surtout pour avoir été un vrai ami. Je ne trouve pas assez des mots pour exprimer ma gratitude envers lui. J'adresse également mes remerciements, pour leur sympathie et leurs conseils à tous les anciens membres de l'équipe Filovirus (Sébastian, Mikel, St Patrik, Jennifer, Cidney, Ilona, Nolwen, Ludo, Amandine) ainsi qu'à nos voisins, l'équipe Pasteur. Je voudrais aussi remercier à toutes les personnes de la TOUR que j'ai croisé à un moment où à un autre et avec qui nous avons partagé des bons moments (Ludovic, Joel, Chloé…).

Un grand merci aussi à mon copain Joévin, pour son inestimable soutien (je me demande encore comment il fait pour me supporter tout le temps surtout en fin de thèse), je le remercie aussi pour notre passion commune des voyages qui me rappelle qu'il y a un monde parallèle à / en dehors de la thèse que je hâte d'explorer. Merci de me rappeler de temps en temps que le travail ce n'est pas tout dans la vie et mercie de t'être toujours adapté à moi et à mes ambitions professionnelles.

A les meves amigues Cèlia i Irene, amb qui he pogut mantenir una veritable amistat al llarg dels anys tot i la llarga distància que ens separa. Els hi agreixo molt el seu suport, la seva forma de ser i el fet que em fassin sentir que malgrat el temps que no ens veiem, encara podem comptar les unes amb les altres i compartir moments genials. També ocupades per la seva tesis, estic contenta d'haver pogut compartir aquesta difícil experiència amb elles i espero que l'acabem aviat i ens poguem veure més sovint.

A Laura, por su apoyo, su alegría, su sonrisa, los viajes rélampago y su amistad.

A Richard Preston, por haberme seducido con su libro Zona Caliente y haber despertado mi interés por Ebola a la temprana edad de 8 años.

A toda mi familia, en especial a mis padres y a mi hermana. A mi hermana, Raquel Escudero Pérez, por haberme hecho sentir que no estaba sola, que siempre podría contar con ella y, a pesar de ser una de las personas mas inteligentes y trabajadoras que conozco, considerarme capaz de sacarme la tesis.

Finalmente, a mis padres, a quien les debo todo. Quisiera agradeceros por vuestra eterna paciencia conmigo, por haber sido los únicos que me habeis escuchado, entendido y luchado a mi lado por mis sueños y mi persona. Os agradezco por haberme dado los valores que tengo, porque nunca han sido los conceptos que he aprendido a lo largo de mis estudios los que me han permitido salir adelante sino vuestros principios, vuestra fuerza y vuestro carácter que ahora también son los míos. No sé si la tesis me definirá como una buena científica, pero sí estoy segura que esta tesis me ha puesto a prueba como persona y que sin vosotros, no podría haberla hecho. Vosotros habeis sido los que habeis marcado la diferencia.

Merci à tous, Beatriz Eugenia Escudero Pérez

La verdadera ciencia enseña, por encima de todo, a dudar y a ser ignorante. Ebola virus is the etiologic agent of a highly lethal hemorrhagic fever in humans and nonhuman primates (NHPs) with mortality rates of up to 90% in humans [START_REF] Zampieri | Immunopathology of highly virulent pathogens: insights from Ebola virus[END_REF]). Due to its high mortality rate, its potential for human-to-human transmission and the lack of an approved vaccine or therapy, the virus is classified as a category A pathogen requiring biosafety level 4 (BSL-4) containment.

Miguel de

List of tables

Ebola virus belongs to the family Filoviridae, in the order Mononegavirales which includes Rhabdoviridae and Paramyxoviridae. The family consists of three genera: Marburgvirus, Ebolavirus and Cuevavirus [START_REF] Kuhn | Proposal for a revised taxonomy of the family Filoviridae: classification, names of taxa and viruses, and virus abbreviations[END_REF].

Ebola haemorrhagic fever (EHF) is caused by five genetically distinct members of the Filoviridae family: Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV),

Côte d'Ivoire ebolavirus (CIEBOV), Bundibugyo ebolavirus (BEBOV) and

Reston ebolavirus (REBOV). The species vary in their pathogenicity in humans with ZEBOV being the most pathogenic (up to 90% case fatality rate). For Sudan and Bundibugyo Ebolavirus the mortality rate is lower, 53% and 34%

respectively. The species Côte d'Ivoire is lethal for NHPs (Formenty, Boesch et al. 1999). Ebolavirus Reston is not pathogenic for humans (4 asymptomatic cases have been described), however, it is lethal for NHPs and pigs. All species are endemic in Africa except Ebolavirus Reston, which is endemic in the Philippines (Miranda, Ksiazek et al. 1999[START_REF] Barrette | Discovery of swine as a host for the Reston ebolavirus[END_REF] 2002, Mahanty and Bray 2004, Baize, Pannetier et al. 2014).

The geographical distribution of the different epidemics of filovirus hemorragic fevers corresponds to the equatorial zone of the globe (Figure 1). Ebola haemorrhagic fever remains a plague for the population of equatorial Africa, with an increase in the numbers of outbreaks and cases since 2000. Almost all human cases are due to the emergence or reemergence of Zaire Ebola virus in regions of Gabon, Republic of the Congo, DRC and very recently in west Africa, and of Sudan Ebola virus in Sudan and Uganda. Subsequently, Reston Ebola virus has been found in the Philippines on several occasions, including reports documenting infections in pigs [START_REF] Barrette | Discovery of swine as a host for the Reston ebolavirus[END_REF]). The emergence of Reston Ebola virus in pigs raises important concerns for public health, agriculture, and food safety in the Philippines and could represent a serious issue for parts of Asia [START_REF] Feldmann | Ebola haemorrhagic fever[END_REF]. Adapted from [START_REF] Feldmann | Ebola haemorrhagic fever[END_REF].

A novel filovirus, provisionally named Lloviu virus (LLOV), was detected during the investigation of Miniopterus schreibersii die-offs in Cueva del Lloviu in southern Europe. LLOV is genetically distinct from other marburgviruses and ebolaviruses and is the first filovirus detected in Europe that was not imported from an endemic area in Africa [START_REF] Negredo | Discovery of an ebolavirus-like filovirus in europe[END_REF]. Phylogenetic analysis of LLOV indicates a common ancestor of all filoviruses, dating to at least 150,000 years ago (Figure 2). The discovery of LLOV in M. schreibersii is consistent with filovirus tropism for bats. However, unlike MARV and EBOV, where asymptomatic circulation is posited to be consistent with evolution to avirulence in this long-term host-parasite relationship, several observations suggest that in the case of LLOV, filovirus infection may be pathogenic; LLOV was found in the affected bat population but not in other healthy bats. In addition, RNA sequences were found in lung and spleen of the affected bat population. The discovery of a novel filovirus in western Europe, and the wide geographical distribution of the associated insectivorous bat is a significant concern. While the virus was detected in the north of Spain, simultaneous bat die-offs were also been observed in Portugal and France (Negredo, Palacios et al. 2011). Filoviruses had been suggested to show a geographically related phylogeographic structure [START_REF] Peterson | Ecologic and geographic distribution of filovirus disease[END_REF]). Viruses from particular geographic areas cluster together phylogenetically, suggesting a stable hostparasite relationship wherein viruses are maintained in permanent local pools.

The discovery of a novel filovirus in a distinct geographical niche suggests that the diversity and distribution of filoviruses should be studied further. Recently, a study has shown that additional bat species not previously associated with EBOV such as Epomorphorus gambianus, Nanyecteris veldkampii and Eiodolon helvum contain antibodies against ZEBOV and REBOV (Hayman, Yu et al. 2012). This reinforces the hypothesis of bats being the possible natural reservoir of filoviruses.

Serological evidence of ebolavirus infection in several bat populations in China has also recently been reported [START_REF] Yuan | Serological evidence of ebolavirus infection in bats, China[END_REF]. This, together with the Lloviu finding, suggests that filoviruses have a wider host range and geographical distribution than previously thought.

Ebola virus natural cycle 1.2.1. Ebola virus reservoir

Despite the discovery of this virus family almost 40 years ago, the natural reservoir of these lethal pathogens remains an enigma.

Several studies have been undertaken in order to try and identify the natural reservoir of the filoviruses. Since 1976 these studies have been based on the capture and analysis of plants and wild animals (vertebrates and invertebrates)

suspected to be potential natural hosts of filoviruses.

In outbreaks where information is available, the human index cases have invariably had direct contact with gorillas, chimpanzees, antelope or bats. The search for a reservoir of EBOV has been very aggressive. Although great apes are generally involved in EHF outbreaks, NHPs are not thought to be natural reservoirs but, rather, susceptible hosts, based on the sudden sharp decline in populations of the great apes in Gabon and the Republic of Congo which coincided with EBOV outbreaks in humans (Pourrut, Kumulungui et al. 2005).

In one study, short EBOV-specific genetic sequences were amplified from organs of six mice (Mus setulosus and Praomys sp) and a shrew (Sylvisorex ollula). However no firm conclusions as to the EBOV reservoir status of these animals can be drawn, given the lack of specific serologic responses, the lack of nucleotide specificities in the amplified viral sequences, the failure of virus isolation attempts and the non-reproducible nature of the results. Also several attempts were made to infect various plant and animal species, including rodents (mice, shrews, rats, guinea pigs, etc.), birds (mainly pigeons), reptiles (tortoises, snakes, geckos, frogs), molluscs (mainly snails), arthropods (spiders, cockroaches, ants, centipedes, mosquitoes and butterflies) and more than 33 plant varieties belonging to 24 species (tomatoes, cucumbers, wheat, cotton, lupin, corn, tobacco, etc.). All attempts to inoculate these species failed to yield evidence of virus replication (Pourrut, Kumulungui et al. 2005).

In contrast, EBOV infection of vertebrates and invertebrates gave the first evidence that both insectivorous and frugivorous bats can support the replication and circulation of EBOV (Swanepoel, Leman et al. 1996). This evidence, along with reports of bat exposures for some of the Ebola index cases, directed research towards bats as potential reservoirs. Indeed, an ecological survey revealed the presence of ZEBOV-specific antibodies in six bat species caught in the field (Epomops franqueti, Hypsignathus monstrosus, Myonycteris torquata, Micropteropus pusillus, Mops condylurus and Rousettus aegyptiacus) (Pourrut, Kumulungui et al. 2005). Since 2000, trapping expeditions have been undertaken in areas close to outbreaks in order to identify the viral reservoir. In total, in one study 1,030 animals were captured and were tested for evidence of infection by Ebola virus. Immunoglobulin G (IgG) specific for Ebola virus was detected in serum from three different bat species: Hypsignathus monstrosus, Epomops franqueti and Myonycteris torquata (Figure 3). Viral nucleotide sequences, some of them clustering phylogenetically within the Zaire clade, were detected in liver and spleen in other bats from the same populations. Surprisingly, none of the IgG-positive animals was PCR-positive, and none of the PCR-positive animals was IgGpositive. Moreover it was not possible to isolate the virus itself (Leroy, Kumulungui et al. 2005).

Figure 3.-Possible Ebola virus reservoirs

The unsuccessful identification of ebolavirus-related genes in the samples is likely attributable to the often low-level of virus replication, the similarly transient nature of the infection in bats or the sequence mis-match of the PCR primers used and the possibly divergent target sequence of the potential unknown ebolavirus genomes.

There are several possibilities to account for the failure to detect neutralizing antibodies. In general, bats seem to produce lower level of neutralizing antibodies in response to viral infection, possibly due to the lower affinity of the bat antibodies [START_REF] Baker | Immunoglobulin heavy chain diversity in Pteropid bats: evidence for a diverse and highly specific antigen binding repertoire[END_REF]). Alternatively, it is possible that one or more as-yet-unknown ebolaviruses are circulating among the bat populations sampled.

In addition, investigations are usually implemented retrospectively, several weeks or months after the index case has been infected from a putative reservoir. It is possible that by that time the putative reservoirs may have moved to another site. A surveillance system capable of early detection of Ebola cases could allow animal reservoir studies in 'real time', which is not always easy in remote places in African forests.

Bats as possible reservoir

Numerous publications in the past few years have reinforced the observation that bats carry a wide range of novel RNA and DNA viruses. One might hypothesize that they could play an important role in facilitating the dispersal of these viruses to different geographical locations and different hosts. The recent surge of interest in bats as a reservoir of viruses was driven by two factors.

First, in the last 20 years, several high profile viral pathogens have been proven

or hypothesized to have a bat origin (Hendra virus (Halpin, Young et al. 2000),

Nipah virus [START_REF] Yadav | Detection of Nipah virus RNA in fruit bat (Pteropus giganteus) from India[END_REF], Severe acute respiratory syndrome (SARS) [START_REF] Field | Bats and emerging zoonoses: henipaviruses and SARS[END_REF], [START_REF] Balboni | The SARS-like coronaviruses: the role of bats and evolutionary relationships with SARS coronavirus[END_REF])) and MERS [START_REF] Kupferschmidt | Emerging infectious diseases. Link to MERS virus underscores bats' puzzling threat[END_REF] .

The second driver for the recent surge in bat virus research has come from advances in modern molecular techniques which have presented opportunities for discovery of novel viruses, that were considered impossible or nonpractical just a decade ago. Using pan-virus-specific primers and next-generation sequencing, it is now possible to detect and characterize novel viral sequences without the need for virus isolation by cell culture or the identification of virions by electron microscopy.

Bats are one of the longest living mammalian orders, and probably constitute the order with the most diversity between species. They originated early, around 50 million years ago, and the different species have changed relatively little over time compared to mammals of other taxa. The wide range of bat species could provide a large 'breeding ground' for viruses. Bat viruses may therefore have co-evolved with or adapted to bats over many millions of years. Besides, bats are the only mammalian species that can fly and some bat species can migrate hundreds of miles to their overwintering or hibernation sites. Thus, bats have more opportunities than terrestrial mammals to have direct or indirect contact with other animal species at different geographical locations, thereby enhancing the opportunity for interspecies virus transmission [START_REF] Calisher | Bats: important reservoir hosts of emerging viruses[END_REF].

Persistence in the absence of pathology or disease appears to be a common characteristic of bat viruses in their natural host population and this is also indicative of a highly evolved relationship (Taylor, Leach et al. 2010). It could be argued, therefore, that the most significant characteristic of viral infections in bats may not be the effectiveness of a highly evolved host immune response, but rather the absence of pathology as the result of an ancient and highly evolved viral survival strategy. For many RNA viruses such as those commonly infecting bats, accessory proteins and evolved secondary functions of other viral proteins play a key role in infection by blocking host innate immune defences, modulating cellular signaling pathways and re-directing normal cellular functions (Frieman and Baric 2008). Highly adapted viruses persistently infecting bat populations might also serve to protect bats at the species or population level from predators, in a sense acting as defensive 'biological weapons'. The best defensive weapons are those that do no harm to the host species and are released only when there is an imminent threat of danger and the emerging bat viruses satisfy these requirements. Henipaviruses are believed to persist in bat populations at a very low viral load and are totally harmless to their natural host.

However, under stress, the viral load increases, facilitating transmission to other animals. Such a mechanism might not be able to protect every individual animal in a population, but it would be an effective way to preserve the species.

In principle, such a symbiotic relationship with viruses would benefit any animal species and there is evidence that such relationships do exist in very different hosts including species ranging from humans to mice to fungi and bacteria. It is perhaps the long period of co-evolution and some unique selective pressures that have driven viral emergence and dominance in bats. Bats have a relatively low reproductive rate compared to other animals such as rodents and bats tend to live in very large and dense populations. These biological and behavioral characteristics may demand far more robust mechanisms to fight infection and predation in order to avoid extinction.

Ebola virus transmission

Filovirus transmission among humans occurs through mucosal surfaces, breaks and abrasions in the skin, or by parenteral introduction. Most human infections in outbreaks seem to occur by direct contact with infected patients (particularly in the late stages of infection) or cadavers, when viral loads are highest.

Transmission can also happen through contaminated needle reuse following a 4

to 16 day incubation period (the period between infection and onset of symptoms).

Infectious virus particles or viral RNA have been detected in semen, genital secretions [START_REF] Yu | Detection of Ebola virus envelope using monoclonal and polyclonal antibodies in ELISA, surface plasmon resonance and a quartz crystal microbalance immunosensor[END_REF], and in skin of infected patients (Zaki, Shieh et al. 1999); they have also been isolated from body fluids, and nasal secretions of experimentally infected non-human primates (Geisbert, Young et al. 2003).

The natural reservoir of infection remains unknown, but the virus clearly has a zoonotic origin. As discussed above, infection cases of people having visited caves containing bats suggest a possible transmission of filoviruses from bats to humans. Despite the fact that this type of transmission has not been demonstrated, transmission between monkeys and humans has clearly been established. In most outbreaks, Ebola virus is introduced into human populations via the handling of infected animal carcasses. In these cases, the first source of transmission is an animal found dead or hunted in the forest, followed by person-to-person transmission from the index case to family members or health-care staff. Animal-to-human transmission occurs when people come into contact with tissues and bodily fluids of infected animals, especially with infected nonhuman primates [START_REF] Leroy | Multiple Ebola virus transmission events and rapid decline of central African wildlife[END_REF]). Virus transmission is also amplified during funerary rituals typical of some regions of Africa. Although proper cooking of foods should inactivate infectious Ebola virus, ingestion of contaminated food cannot wholly be ruled out as a possible route of exposure in natural infections. Notably, handling and consumption of freshly killed bats was associated with an outbreak of Zaire Ebola virus in Democratic Republic of Congo [START_REF] Leroy | Human Ebola outbreak resulting from direct exposure to fruit bats in Luebo, Democratic Republic of Congo, 2007[END_REF].

Organ infectivity titres in non-human primates infected with Ebola virus are frequently in the range of 10 7 to 10 8 pfu/g; thus, exposure through the oral route could be associated with very high infectious doses. Transmission of Zaire Ebola virus to uninfected macaques housed in the same room as experimentally infected macaques has been reported; however, the study did not exclude the possibility that exposure had occurred from excreted virus that was aerosolized during cleaning of the cages rather than "true" primate-toprimate transmission [START_REF] Johnson | Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus[END_REF]. In fact, studies have shown that filoviruses are relatively stable as aerosols, retain virulence after lyophilisation and can persist for long periods on contaminated surfaces [START_REF] Belanov | Survival of Marburg virus infectivity on contaminated surfaces and in aerosols[END_REF]. Another study shown that Zaire Ebola virus is highly lethal when given orally to Rhesus monkeys causing a pathology close to the one observed through intraperitoneal infection and also leads to death of the animal [START_REF] Johnson | Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus[END_REF]. The possibility to generate aerosol containing filoviruses is a possible bioterrorism threat. This form of transmission has been observed with REVOB. In 1990 all monkeys from Reston animal facility were contaminated, including those who were separated from infected macaques, which could be explained by an aerosol inter-simian transmission.

However, there is no evidence that primate-to-primate transmission by aerosol actually occurred [START_REF] Jahrling | Preliminary report: isolation of Ebola virus from monkeys imported to USA[END_REF]).

Even though aerosolised filoviruses are highly infectious for non-human primates in the laboratory, transmission patterns during epidemics indicate that the virus does not spread naturally among human beings by the respiratory route, which suggests that it is not efficiently aerosolised by humans. Thus, aerogenic transmission of filovirus has still not been demonstrated in humans.

In human beings, the route of infection seems to affect the disease course and outcome. The mean incubation period for cases of Zaire Ebola virus infection known to be due to injection is 3-6 days, versus 5-9 days for contact exposures.

Moreover, the case-fatality rate in the 1976 outbreak of Zaire Ebola virus was 100% (85 of 85) in cases associated with injection compared with about 80%

(119 of 149) in cases of known contact exposure. For nonhuman primates infected with Zaire Ebola virus the disease course seems to progress faster in animals exposed by intramuscular or intraperitoneal injection than in animals exposed by aerosol droplets [START_REF] Geisbert | Vesicular stomatitis virus-based vaccines protect nonhuman primates against aerosol challenge with Ebola and Marburg viruses[END_REF].

Currently there is no treatment or vaccine approved to use in humans against Ebola virus.

Clinical aspects of Ebola virus haemorrhagic fever

Ebola viruses (EBOV) are among the most pathogenic viruses known to humans with a high case fatality rate of up to 90% that is rarely observed in viral diseases. EBOV hemorrhagic fever (EHF) is often characterized by the sudden onset of fever, weakness, muscle pain, headache, and a sore throat, which are followed by vomiting, diarrhea, rash, kidney and liver dysfunction, and often internal and external bleeding (Sadek, Khan et al. 1999).

After an incubation period of 1-21 days, patients become abruptly ill. The signs and symptoms evolve in three successive phases, which sometimes overlap (Figure 4). The first phase is characterized by nonspecific symptoms; the second by multiple organ involvement; and the third, terminal phase, by recovery or death, depending on both host and viral factors.

Phase I

Symptoms typically begin with a flu-like syndrome with abrupt-onset fever (39-40ºC), chills, violent headaches and generalized fatigue with myalgia.

Sometimes, myalgia with concomitant neck muscle tension occurs, mimicking meningitis.

Phase II

The second phase begins with severe visceral disorders on day 2-4 after symptom onset, and lasts for 7-10 days. During this phase several sympthoms occur: gastrointestinal problems (stomach pain, vomiting, watery diarrhoea), respiratory disorders (throat and chest pain, cough) and neurological manifestations (prostration, confusion, delirium), indicating systemic viral dissemination and multi-organ failure. Haemorrhagic manifestations vary in severity and location, and often include skin rashes, nosebleeds, melaena, haematemesis and bleeding at venepuncture sites. There is also a loss of appetite leading to weight loss and marked dehydration.

Patients have ''a ghostly appearance'' with deep-set eyes, an expressionless face, and extreme lethargy. Respiratory symptoms appear at the same time, with violent sore throat accompanied by chest pain and dry cough, resulting from pharyngeal damage. Swallowing is hindered, further aggravating nutritional status. A non-itchy rash appears between the second and seventh day, followed by fine scaling. Bleeding is frequent, with petechiae at various sites, an ocular burning sensation, reddening, melena, hematemesis, hematuria, epistaxis, hemoptysis, and bleeding at puncture sites. Ultimately, the nervous system can be involved, leading to behavioural disorders (aggressiveness, confusion, delirium), paresthesia, hyperesthesia, and seizures.

Phase III

The terminal phase results in recovery or death. Tachypnea may occur with hiccups, followed by anuria. Death occurs after 2-3 days due to plasmatic shock with capillary extravasation and multiorgan failure. Survivors experience a lengthy period (one month or more) of painful convalescence with intense fatigue, loss of appetite, profound prostration, weight loss, and migratory arthralgia. tachypnoea, anuria, delirium, or coma signal the onset of irreversible shock [START_REF] Kortepeter | Basic clinical and laboratory features of filoviral hemorrhagic fever[END_REF].

INFECTION DECLARATION DEATH

Convulsions

Biological parameters include mild and early leucopenia, associated with lymphopenia, thrombocytopenia (< 100 000 platelets/mm 3 ), hyperproteinaemia, and marked transaminase elevation. The prothrombin time is always prolonged and fibrin split products are often detected, indicating disseminated intravascular coagulation [START_REF] Leroy | Ebola and Marburg haemorrhagic fever viruses: major scientific advances, but a relatively minor public health threat for Africa[END_REF].

Fatal illness is associated with high and increasing amounts of virus in the bloodstream. By contrast, patients who survive infection begin to show a decrease in amounts of circulating virus and clinical improvement around day 6-11. In most cases, this improvement coincides with the appearance of ebolavirus-specific antibodies. Patients with non-fatal or asymptomatic disease increase specific IgM and IgG responses that seem to be associated with a temporary, early and strong inflammatory response, including interleukin β, interleukin 6, and tumour necrosis factor α (TNFα). However, whether this is the mechanism for protection from fatal disease remains to be proven [START_REF] Feldmann | Ebola haemorrhagic fever[END_REF]. The humoral response can result in the formation of antigen-antibody complexes, and some recovering patients develop acute arthralgia and other symptoms consistent with immune complex disease.

Although virus disappears quickly from the bloodstream of survivors, it may persist in particular immunologically privileged sites and has been recovered from the semen of a few patients weeks after the acute illness. Some survivors are unable to recall the most severe period of illness. Thus, convalescence is extended and often associated with sequelae which may include orchitis, recurrent hepatitis, transverse myelitis, and uveitis. Psychiatric sequelae include confusion, anxiety, and restless and aggressive behaviour [START_REF] Nkoghe | Cutaneous manifestations of filovirus infections[END_REF]).

1.4.Diagnostic techniques to identify Ebola virus

Ebola haemorrhagic fever can be suspected in acute febrile patients with a history of travel to an endemic area, if they present with fever and constitutional symptoms. Identification might be difficult because severe and acute febrile diseases can have a wide range of causes in areas endemic for Ebola virus, with the most prominent being malaria and typhoid fever followed by others The development of sensitive diagnostic assays capable of detecting EBOV in human and nonhuman primates is important for identifying outbreaks and supporting ensuing epidemiologic investigations.

Several diagnostic assays for Ebola infection are currently used (Table 2). Virus isolation, the most definitive but not necessarily the most sensitive method, requires high level biocontainment, and regardless is too slow for outbreak detection and management (Leroy, Baize et al. 2000). Similarly electron microscopy and histological techniques are sensitive methods particularly reliable for post-mortem diagnosis but require sophisticated equipment and reagents (Zaki, Shieh et al. 1999). Both methods have proved to be very effective as field diagnostic tools for the detection of EBOV antigen and nucleic acids in patient serum, plasma, and whole blood [START_REF] Onyango | Laboratory diagnosis of Ebola hemorrhagic fever during an outbreak in Yambio, Sudan, 2004[END_REF].

Moreover these assays can only be done on materials that have been rendered non-infectious. An efficient way to inactivate the virus for antigen and antibody detection is the use of gamma irradiation or heat inactivation. Similarly, nucleic acid can be amplified by purification of the virus, such as RNA from materials treated with guanidinium isothiocyanate -a chemical chaotrope that denatures the proteins of the virus and renders the sample non-infectious.

1.5.Ebola virus animal models

Development of animal models is critical to understanding Ebola virus pathogenesis. Several animal models have been developed in order to study pathophysiology, vaccines and therapeutics of Ebola hemorrhagic fever including mice, guinea pigs, hamsters and NHPs [START_REF] Connolly | Pathogenesis of experimental Ebola virus infection in guinea pigs[END_REF][START_REF] Bente | Disease modeling for Ebola and Marburg viruses[END_REF], Bradfute, Warfield et al. 2012). However, they do not all show the same pathological features when infected by Ebola virus (Table 3).

-Mouse model: development of mice, in contrast to guinea pigs and NHP models, has been unsuccessful due to the fact that serial passages of wild-type EBOV in mice are needed for adaptation. The adapted virus resulted in 8 amino acid changes in both coding and non-coding regions of the virus genome compared to the original wild-type virus; nucleotide substitutions leading to amino acid changes were found in VP35, VP24, NP and L polymerase (Ebihara, Takada et al. 2006). Moreover, the route of viral inoculation is determinant to observe or not infection: intraperitoneal inoculation causes lethal infection whereas subcutaneous inoculation does not cause a symptomatic illness [START_REF] Gibb | Pathogenesis of experimental Ebola Zaire virus infection in BALB/c mice[END_REF]). This phenomenon is not observed in NHP models, which are susceptible to wild-type infection. Infected mice become acutely ill with symptoms of ruffled fur, reduced activity and loss of weight. However, mice infected with mouse-adapted filovirus show differences when compared to infection in human disease or NHP models in terms of a lack of severe coagulation disorder and fibrin deposition; in addition, the cytokine profile observed during infection is not the same as in humans and NHP.

Nevertheless, mouse models can be useful tools for studying basic aspects of replication, pathogenesis, and immune responses and also serve to evaluate candidate vaccines and therapeutic agents [START_REF] Nakayama | Animal models for Ebola and Marburg virus infections[END_REF].

-Guinea pig model: Infection of guinea pigs with wild-type EBOV causes only a transient febrile illness; Ebola virus needs to be serially passaged in guinea pigs to acquire the ability to cause lethal infection in this model (Ryabchikova, Strelets et al. 1996). The lack of virulence of wild-type virus in guinea pigs is associated with an inability of the virus to replicate/or be released from macrophages and hepatocytes. Guinea pigs inoculated with guinea pigadapted virus showed similar symptoms to those reported in humans and NHP: fever, anorexia, dehydratation, lymphopenia, fibrin deposition and a decrease in platelet count during infection [START_REF] Nakayama | Animal models for Ebola and Marburg virus infections[END_REF]. Guinea pig adapted virus resulted in 8 nucleotide changes that lead to 5 amino acids changes found in NP and L (single mutations) and VP24 (with three substitutions) (Volchkov, Chepurnov et al. 2000). Guinea pig models are useful tools for studying basic aspects of replication, pathogenesis and coagulation disorders. The guinea pig provides a more restringent evaluation of vaccine efficacy and is more predictive of success in NHP model than the mouse model. Unfortunately, significantly fewer tools are available for immunological research using this animal model.

-Syrian Golden hamster model: this model is based on infection of adult hamsters with mouse-adapted Ebola virus and shows an uniformly lethal outcome. In contrast to other rodent models, hamsters accurately reproduce the severe coagulation abnormalities associated with Ebola virus infection, including fibrin deposition, thrombocytopenia and reduction of Protein C levels.

Importantly, this model also shows cytokine dysregulation and suppression of IFN type I responses, which are important for EBOV pathogenesis [START_REF] Ebihara | A Syrian golden hamster model recapitulating ebola hemorrhagic fever[END_REF]. Differently from guinea pigs, in the hamster model prolonged thrombin time (TT) and severe lymphopenia is observed. However, as with guinea pigs, immunological tools are limited.

-Non-human primate (NHP) model: African green monkeys, cynomolgus macaques and hamadryas baboons have been employed as models for EBOV infection [START_REF] Baskerville | The pathology of experimental Ebola virus infection in monkeys[END_REF], Fisher-Hoch, Platt et al. 1985[START_REF] Jaax | Lethal experimental infection of rhesus monkeys with Ebola-Zaire (Mayinga) virus by the oral and conjunctival route of exposure[END_REF]). The NHP model has been proven to be valuable in providing new information regarding filoviral pathogenesis; the differences in the disease pathology observed in other models (Table 3) means that the NHP model remains the most useful and closest to haemorrhagic fever observed in humans despite the practical, and especially ethical, considerations that lead to the restriction of experiments (Geisbert, Young et al. 2003). NHP is the best model for vaccine and drug testing, since it shows both the characteristic clinical and pathological features seen in human EBOV infection.

It should be taken into account that genetic differences, even among the same animal species, and the origin of the species may influence disease presentation and progression. In order to fight against an illness two strategies are generally followed: (i) fight the virus by directly inhibiting its entry into cells or its replication (antiviral treatment) or (ii) fight the inappropriate immune response of the host (symptomatic treatment).

For the moment, the only treatment provided to patients during outbreaks is palliative to limit dehydration and the development of certain symptoms (nausea, fever and pain principally) and control of secondary infections.

However, several treatments have been tested in animal models (Table 4).

Some treatments also target coagulation impairment by using the anticoagulant protein derivative from nematodes (rNAP C2 ) or recombinant protein C, both having given encouraging results in infected monkeys (Geisbert, Hensley et al. 2003). Contrary to ribavirin, which has not shown to be very efficient as an EBOV infection treatment (Moss andWilson 1992, Gunther, Asper et al. 2004), the administration of oligonuclotides targeting viral mRNAs seems to be a promising technique (Warren, Warfield et al. 2010).

Therapy trials injecting EBOV immunized serums have given controversial results in hospitals and NHPs. In fact, during Ebola virus infection, infected patients develop a very weak antibody response after serum administration thus suggesting that administration of serum did not provide protection.

Concentrated immune serum produced in horses seemed very efficient in baboons and guinea pigs infected with Ebola virus. However, their effectiveness has not been proved in Rhesus monkeys or mice models (Jahrling, Geisbert et al. 1996). During the outbreak in 1995 in the Democratic Republic of the Congo blood transfusions from convalescent patients containing IgG against Ebola virus improved the survival of EBOV infected patients when compared to nontreated/transfused patients [START_REF] Mupapa | Treatment of Ebola hemorrhagic fever with blood transfusions from convalescent patients. International Scientific and Technical Committee[END_REF]). However, the real involvement of these transfusions in the illness has not been proved and is probably negligible. The use of monoclonal antibodies against EBOV-GP has been shown to be efficient in EBOV infected mice (Wilson, Hevey et al. 2000). [START_REF] Sullivan | Correlates of protective immunity for Ebola vaccines: implications for regulatory approval by the animal rule[END_REF].

Several studies done in mice and later also in NHP have shown that protection against Ebola virus infections seems to correlate with a humoral immune response [START_REF] Konduru | Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice[END_REF]). However, this is not always the case, and it happens that a high titer of antibodies does not result in a protective immune response (Warfield and Olinger 2011); inversely, sometimes protective responses do not induce neutralizing antibodies [START_REF] Sullivan | Correlates of protective immunity for Ebola vaccines: implications for regulatory approval by the animal rule[END_REF].

Finally, according to Sullivan, there is a correlation between the antibody titer (IgG) and survival [START_REF] Sullivan | Correlates of protective immunity for Ebola vaccines: implications for regulatory approval by the animal rule[END_REF], and according to Warfield, CTL responses play an essential role in controlling viral replication and clearance of the virus (Warfield and Olinger 2011). Altogether data suggests that an effective vaccine against Ebola virus infection should be able to induce a complete immune response including the humoral immune response and specific immune cellular response (CD4 + , CD8 + and CTL).

At the present time, there are no vaccines authorized against Ebola virus.

Several vaccination strategies have been generated and several candidate vaccines are still being tested on animal models (Table 5).

Various studies were first made with inactivated viral particles showing that guinea pigs could be partially protected against Ebola virus infection (Lupton, Lambert et al. 1980). It was then also shown that it was possible to obtain protective immunity during baboon infection using viral particles previously fixed with paraformaldehyde, whereas, conversely Chepurnov's team did not manage to induce an immunological response strong enough to offer protection in NHPs vaccinated with gamma-irradiation inactivated virus (Geisbert, Bausch et al. 2010).

For a long time, the idea of using attenuated virus as a candidate of vaccine remained in the state of "theory" due to the high risk that virus could mutate and become pathogenic for humans [START_REF] Vanderzanden | DNA vaccines expressing either the GP or NP genes of Ebola virus protect mice from lethal challenge[END_REF]). In the hope of amplifying humoral and cellular immune responses in animal models, encapsulated liposomes were used as a tool to release inactivated particles but the strategy failed to protect NHPs against a lethal challenge [START_REF] Geisbert | Evaluation in nonhuman primates of vaccines against Ebola virus[END_REF]. Nowadays, all these strategies have been abandoned. Virus-likeparticles expressing GP, NP and VP40 have also been tested but this approach remains difficult and expensive.

Research has focused henceforth on the use of recombinant viral vectors expressing one or several Ebola viral proteins, principally EBOV glycoprotein.

Viral vectors concern non-pathogenic or non-replicative recombinant virus.

Several viral vectors have been used in order to generate a vaccine against Ebola virus: alphavirus (Venezuelan equine encephalitis virus), vesicular stomatitis virus (Garbutt, Liebscher et al. 2004), parainfluenza human virus type 3 (Bukreyev, Rollin et al. 2007) or human adenovirus serotype 5 [START_REF] Sullivan | Accelerated vaccination for Ebola virus haemorrhagic fever in nonhuman primates[END_REF]. The attenuated recombinant VSV expressing EBOV-GP was shown to be protective against 50% of EBOV infected guinea pigs and 100% of mice 24 hours after lethal challenge and 50% of NHP treated 30 minutes after a lethal challenge [START_REF] Feldmann | Effective post-exposure treatment of Ebola infection[END_REF].

Since viral glycoprotein GP is the only viral protein exposed on the surface of

Molecular biology of Ebola virus

Morphology and genome organisation

The morphology of EBOV is unique among viruses. The virion is pleomorphic, producing 'U'-shaped, '6'-shaped, or circular forms but the predominant forms of the virion most frequently seen by electron microscope are long tubular structures about 14 000 nm long and 80 nm wide. These characteristic filamentous particles give the virus family its name [START_REF] Kiley | Filoviridae: a taxonomic home for Marburg and Ebola viruses?[END_REF].

The filovirus genome consists of a single 19 kb strand of negative-sense RNA.

Transcription begins at the 3' end and produces seven individual mRNAs: EBOV differs from MARV and from other members of the Mononegavirales in that infected cells secrete large amounts of a non-structural glycoproteins: shed GP and sGP, this last being the primary product of the GP gene, and which is expressed from non-edited mRNA species (Volchkov, Volchkova et al. 1998).

The linear arrangement of the seven viral genes and the replication strategy in infected cells resemble those of the better characterised rhabdoviruses and paramyxoviruses. Because the genome cannot be transcribed or copied by host cell enzymes, all of the components required for viral replication must be carried within the virion (Mahanty and Bray 2004).

2.2.EBOV proteins and their role during pathogenesis

2.2.1.Nucleoprotein (NP)

The first reading frame in EBOV genome will allow the transcription of an mRNA which codes for the viral nucleoprotein (NP). It is the most abundant protein of the RNP complex and it is responsible for genomic RNA and antigenome encapsidation after transcription and replication, respectively. Associated to RNA, it represents the essential link of RNP that will allow L polymerase, RNA and the other viral proteins of the nucleocapsid to interact [START_REF] Becker | Interactions of Marburg virus nucleocapsid proteins[END_REF]. Similarly to other enveloped, negative stranded RNA virus, the aminoterminal domain of NP protein, rich in hydrophobic amino acids, is responsible for the association between NP and viral genomic RNA. The carboxy terminal domain, which is less conserved, is very rich in negatively charged amino acids and its hydrophobicity indicates a possible implication in protein-protein interactions, especially during viral assembly [START_REF] Muhlberger | The nucleotide sequence of the L gene of Marburg virus, a filovirus: homologies with paramyxoviruses and rhabdoviruses[END_REF].

EBOV NP protein contains 739 amino acids with 7 phosphorylated domains rich in serine and threonine distributed in the carboxy terminal domain.

Phosphorylation of these residues seems to be essential for NP activity; in fact, in viral particles NP is only present in a phosphorylated form [START_REF] Becker | The nucleoprotein of Marburg virus is phosphorylated[END_REF]).

In the absence of other viral proteins, NP is capable of auto-assembly and of generating RNP-like helïcoidal structures. However, these structures show a larger diameter and a more irregular organisation compared to regular RNPs.

This indicates the requirement of the viral proteins in order to assemble mature RNPs [START_REF] Mavrakis | Morphology of Marburg virus NP-RNA[END_REF].

2.2.2.VP35

The VP35 gene position in EBOV genome corresponds to that of phosphoprotein P in Paramyxo-and Rhabdoviruses. Even if no significant sequence homology with P protein in other Mononegavirales has been identified, a functional analogy has been observed [START_REF] Muhlberger | Three of the four nucleocapsid proteins of Marburg virus, NP, VP35, and L, are sufficient to mediate replication and transcription of Marburg virusspecific monocistronic minigenomes[END_REF].

VP35 is known to bind NP protein and to condense RNPs, obtaining a regular helicoidal structure during mature RNP assembly [START_REF] Becker | Interactions of Marburg virus nucleocapsid proteins[END_REF].

VP35 also interacts with viral polymerase L; in fact, VP35 is the cofactor that allows, in association with NP, recruitment of the polymerase to viral genomic RNA and antigenomes in order to carry out transcription and replication [START_REF] Muhlberger | Three of the four nucleocapsid proteins of Marburg virus, NP, VP35, and L, are sufficient to mediate replication and transcription of Marburg virusspecific monocistronic minigenomes[END_REF]).

VP35 has also been shown to be an antagonist of the type I interferon response. It is capable of inhibiting IFN α/β in infected cells by blocking phosphorylation of IRF-3 regulation factor [START_REF] Basler | The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3[END_REF]) and protein kinase PKR [START_REF] Feng | The VP35 protein of Ebola virus inhibits the antiviral effect mediated by double-stranded RNAdependent protein kinase PKR[END_REF]).

2.2.3.VP40

EBOV VP40 has 326 amino acids and is the protein responsible for assembly and virus budding. The matrix protein VP40 is the most abundant protein in virions; it covers the inner part of the viral membrane and it allows the cohesion between RNP and the viral envelope. Like VP35, this protein does not share homology when compared to other matrix proteins in Mononegavirales, however, a functional analogy has been identified (Timmins, Ruigrok et al.

2004).

Ebola virus VP40 is sufficient for virus assembly and budding from the plasma membrane (Timmins, Scianimanico et al. 2001). However, the addition of GP protein increases VP40-induced viral production (Licata, Johnson et al. 2004).

Interestingly, EBOV VP40 contains two overlapping late domains that interact with Tsg101, a component of the export complex in endosomes, which plays a role during the late state of viral budding [START_REF] Hoenen | Ebola virus: unravelling pathogenesis to combat a deadly disease[END_REF]). The efficiency of viral production is considerably reduced when these two domains are altered (Harty, Brown et al. 2000). However, the interaction between late domains and endosomal cellular proteins is not essential for EBOV replication, it only implies a reduction in viral titer [START_REF] Neumann | Ebola virus VP40 late domains are not essential for viral replication in cell culture[END_REF]).

2.2.4.GP

In EBOV, four variants of the envelope glycoprotein are synthesized as a result of transcriptional stuttering or posttranslational processing (Figure 6A): the surface glycoprotein GP and three soluble glycoproteins (sGP, shed GP and ssGP). In fact, Ebola virus GP gene contains two overlapping reading frames.

The majority of mRNAs are transcribed from the first reading frame leading to the synthesis of soluble glycoprotein sGP (or secreted GP). The expression of Ebola virus GP requires an editing phenomenon during transcription. About 25% of transcripts from GP gene will result from the addition of an extra Adenosin (A) in the editing site, already containing 7A; this will lead to the synthesis of mRNA transcribed from the totality of the two reading frames and leads to the production of surface GP, which has 767 amino acids (Figure 6A). The disulfide bond between GP1 and GP2 is indicated, as well as the locations of N-linked glycans (marked with "Y"s) in the GP1 and GP2 domains, and the known protease cleavage sites are indicated. SP, signal peptide; RBD, receptor-binding domain; MLD, mucin-like domain; IFL, internal fusion loop;

HR1 and HR2, heptad repeats 1 and 2; TM, transmembrane domain [START_REF] Lennemann | Comprehensive functional analysis of N-linked glycans on Ebola virus GP1[END_REF].

The N-terminal region of GP1 (residues 57 to 149) has been defined as a receptor-binding domain (RBD). Although Ebola viruses are antigenically distinct, the RBD is highly conserved, with an average of 87% amino acid identity. This conservation suggests an absence of selective pressures driving diversification of this region of the protein. In contrast, the glycan cap and MLD have extensive sequence diversity between different Ebola virus species. GP2 contains the hydrophobic fusion peptide (residues 524-539) and heptad repeats that mediate membrane fusion (Dube, Brecher et al. 2009, Lee and[START_REF] Lee | Ebolavirus glycoprotein structure and mechanism of entry[END_REF]. GP2 also contains a sequence of 26 hydrophobic amino acids that shares 40% similarity with the immunosupressive domains of some retrovirus;

however, the role of this domain in mature GP during infection is still not known.

In the carboxy-terminal extremity, residues from 651-672 have been characterised as a protein anchor to the plasma membrane on the surface of cells. Finally, a short cytoplasmic tail containing 4 amino acids has also been identified (Volchkov, Volchkova et al. 1998) (Figure 6A).

Surface GP is heavily N-and O-glycosylated. Depending on the EBOV species, envelope GP contains 11-18 N-linked glycan sites. Both the glycan cap and the MLD are highly glycosylated, resulting in the majority of the protein being masked by glycans. The glycan cap contains only N-linked glycan sites and, even if the glycan cap is generally poorly conserved, these sites are well conserved among Ebola viruses, suggesting functional significance. In contrast, the 150-residue unstructured MLD is highly variable between the different filoviruses and include both N-and O-linked glycans [START_REF] Jeffers | Covalent modifications of the ebola virus glycoprotein[END_REF]) (Figure 6B). The N-linked glycans are a heterogeneous mixture of highmannose, hybrid, and complex oligosaccharides, while the O-linked glycans are primarily smaller trisaccharide structures that contain varying amounts of sialic acids [START_REF] Ritchie | Identification of N-glycans from Ebola virus glycoproteins by matrixassisted laser desorption/ionisation time-of-flight and negative ion electrospray tandem mass spectrometry[END_REF].

High glycosylation of Ebola virus surface GP masks essential epitopes on EBOV GP (Figure 6B) and creates an unfavorable environment for the interaction of otherwise neutralizing antibodies. No antibodies have been identified that target the receptor-binding site, however a number of neutralizing antibodies have been generated against the more variable mucin-like domain (Wilson, Hevey et al. 2000). The mucin-like domain is not required for pseudotyped EBOV entry in Vero E6 cells [START_REF] Kaletsky | Proteolysis of the Ebola virus glycoproteins enhances virus binding and infectivity[END_REF], Hood, Abraham et al. 2010). However, the mucin-like domain could play a more important role in filovirus entry into natural target cells such as endothelial cells (ECs), hepatocytes, and antigen-presenting cells. In mice, removal of the the mucin-like domain of EBOV GP leads to the production of cross-species antibodies directed at the conserved glycoprotein core structure (Hood, Abraham et al. 2010). A small non-glycosylated area near the base of the EBOV GP core is available for antibody recognition (Figure 6B). This region is vey conserved in both Zaire and Sudan EBOV species, and the neutralizing antibodies KZ52 and 16F6-1 bind to this hotspot [START_REF] Dias | A shared structural solution for neutralizing ebolaviruses[END_REF].

Several studies have shown that EBOV glycoprotein GP1,2 is responsible of the cytopathc effects observed in EBOV infected cells. In fact, expression of GP1,2 causes cell rounding in different cell lines, including human embryonic kidney cells (293T), monkey kidney cells (VeroE6), macrophages and endothelial cells (Chan, Ma et al. 2000). EBOV mucin-like domain of surface GP also has the unique ability to provide a "glycan umbrella" that shields surface epitopes and inhibits surface protein recognition by masking the function of host cellular proteins that are important in response to viral pathogens [START_REF] Reynard | Ebolavirus glycoprotein GP masks both its own epitopes and the presence of cellular surface proteins[END_REF]. Indeed, transient expression of EBOV GP results in low detectable levels of various cell surface proteins such as major histocompatibility complex (MHC) class I proteins and β-integrins. Initially, it was thought that EBOV GP downregulated expression or degraded these proteins from the cell surface [START_REF] Simmons | Ebola virus glycoproteins induce global surface protein downmodulation and loss of cell adherence[END_REF]. However, MHC class I and β-integrins are not removed from the cell surface but masked by a "glycan-umbrella" [START_REF] Reynard | Ebolavirus glycoprotein GP masks both its own epitopes and the presence of cellular surface proteins[END_REF], Francica, Varela-Rohena et al. 2010) (Figure 6B). This represents a novel mechanism of disrupting immune function that does not involve downregulation or degradation of surface proteins.

Ebola virus GP protein is responsible for viral tropism. The use of retroviral vectors has shown that GP1 subunit is responsible for viral attachment to the surface of target cells (Takada, Robison et al. 1997). After binding, internalisation and trafficking of EBOV particles into late endosomes, surface GP in the virion is cleaved in two steps, first by cathepsin L (into 20kDa species)

and further by cathepsin B (into 19 kDa species) at the β13-β14 loop of GP1 [START_REF] Schornberg | Role of endosomal cathepsins in entry mediated by the Ebola virus glycoprotein[END_REF][START_REF] Dube | The primed ebolavirus glycoprotein (19-kilodalton GP1,2): sequence and residues critical for host cell binding[END_REF], Hood, Abraham et al. 2010). This cleavage implies the removal of approximately 60% of GP1 amino acids including the mucin-like domain, glycan cap and a part of RBD [START_REF] Manicassamy | Comprehensive analysis of ebola virus GP1 in viral entry[END_REF], Mpanju, Towner et al. 2006). Low pH is required to facilitate conformational changes in GP and allow fusion to endosome membrane [START_REF] Gregory | Structure and function of the complete internal fusion loop from Ebolavirus glycoprotein 2[END_REF] Because viral RNA-dependent RNA polymerases typically have a high error rate, such lack of variation appears unusual (Mahanty and Bray 2004).

2.2.5.Ebola virus soluble glycoproteins

Many enveloped viruses, including EBOV, Lassa, respiratory syncytial and herpes simplex produce free glycoproteins that act as either ''antibody sinks'' or decoys of host immunity [START_REF] Bukreyev | The secreted form of respiratory syncytial virus G glycoprotein helps the virus evade antibody-mediated restriction of replication by acting as an antigen decoy and through effects on Fc receptor-bearing leukocytes[END_REF], Branco, Grove et al. 2010[START_REF] Cook | The secret life of viral entry glycoproteins: moonlighting in immune evasion[END_REF]. The shedding or secretion of soluble viral glycoproteins exemplifies a viral strategy of humoral misdirection.

2.2.5.1.Secreted GP

EBOV-infected cells secrete two glycoproteins (secreted GP and shed GP) into an infected person's sera in very high amounts (Sanchez, Yang et al. 1998, Dolnik, Volchkova et al. 2004). 70% of GP gene transcripts code for secreted GP (sGP), which is a dimeric 110kDa protein that has 364 amino acids (Figure 6A). Whereas the first 295 amino acids of sGP are identical to the GP1 subunit of surface GP, due to transcriptional stuttering the 69 amino acids forming the carboxy-terminal region are unique and form different disulfide linkages leading to a homodimeric rather than a trimeric assembly. As a result, sGP lacks regions found in GP that have been shown to be important in the neutralization of the virus [START_REF] Dias | A shared structural solution for neutralizing ebolaviruses[END_REF]. sGP protein is synthesized as a precursor: pre-sGP, which is the post-traductionally modified, similarly to surface GP, leading to mature sGP protein; its post-traductional modifications include: cleavage of signal peptide, N-glycosylation and oligomerization in the endoplasmic reticulum and then, glycosylation and post-traductional cleavage in the Golgi (Volchkova, Feldmann et al. 1998). Mature sGP and pre-sGP precursor are anti-parallel-orientated or parallel homodimers linked by two disulfide bridges between cysteines 53 and 306 (Volchkova, Feldmann et al. 1998, Falzarano, Krokhin et al. 2006). The cleavage of sGP by furin leads to the release of a small highly glycosylated peptide of about 10 to 14kDa: Δ-peptide (Volchkova, Klenk et al. 1999). It has been shown that Δ-peptide could have a role in viral replication by its ability to inhibit entry of retroviruses pseudotyped with Marburg virus GP as well as Marburg virus and Ebola virus infection in a dose-dependent manner [START_REF] Radoshitzky | Ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry[END_REF]). Several roles have been shown for sGP: first, sGP synthesis allows a reduction in surface GP production thus limiting its cytotoxicity for cells; this would allow therefore better replication and propagation of Ebola virus [START_REF] Volchkov | Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity[END_REF]. In addition, it has been shown that sGP has a protective role on vascular endothelium integrity; in fact, sGP would inhibit the effects of TNF-α on endothelial barrier permeability thus being a regulation factor of the inflammatory response in endothelial cells [START_REF] Wahl-Jensen | Effects of Ebola virus glycoproteins on endothelial cell activation and barrier function[END_REF]. Moreover, secretion of sGP protein by infected cells suggests that sGP could be a molecular lure that would allow the virus to escape from the immunosurveillance system (Sanchez, Ksiazek et al. 1999). Indeed, it has been demonstrated that sGP subverts immune responses by acting as a decoy antibody; in fact, sGP induces a host antibody response that focuses on epitopes it shares with GP1,2 thereby allowing it to bind and compete for anti-GP1,2 antibodies. Distinct from previously proposed "decoy" mechanisms in which secreted glycoprotein simply passively absorbs anti-glycoprotein antibodies, is has been demonstrated that sGP can also subvert an existing anti-GP1,2 immune response that was only weakly cross-reactive with sGP (Mohan, Li et al. 2012).

2.2.5.2.Shed GP

Cleavage at the membrane proximal external region by the tumor necrosis factor-a converting enzyme (TACE) releases the trimeric glycoprotein, termed shed GP (Figure 6A). In general, the process of shedding affects different cellular proteins both structurally and functionally; this is the case for a range of secreted cellular proteins including receptors, precursors of cytokines, growth factors, cell adhesion molecules, etc. [START_REF] Edwards | The ADAM metalloproteinases[END_REF].

Cleavage usually occurs close to the extracellular face of the membrane, providing a mechanism for down-regulating the protein at the cell surface or releasing physiologically active proteins (Hooper, Karran et al. 1997). It should be mentioned that a classical consensus cleavage motif for the metalloprotease TACE does not exist [START_REF] Ehlers | Proteolytic release of membrane-bound angiotensin-converting enzyme: role of the juxtamembrane stalk sequence[END_REF]. However, there are certain preferences for amino acids at positions P1', P1, P2, P3 and P5 within the sequences surrounding cleavage sites. Indeed TACE was shown to have strong preference for hydrophobic residues at P1' position, for alanine in P1 and P2

positions and for proline in P3 or P5 positions (Figure 8) [START_REF] Caescu | Active-site determinants of substrate recognition by the metalloproteinases TACE and ADAM10[END_REF]. Therefore three parameters seem to be important for TACE cleavage efficiency: the amino acid sequence, the amino acid conformation and the distance between the cleavage site and the transmembrane anchor. indicate no selectivity, while residues having a value greater than 1 (red) are positively selected by the protease. Adapted from [START_REF] Caescu | Active-site determinants of substrate recognition by the metalloproteinases TACE and ADAM10[END_REF] Overall, shed GP's structure is practically identical to that of surface GP. It has also been demonstrated that shed GP and surface GP have the same glycosylation pattern by treatment with Endo H or PNGase F. Furthermore, it has been showed that shed GP is present in significant amounts in the blood of virus-infected animals and that, in a guinea pig model, it efficiently blocks the activity of KZ52, an Ebola virus-neutralizing antibody. It has also been hypothesized that the release of surface GP in a soluble form could be another mechanism, besides sGP production, to reduce cellular cytotoxicity (Dolnik, Volchkova et al. 2004).

Besides its neutralising antibody function, no other role has yet been found for shed GP in other studies (Wahl-Jensen, Kurz et al. 2005). However, due to its similar structure to surface GP and the high quantity in which it is released in infected animals and humans, it is be possible that other roles for this protein have still not been identified.

2.2.5.3.Small secreted GP

In 5% of transcripts, the insertion of two adenosines produces another editing product of the GP gene of Ebola virus: small secreted glycoprotein (ssGP) 6A), which shares 295 amino-terminal amino acid residues with sGP. It is secreted from cells in a monomeric form due to the lack of the carboxylterminal part (present in sGP), including a cysteine at position 306. The role of this 298-residue secreted ssGP protein is still unknown (Volchkova, Feldmann et al. 1998).

(Figure

2.2.6.VP30

Contrary to other Mononegavirales, EBOV RNP contains a forth protein: VP30.

It has 288 amino acids and its amino-terminal region contains six serines and one threonine suitable to be phosphorylated by cellular kinases and phosphatases (Modrof, Muhlberger et al. 2002).

The use of a minigenome system has shown that VP30 is an essential transcription factor. In fact, non-phosphorylated VP30 has a role as a transcription factor of Ebola virus but it cannot bind NP protein; when it is completely phosphorylated, only its association function to the nucleoprotein is kept. In contrast, when one or two serine residues are phosphorylated, VP30 maintains both activities: as an assembly and as a transcription factor.

Particularly, phosphorylation of serine residues at position 40 and 42 is essential for VP30 binding to NP protein, whereas a Zinc finger structure in the amino-terminal region is required for its transcriptional function (Modrof, Muhlberger et al. 2002).

2.2.7.VP24

The sixth gene of Filovirus genome codes for VP24 protein, which has 253 amino acids and shares 35% of homology with Marburg VP24 (Bukreyev, Belanov et al. 1995). Its high content of hydrophobic amino acids and the detection of its incorporation into virions has led to its definition as a secondary matrix protein [START_REF] Elliott | Descriptive analysis of Ebola virus proteins[END_REF]. EBOV VP24 has been shown to have biochemical and functional properties as a matrix protein, thus suggesting a role in assembly and budding of virus. However, the absence of budding in viral pseudo particles in the presence of VP24 alone and its absence in EBOV VLPs represent two arguments against its possible role as a strict matrix protein.

It has also been shown that VP24 is an interferon antagonist. It is capable of blocking STAT-1 translocation by binding to karyopherin α1 (an importin), a mechanism that has as a consequence the blocking of an antiviral response (Reid, Leung et al. 2006), as will be explained below in the section detailing

Ebolavirus pathogenesis. Recently it has also been shown that it can directly bind STAT-1 (Zhang, Bornholdt et al. 2012).

Interestingly, Ebola virus infection of guinea pigs with wild-type EBOV virus results in an asymptomatic illness. However, sequential passaging of wild-type virus in guinea pigs can lead to the generation of highly pathogenic variants of the virus. These viruses present mutations in their genome; for instance, variant 8mc has, after 8 passages in guinea pigs, 5 mutations: 1 in L polymerase, one in NP and 3 in VP24 (Volchkov, Chepurnov et al. 2000). By generating several recombinant virus containing different combinations of these mutations, it has been demonstrated that mutations in VP24 were sufficient to acquire virulence (Mateo, Carbonnelle et al. 2011).

L polymerase

EBOV L protein is the largest (2212 amino acids, 260 KDa) but the least abundant protein in virions and infected cells. It contains three conserved domains: A (putative binding domain), B (RNA recognition domain and formation of phosphodiester bridges) and C (ATP and tri-phosphate ribonucleotide binding domain) [START_REF] Muhlberger | The nucleotide sequence of the L gene of Marburg virus, a filovirus: homologies with paramyxoviruses and rhabdoviruses[END_REF], Volchkov, Volchkova et al. 1999). L protein is the RNA-polymerase RNA dependent which contains all of the enzymatic activities necessary to synthesize viral RNA and antigenomes during transcription and replication, respectively; however, it is not functional without the other nucleocapsid proteins.

2.3.Ebola virus replication cycle

EBOV replication cycle is similar to the other Mononegavirales. Host cell entry is the first essential step in EBOV infection. EBOV has a very broad cell tropism including primary targets such as monocytes, macrophages and dendritic cells and secondary targets including hepatocytes, adrenal cortical cells, fibroblasts and endothelial cells. EBOV binds to multiple attachment factors, notable among which are:

-C-type lectins. The highly glycosylated GP of EBOV contains a set of N-and O-linked glycans that, depending on their structure, can be recognised in a calcium-dependent manner by different C-type lectins including: DC-SIGN and macrophage galactose lectin, MGL, on macrophages and immature DCs, and L-SIGN and LSECtin on endothelial cells in liver and lymph nodes [START_REF] Alvarez | C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans[END_REF][START_REF] Simmons | DC-SIGN and DC-SIGNR bind ebola glycoproteins and enhance infection of macrophages and endothelial cells[END_REF], Takada, Fujioka et al. 2004[START_REF] Gramberg | LSECtin interacts with filovirus glycoproteins and the spike protein of SARS coronavirus[END_REF]. It has also been shown that MBL (Mannose binding lectin) enhances EBOV infection at low complement levels [START_REF] Brudner | Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane C-type lectin receptors[END_REF]. However other studies have shown that MBL blocks EBOV infection (Ji, Olinger et al. 2005, Michelow, Dong et al. 2010[START_REF] Michelow | High-dose mannosebinding lectin therapy for Ebola virus infection[END_REF]. Interestingly, very recently it has been shown that removal of N-glycans in GP1 subunit does not affect Ebola virus pseudovirion (VSV-GP) entry into murin peritoneal macrophages [START_REF] Lennemann | Comprehensive functional analysis of N-linked glycans on Ebola virus GP1[END_REF]).

-Niemann-Pick C1 (NPC1) is an endosomal and lysosomal membrane protein which is known to be a cholesterol transporter. Mutations in NPCI gene result in cholesterol accumulation that leads to altered protein and lipid trafficking [START_REF] Carstea | Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis[END_REF]. Cathepsins B and L process EBOV GP thus exposing its RBR domain which leads to binding to NPC1 [START_REF] Carette | Ebola virus entry requires the cholesterol transporter Niemann-Pick C1[END_REF][START_REF] Cote | Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection[END_REF][START_REF] Shoemaker | Multiple cationic amphiphiles induce a Niemann-Pick C phenotype and inhibit Ebola virus entry and infection[END_REF]. It is considered as the intracellular receptor for EBOV since it is the only molecule that has been shown to be essential for EBOV entry after uptake into the cell and proteolytic processing of EBOV-GP.

-TAM family includes tyrosine kinase receptors Tyro3, Axl and Mer (TAM), which play important roles in different cellular process such as cell proliferation, cell adhesion and cytokine release (Linger, Keating et al. 2008).

Axl, which is highly expressed on macrophages, has been shown to be an entry factor during EBOV infection [START_REF] Shimojima | Tyro3 familymediated cell entry of Ebola and Marburg viruses[END_REF][START_REF] Kondratowicz | T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus[END_REF]. Even if all studies failed to demonstrate an interaction between EBOV-GP and Axl, it was clearly shown that Axl augmented EBOV pseudovirion internalisation suggesting that endosomal uptake of filoviruses is facilitated by Axl.

-β1 integrins are expressed in a wide range of cells and are responsible for very diverse functions (cell-cell adhesion, migration, differentiation, proliferation...). They are implicated in EBOV-GP pseudotyped VSV entry (Takada, Watanabe et al. 2000[START_REF] Schornberg | Alpha5beta1-integrin controls ebolavirus entry by regulating endosomal cathepsins[END_REF]). However, direct binding of EBOV has still not been demonstrated.

-T-cell immunoglobulin mucin domain 1 (TIM-1) is expressed on the surface of activated T-cells, epithelial cells, renal tissue and some cell lines such as Huh7 (liver cell line) and Vero cells (monkey kidney cell line) (Hofmann-Winkler, Kaup et al. 2012). TIM-1 has been shown to promote EBOV infection of certain cells lines and soluble TIM-1 inhibits virus entry into permissive cells [START_REF] Kondratowicz | T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus[END_REF]). However, monocytes, macrophages and DCs do not express TIM-1 meaning that their cognate receptor has yet to be identified.

-Toll-Like receptor 4 (TLR4) is principally expressed on polymorphonuclear cells, monocytes, macrophages and DCs (Muzio, Bosisio et al. 2000). It is responsible of binding of LPS through LBP (lipopolysaccharidebinding protein) that will transfer LPS to CD14, which, in turn, will transfer LPS to TLR4-MD2 heterodimers (Triantafilou and Triantafilou 2002). TLR4 has been shown to bind GP-EBOV VLPs [START_REF] Okumura | Interaction between Ebola virus glycoprotein and host toll-like receptor 4 leads to induction of proinflammatory cytokines and SOCS1[END_REF]. Moreover, EBOV-VLPs stimulate the expression of proinflammatory cytokines but also type I interferons (IFNs) in murine DCs (Ayithan, Bradfute et al. 2014).

The filovirus replication cycle is depicted in Figure 9. EBOV binds to the previously described attachment factors on the cell surface (1) and enters target cells either by clathrin/caveolae endocytosis [START_REF] Bhattacharyya | Ebola virus uses clathrin-mediated endocytosis as an entry pathway[END_REF][START_REF] Aleksandrowicz | Ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis[END_REF] or by macropinocytosis [START_REF] Nanbo | Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner[END_REF], Saeed, Kolokoltsov et al. 2010), depending on the type of target cell involved and the viral system used to study viral entry. Virions are then trafficked through early and late endosomes where, under acid pH, cathepsin L and B will process EBOV-GP thus exposing RBR domain by proteolysis in acidified endocytic vesicles leading to EBOV-GP binding to NPC1 receptor and subsequent release of genetic material into the cytoplasm (2). There, the nucleocapsid serves as a template for both transcription (3) and replication (4).

During transcription, the seven viral genes are transcribed from 3'-5' by the viral polymerase L into 7 monocystronic mRNAs, all capped and polyadenylated, which will be translated into viral proteins by cellular ribosomes. Transcription occurs in a sequential gradient from 3'-5', inducing a strong expression of NP protein and weaker for L polymerase.

During replication, RNA antigenomes (positive-sense mRNA) are generated and encapsidated. These antigenomes are used for the synthesis of negative mRNA for the new viral progeny, which will accumulate in inclusion bodies. In inclusion bodies, viral proteins and nucleoproteins get assembled to form nucleocapsids which will be transported to the plasma membrane (5), where nucleocapsids will be wrapped with the cellular membrane. Virions will then bud mostly in lipid rafts (microdomains in the plasma membrane containing sphingolipids and cholesterol) (6).

. where budding takes place (6). Soluble glycoproteins sGP and shed GP are also released from infected cells (7).

Ebola virus pathogenesis

Ebola virus pathogenesis is different depending on the viral species. Besides that, they have in common that the high lethality of Ebola virus is due to a combination of the deleterious effects of high viral titers and direct viral damage and a nonspecific and abnormally sustained innate immune response.

3.1.Host immune response to Ebola virus infection

3.1.1.Innate immune response

Innate immunity is the first line of defense against infectious agents and pathogens. It begins very early during infection and is functional for several days. The innate immune response acts in two different ways:

1.-The specific recognition of the "non-self": it involves pattern recognition receptors (PRR), which will recognise "non-self" patterns on the surface of pathogens (Pathogen associated molecular patherns: PAMPs). Recognition of the "non-self" also induces the intervention pf pro-inflammatory cytokines and chimiokines and starts the antiviral cell function by inducing the recruitment of innate immune cells and apoptosis of the infected cell.

2.-Induction and organisation of the antigen-specific immune response involving T cells and antibodies (Ab).

During Ebola infection, both processes are affected.

3.1.1.1.Ebola virus increases the cytokine response

Ebola virus has a broad cell tropism, infecting a wide range of cell types. In-situ hybridisation and electron microscope analyses of tissues from patients with fatal disease or from experimentally infected non-human primates show that monocytes, macrophages, dendritic cells (DCs), endothelial cells, fibroblasts, hepatocytes, adrenal cortical cells, and several types of epithelial cells all lend support to replication of Ebola virus (Zaki, Shieh et al. 1999).

Studies in non-human primates experimentally infected with Zaire Ebola virus suggest that monocytes, macrophages, and dendritic cells are the early and preferred replication sites for this virus (Geisbert, Hensley et al. 2003). These antigen presenting cells (APCs) seem to have a role in the dissemination of the virus by supporting viral replication but also by spreading the virus from the initial infection site to regional lymph nodes, probably through the lymphatic system, and to the liver and spleen through the blood. Monocytes, macrophages, and dendritic cells infected with Ebola virus migrate out of the spleen and lymph nodes to other tissues including the liver and kidneys, thus amplifiying and disseminating the infection (figure 10). Monocytes can present antigens and also, especially under inflammatory conditions, go to tissues to differentiate into macrophages or dendritic cells [START_REF] Geissmann | Development of monocytes, macrophages, and dendritic cells[END_REF] Normally, the release of proinflammatory cytokines recruits other monocytes, macrophages or dendritic cells, all of which are sensitive to Ebola virus infection; however, during Ebola virus infection the inflammatory response will be uncontrolled and will lead to what is called a "cytokine storm". Overall, this 'cytokine storm', particularly prominent in the terminal stages of the disease in humans and non-human primates, has dramatic consequences. Virus-induced expression of these mediators results in an immunological imbalance that partly contributes to the progression of disease. In addition, some of these mediators may induce the expression of adhesion molecules on the surface of endothelial cells, allowing neutrophils and monocytes to invade sites of infection; this results in the release of further inflammatory cytokines, such as TNF-α, chemokines and other species such as nitric oxide that will in turn mobilise immature circulating neutrophils and will also facilitate the exit of inflammatory molecules and proteins from the blood that will contribute to vasodilatation and increased vascular permeability (causing tissue damage) [START_REF] Bray | Ebola virus: the role of macrophages and dendritic cells in the pathogenesis of Ebola hemorrhagic fever[END_REF]. In addition, infected monocytes and macrophages express cell surface tissue factor (TF), which may be involved in the development of coagulopathies and in the induction of disseminated intravascular coagulation (DIC).

Proinflammatory responses recorded in fatal cases of Ebola haemorrhagic fever are dysregulated, whereas early and well regulated inflammatory responses have been associated with recovery (Baize, Leroy et al. 2002).

Like monocytes and macrophages, immature dendritic cells are targets of Ebola virus. Dendritic cells are among the most effective antigen-presenting cells of the immune system. They express a large number of MHCII and are considered relatively less phagocytic than macrophages but they are highly stimulatory for T cells by secreting important interleukins and cytokines that provide a critical link between innate and adaptive immune responses to many pathogens [START_REF] Martinez | The role of antigenpresenting cells in filoviral hemorrhagic fever: gaps in current knowledge[END_REF]). DCs infected with Ebola virus are severely compromised in these critical functions. Human myeloid DCs infected with live virus fail to secrete the normal profile of proinflammatory cytokines and costimulatory molecules [START_REF] Mahanty | Cutting edge: impairment of dendritic cells and adaptive immunity by Ebola and Lassa viruses[END_REF][START_REF] Bosio | Ebola and Marburg virus-like particles activate human myeloid dendritic cells[END_REF]). These cells do not become mature or activated and are unable to upregulate major histocompatibility complex (MHC) molecules and thus to stimulate T cells. By contrast, treatment with noninfectious Ebola virus-like particles (VLPs) activates DCs and stimulates a robust inflammatory response [START_REF] Bosio | Ebola and Marburg virus-like particles activate human myeloid dendritic cells[END_REF]). 11). Receptor binding activates the Jak-STAT signaling cascade by recruiting the Janus kinases (JAK1 and TyK2). JAKs phosphorylate STAT1 and STAT2, which then dimerize and interact with IFN regulatory factor 9 (IRF9), forming a complex named ISGF3.

ISGF3 binds to IFN-stimulated response elements (ISRE) in the promoters of IFN-stimulated genes (ISG) to regulate their expression [START_REF] Jewell | Lambda interferon is the predominant interferon induced by influenza A virus infection in vivo[END_REF]. Interferon production is blocked in macrophages, peripheral blood mononuclear cells and DCs by Ebola virus infection in vitro and in vivo (Bosio, Aman et al. 2003[START_REF] Mahanty | Protection from lethal infection is determined by innate immune responses in a mouse model of Ebola virus infection[END_REF]. In addition, the expression of interferonstimulated genes important in the type-I interferon response is decreased in Ebola virus-infected cells [START_REF] Kash | Global suppression of the host antiviral response by Ebola-and Marburgviruses: increased antagonism of the type I interferon response is associated with enhanced virulence[END_REF]. Like some other viruses, Ebola encodes specific viral proteins that antagonize the interferon response. Two virally encoded proteins, VP24 and VP35, have been shown to interfere with the induction of the interferon response [START_REF] Basler | The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3[END_REF], Reid, Leung et al. 2006). VP35 blocks the IFN independent pathway and VP24 blocks the IFN dependent pathway (Figure 13). Ebola VP35 blocks the activity of the interferon regulatory factor 3 (IRF-3), thus decreasing interferon responses [START_REF] Basler | The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3[END_REF]. More recently, VP35 was shown to counteract the activity of the double-stranded RNA-dependent protein kinase (PKR) [START_REF] Feng | The VP35 protein of Ebola virus inhibits the antiviral effect mediated by double-stranded RNAdependent protein kinase PKR[END_REF]. Translocation of phosphorylated STAT1 to the nucleus is necessary for transcriptional activation of multiple genes induced by type I interferon (IFN-I). This translocation is triggered by its interaction with karyopherin α1 (KPNα1) (Najjar and Fagard 2010). EBOV VP24 protein interacts with KPNα1 thereby preventing translocation of STAT-I [START_REF] Reid | Ebola virus VP24 proteins inhibit the interaction of NPI-1 subfamily karyopherin alpha proteins with activated STAT1[END_REF], and also interacts with heterogeneous nuclear ribonuclear protein complex C1/C2 (hnRNP C1/C2) partially altering its nuclear transport [START_REF] Shabman | The Ebola virus VP24 protein prevents hnRNP C1/C2 binding to karyopherin alpha1 and partially alters its nuclear import[END_REF]. VP24 also blocks the phosphorylation of p38 In combination, these studies suggest that virus-induced inhibition of the interferon pathway not only decreases interferon-stimulated gene transcription to prevent an antiviral response state, but also contributes to lower numbers of mature and activated myeloid DCs, which in turn hinders the activation of the adaptive immune response.

(A) VP35 inhibition of IFN independent pathway (B) VP24 inhibition of IFN dependent pathway

3.1.2.Adaptative immune response

DCs play an important role in activating cellular adaptative immune responses.

DCs present pathogen-derived antigens to T H lymphocytes through major histocompatibility complex II (MHCII), which are present on the surface of APCs, and this will stimulate lymphocytes and polarize the immune response towards a cellular TH 1 response (T lymphocytes CD4 + and T lymphocytes CD8 + ) or towards a humoral TH 2 response (B lymphocytes, antibodies).

3.2.2.1.Cellular immune response

Patients who succumb to Ebola virus infection show little evidence of an activated adaptive immune response. Adaptive immunity is severely compromised not only because of a lack of functional DCs and the decrease of APCs due to infection, but also because lymphocytes undergo massive apoptosis in infected humans and nonhuman primates (phenomena known as lymphopenia) [START_REF] Baize | Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients[END_REF], Gupta, Spiropoulou et al. 2007).

Indeed, although lymphocytes are not targets of the virus, substantial numbers -except for B lymphocytes -undergo apoptosis during the illness [START_REF] Geisbert | Apoptosis induced in vitro and in vivo during infection by Ebola and Marburg viruses[END_REF]; as a result, CD4 + and CD8 + T lymphocytes and NK cells [START_REF] Reed | Depletion of peripheral blood T lymphocytes and NK cells during the course of ebola hemorrhagic Fever in cynomolgus macaques[END_REF]) are considerably reduced in fatal human and nonhuman primate infections before death [START_REF] Baize | Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients[END_REF][START_REF] Reed | Depletion of peripheral blood T lymphocytes and NK cells during the course of ebola hemorrhagic Fever in cynomolgus macaques[END_REF]). Lymphocyte apoptosis is also a common manifestation of other viral hemorrhagic fevers and is frequently observed during septic shock [START_REF] Geisbert | Exotic emerging viral diseases: progress and challenges[END_REF].

Studies with lymphocytes in vitro indicate that several molecules involved in triggering apoptosis are present on these cell populations, including TNF related apoptosis-inducing ligand (TRAIL) and FasL (Gupta, Spiropoulou et al. 2007).

However, the mechanisms responsible for this apoptosis are still under investigation. It may be that inflammatory mediators and other factors, such as the pro-apoptotic soluble factor nitric oxide (NO) secreted by infected macrophages, are capable of inducing the observed lymphocyte apoptosis.

Alternatively, impaired DC function and an overall immunosuppressive state may contribute to the phenomenon. Also, the fact that a part of monocytes, macrophages and DCs die during infection, could reduce antigen presentation to lymphocytes. Yet another possibility is that cell death is actively triggered by direct interactions between lymphocytes and Ebola virus or soluble Ebola virus proteins. The recognition of an immunosuppressive motif in the carboxylterminal region of the virus glycoprotein lends support to the notion that virus particles or proteins might partly contribute to the dysfunction or the loss of lymphocytes, or both [START_REF] Zampieri | Immunopathology of highly virulent pathogens: insights from Ebola virus[END_REF].

The importance of early responses involving cells of the innate immune system and/or a rapid adaptive antibody response is highlighted by a study showing protection of nonhuman primates with post-exposure vaccine administration [START_REF] Feldmann | Effective post-exposure treatment of Ebola infection[END_REF]). However, massive apoptosis of lymphocytes does not seem to be a major factor of Ebola virus pathogenesis in mice; in fact, transgenic mice highly expressing the anti-apoptotic factor bcl-2 showed that, inhibition of lymphocyte apoptosis did not protect animals against a lethal challenge with mouse adapted Ebola virus (Bradfute, Swanson et al. 2010).

This would suggest that lymphocyte apoptosis would be a secondary effect due to cytokine overexpression rather than a direct consequence of infection. In contradiction to this, it has previously been shown that individuals having survived Sudan Ebola virus infection had an increased CD8 + cellular profile when compared to patients that had not survived [START_REF] Baize | Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients[END_REF]).

However, another study showed that it was possible to protect naive mice against mouse adapted Ebola virus infection by transferring purified CD8 + lymphocytes (Bradfute, Warfield et al. 2008).

3.2.2.2.Humoral immune response

During fatal Ebola virus infection, antibody production is normally weak or even absent, suggesting that antibody titer might be correlated with protection to EBOV infection. A humoral immune response is sometimes generated during lethal EBOV infection, meaning that antibodies produced by survivors were more efficient than the ones synthesized by the patients who did not survive.

Such a humoral immune response seems to be long lasting: 20 patients having survived Zaire Ebola virus infection from 3 different outbreaks in Gabon, still had IgG antibodies against Zaire Ebola virus 11 years after infection (Wauquier, Becquart et al. 2009).

A neutralising antibody was isolated from a survivor's bone marrow during an outbreak of Zaire Ebola virus in 1995. This antibody, named "KZ52", is capable of neutralising 90% of viral particles [START_REF] Maruyama | Ebola virus can be effectively neutralized by antibody produced in natural human infection[END_REF]. It specifically recognizes a 15 amino acid long sequence of GP; it neutralises the virus by preventing fusion loop rearrangement thus precluding the fusion of viral and cellular membrane in the endosome. Moreover, it appears that KZ52 fixation to its epitope, generates a steric bulk that would hide the binding domain from the cellular receptor (Lee, Fusco et al. 2008). Treatment with this antibody has been tested in NHP but it did not protect animals neither before, nor after exposure to Ebola virus; this had put into question the idea of using antibodies to treat infected patients [START_REF] Oswald | Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys[END_REF]).

However, a more recent study has shown that it was possible to protect NHP Although filoviruses are among the most virulent and fatal pathogens known, as mentioned before, some patients infected with Ebola virus recover from infection. Identifying the differences in the immune response between fatal and nonfatal cases is important for future development of effective therapies and vaccines. Specific differences in clinical presentation and immune responses have been noted in those who succumb contrasted with those who recover from Ebola virus infection (Table 6) [START_REF] Zampieri | Immunopathology of highly virulent pathogens: insights from Ebola virus[END_REF]).

Non-lethal infection Lethal infection

Prominent [START_REF] Zampieri | Immunopathology of highly virulent pathogens: insights from Ebola virus[END_REF]).

This comparison shows that the development of an antigen specific cellmediated immune response correlates with clearance of the virus. Studies demonstrating antigen-specific cellular immune responses in vaccinated nonhuman primates that survived infectious Ebola virus challenge support this finding [START_REF] Sullivan | Development of a preventive vaccine for Ebola virus infection in primates[END_REF][START_REF] Sullivan | Accelerated vaccination for Ebola virus haemorrhagic fever in nonhuman primates[END_REF]. Based on these considerations, it is possible that an early and robust, but transient, innate immune response and the subsequent activation of adaptive immune response are necessary to protect against fatal infection.

3.1.3.Coagulation impairment

In vertebrates, the role of the coagulation cascade is to repair damage and protect the cardiovascular system. Coagulation prevents blood loss through haemostatic mechanisms and limits the spread of systemic infection by encapsulation of pathogens.

Ebola haemorrhagic fever is characterized by coagulative disorders and haemorrhages. These defects in blood coagulation and fibrinolysis are manifested as petechiae, ecchymoses, mucosal haemorrhages, congestion, and uncontrolled bleeding at venepuncture sites. Massive loss of blood is infrequent and, when present, is mainly limited to the gastrointestinal tract. Even in these cases, the amount of blood that is lost is not substantial enough to cause death [START_REF] Feldmann | Ebola haemorrhagic fever[END_REF].

Thrombocytopenia, consumption of clotting factors, deposition of fibrin and increased concentrations of fibrin degradation products are other indicators of the coagulopathy that characterises Ebola virus infection. The mechanisms responsible for triggering the coagulation disorders that typify Ebola haemorrhagic fever are not wholly understood. Besides the fact that Ebola virus replicates in endothelial cells, their infection does not extensively disrupt the vascular endothelium in cynomolgus monkeys. Moreover, endothelial cell infection in vivo was only seen at late stages of disease, after the onset of hemorrhagic abnormalities that characterize Ebola virus fever (Geisbert, Hensley et al. 2003), meaning that other non viral factors such as cytokines could be the cause of endothelial barrier disruption. This is in agreement with the fact that this disruption was shown to be not associated with the presence of intracytoplasmic Ebola virus antigens (Geisbert, Young et al. 2003). This data is consistent with the histological observations in fatal human cases from initial outbreaks in 1976, where no vascular lesions were identified [START_REF] Fa | Pathology of Ebola virus infection. Ebola Virus Haemorrhagic Fever[END_REF].

Although it may be a relatively late event in Ebola virus haemorrhagic fever, and not a direct contributor to the hemorrhagic diathesis, late-stage endothelial cell infection may induce other host cell changes that play important roles in the disease and outcome of the infection.

Several studies suggest that the coagulation abnormalities caused during Ebola haemorrhagic fever are generally consistent with disseminated intravascular coagulation (DIC) (Levi 2007). Furthermore, histological and biochemical evidence of disseminated intravascular coagulation during Ebola virus infection in several non-human primate species have been shown (Bray, Hatfill et al. 2001, Geisbert, Young et al. 2003).

Coagulation is initiated by tissue factor , a structural member of the cytokine receptor family [START_REF] Edgington | The molecular biology of initiation of coagulation by tissue factor[END_REF]. Microbial mediators directly induce rapid upregulation of TF in cells of the innate immune system, principally monocytes and macrophages, whereas endothelial cell expression of TF appears to come later, dependent on secondary mediators and restricted to selective vascular beds. TF triggers coagulation by binding the enzyme coagulation factor VIIa and the substrate factor X. Then, Xa is generated and leads to thrombin formation, fibrin deposition and platelet activation, processes that are tightly controlled by anticoagulant mechanisms of the protein C pathway (Figure 14) [START_REF] Ruf | Emerging roles of tissue factor in viral hemorrhagic fever[END_REF]. Results from several studies strongly suggest that expression or release of tissue factor from monocytes and macrophages infected with Ebola virus are key factors that induce the development of coagulation irregularities reported in Ebola haemorrhagic fever (Geisbert, Young et al. 2003). Overexpression of tissue factor, a glycoprotein of 47kDa that functions as the primary cellular initiator of the coagulation protease cascades, is a leading cause of DIC and thrombosis-related organ failure. During DIC, the tissue factor (TF)-dependent pathway is the dominant route to thrombin generation that leads to fibrin deposition (Figure 15). metabolism and stored in hepatocytes and released into the blood when hepatocytes are acutely damaged).

Adrenocortical infection and necrosis have also been reported in humans and non-human primates infected with Ebola virus (Geisbert, Hensley et al. 2003).

The adrenal cortex plays an important role in the control of blood pressure homoeostasis. Impaired secretion of enzymes that synthesis steroids leads to hypotension and sodium loss with hypovolaemia, which are important elements that have been reported in nearly all cases of Ebola haemorrhagic fever.

Impairment of adrenocortical function by Ebola virus infection could therefore significantly contribute to the shock that typifies the late stages of Ebola haemorrhagic fever.

Together, the data so far suggest that an impaired and ineffective host response leads to high concentrations of virus and proinflammatory mediators in the late stages of disease, which is important in the pathogenesis of haemorrhage and shock. The prevailing hypothesis at this time is that infection and activation of antigen-presenting cells is fundamental to the development of Ebola haemorrhagic fever. The release of proinflammatory cytokines, chemokines, and other mediators, like tissue factor, from antigen presenting cells and other cells, causes impairment of the vascular and coagulation systems leading to multiorgan failure and a syndrome that in some ways resembles septic shock [START_REF] Feldmann | Ebola haemorrhagic fever[END_REF].

Disseminated Intravascular Coagulation (DIC)

Coagulation abnormalities caused during Ebola haemorrhagic fever are generally consistent with disseminated intravascular coagulation (DIC) (Levi 2007). Disseminated Intravascular Coagulation (DIC) is a syndrome characterized by systemic activation of the blood coagulation system, which results in the generation and deposition of fibrin, leading to microvascular thrombi (Figure 16A) in various organs and contributing to the development of multi-organ failure. Moreover, clotting can lead to an increased consumption of platelets and of some clotting factors which may induce severe bleeding complications (Figure 16B) (Levi and Ten Cate 1999). These changes, which may occur rapidly or at a more chronic rhythm, can induce abnormal bleeding, small-vessel obstruction and single or multiple organ dysfunction. Hence, a patient with DIC can present simultaneously thrombotic and bleeding problems.

DIC pathogenesis

Two principal simultaneously mechanisms play a role in the pathogenesis of DIC (Figure 17), fibrin deposition and impairment of fibrin removal. Fibrin deposition is a result of tissue factor-mediated thrombin generation that is insufficiently balanced by dysfunctional physiologic anticoagulant mechanisms (such as the antithrombin system and the protein C system). In addition, impairment of fibrin removal is a result of the impairment of the fibrinolytic system, mainly caused by high circulating levels of PAI-1, a fibrinolytic inhibitor.

During Ebola virus infection, the appearance in the tissues of fibrin deposits, coinciding with detectable viremia and infectious virus, strongly supports a direct link between DIC and viral infection (Geisbert, Young et al. 2003).

Proinflammatory cytokines

Vascular endothelial cells

TF

Tissue factor expression

Impairment of anticoagulant mechanisms

Inhibition of fibrinolysis

Protein C In somewhat more detail, the following processes are involved:

1. Tissue factor expression 2. Impairment of anticoagulant mechanisms 3. Impairment of fibrin removal

Tissue factor expression

TF is best known as the key initiator of coagulation in vivo, but it is also a member of the cytokine receptor superfamily and functions as a signaling receptor. Not only vascular injury, but also inflammatory cytokines and other factors can lead to TF expression, thus linking inflammation and coagulation.

Monocytes are the predominant cell type to express TF after appropriate stimulation and are partially responsible for the activation of coagulation in endotoxemia. LPS and the subsequent host response also induce endothelial cells to express TF. Indeed, monocytes and ECs do not express TF constitutively but only after stimulation by endotoxin immune complexes, certain cytokines, and platelets. ECs have also been shown to release procoagulant TF in response to TNFα and IL-6. Recent studies have shown that activated platelets release TF from their alpha granules and provide another source of intravascular TF. Other leukocytes do not express TF but can acquire TF by binding with monocyte-derived microparticles (MPs).

The initiation of coagulation requires the exposure of TF to VIIa, which is circulating at low concentration in the blood. The TF/FVIIa complex activates both IX and X, leading to generation of fibrin clots (Figure 14). In line with this observation, abrogation of the tissue factor/VIIa pathway by monoclonal antibodies resulted in a complete inhibition of thrombin generation in endotoxinchallenged chimpanzees and prevented the occurrence of DIC and mortality in baboons that were infused with E. coli (ten Cate, Schenk et al. 1994[START_REF] Biemond | Complete inhibition of endotoxin-induced coagulation activation in chimpanzees with a monoclonal Fab fragment against factor VII/VIIa[END_REF], Taylor 2001). Importantly, a study showed that samples obtained from Ebola virus infected macaques contained increased levels of TF associated with lymphoid macrophages. Plasma from these macaques also contained increased numbers of TF-expressing membrane microparticles (Geisbert, Young et al. 2003). Interestingly, another study showed that administration of recombinant nematode anticoagulant protein c2 (rNAPc2), a potent inhibitor of tissue factor-initiated blood coagulation, to Ebola virus infected macaques prolonged survival time, with a 33% survival rate. Moreover, rNAPc2 also attenuated the coagulation in all treated animals (Geisbert, Hensley et al. 2003).

As seen before, tissue factor can also activate thrombin, which itself has several roles in coagulation, including:

 The conversion of fibrinogen to fibrin, activation of factors V, VIII, XIII, protein C, and thrombin-activatable fibrinolysis inhibitor (TAFI). Therefore, it should be noted that circulating thrombin itself has several potent pro-inflammatory properties (thus contributing to coagulation): from activation of proinflammatory cytokines to induction of clotting through the conversion of fibrinogen into fibrin. Pro-inflammatory cytokines also contribute to decreased production of endothelial HSPGs, reducing the efficacy of AT.

 Mediation of inflammation, for example, by direct binding to leukocytes and thereby attenuation of cytokine receptor expression.

Thrombomodulin (TM) is a membrane protein synthesized predominantly by

ECs and is ubiquitously expressed. Some of its functions include:

 TM binding to thrombin and generation of thrombin-TM complexes which activate protein C by a 100-fold. This complex also blocks the thrombinmediated conversion of fibrogen into fibrin and inhibits the binding of thrombin to other cellular receptors on platelets and inflammatory cells. As with thrombin, most of PCA's actions are mediated through its interaction with PAR-1 and via the endothelial protein C receptor (EPCR). EPCR is expressed on ECs as well as on leukocytes. Endothelial NF-κB activity, reduces thrombomodulin and EPCR expression and increases their shedding from the endothelial surface, thus rendering the endothelium less capable of activating Protein C (White, Schmidt et al. 2000).

Animal experiments based on the study of severe inflammation-induced coagulation activation showed that compromising the protein C system results in increased morbidity and mortality rates, whereas restoring an adequate function of activated protein C improves survival and organ failure (Levi, van der Poll et al. 2004). The analysis of samples obtained from 25 macaques infected with Zaire Ebola virus showed a rapid decrease in plasma levels of protein C concomitant with progression of disease (Geisbert, Young et al. 2003). In agreement with this study, treatment with recombinant activated Protein C (rPCA) of rhesus macaques that had been challenged with a lethal dose of Zaire Ebola improved NHP survival in at an 18% rate [START_REF] Hensley | Recombinant human activated protein C for the postexposure treatment of Ebola hemorrhagic fever[END_REF].

TFPI, a protein produced by ECs, inhibits the TF-VIIa complex to prevent further generation of Xa. TAFI has also been shown to be the primary enzyme responsible for inactivation of complement factor C5a, thus providing protection against complement-mediated injury in the micro-vasculature. Studies evaluating recombinant TFPI found that it significantly blocks inflammationinduced thrombin generation, but no conclusions have been drawn from phase I or II clinical trials (O'Brien 2012). In addition, another study showed that TFPI is capable of preventing mortality during systemic infection and inflammation.

Alltogether data suggest that high concentrations of TFPI are capable of modulating tissue factor-mediated coagulation (de Jonge, [START_REF] De Jonge | Tissue factor pathway inhibitor dose-dependently inhibits coagulation activation without influencing the fibrinolytic and cytokine response during human endotoxemia[END_REF].

Impairment of fibrin removal

Dissolution of the fibrin clot by enzymes with fibrinolytic activity is required to restore integrity to intravascular and extravascular sites of thrombus formation.

The amount of fibrin deposited in tissues is generally highest in the smallest vessels but may be influenced by the local rate of blood flow and the activity of local clearance mechanisms. The effect of fibrin deposition can lead to organ ischaemia and failure. Passage of red cells through fibrin strands can also cause red blood cell distortion and fragmentation and subsequent haemolysis and local platelet aggregation.

There are principally two systems to remove fibrin:

1. Plasmin is the primary fibrinolytic pathway of plasma, but platelets and neutrophils also contain fibrinolytic proteases. The fibrinolytic proteases of leukocytes, such as elastase, are found in secretory granules and are capable of degrading fibrin at neutral pH [START_REF] Plow | Leukocyte elastase release during blood coagulation. A potential mechanism for activation of the alternative fibrinolytic pathway[END_REF]). and evidence of fibrin in tissues (spleen, hepatic sinusoids and in blood vessels of the renal medulla) in association with viral antigen. In agreement with this, blood samples from patients infected with the Sudan species of Ebola virus (EBOV), obtained during an outbreak of the disease in Uganda in 2000, were analysed and d-Dimer levels in blood specimens were drastically increased in both patients with fatal and nonfatal infections but were 4 times higher in patients with fatal cases than in patients who survived. (Geisbert, Young et al. 2003[START_REF] Rollin | Blood chemistry measurements and D-Dimer levels associated with fatal and nonfatal outcomes in humans infected with Sudan Ebola virus[END_REF]. Importantly, in another study, when Ebola virus infected macaques were treated with rNAPc2, the coagulation response was attenuated, as evidenced by modulation of various important coagulation factors, including plasma D dimers, which were reduced in nearly all treated animals; less prominent fibrin deposits and intravascular thromboemboli were observed in tissues of some animals that succumbed to Ebola virus (Geisbert, Hensley et al. 2003).

Inflammatory response and DIC

After pathogen exposure there is increased production of pro-inflammatory cytokines. Cells of the immune system secrete most of these inciting mediators.

Cytokines act on many cell types to modulate the host's immune response.

Under normal circumstances, cytokines exert their actions locally and their effects are restricted to neighboring cells.

During DIC syndrome, the excessive release of pro-inflammatory cytokines overwhelms the host's defenses and contributes to the morbidity and mortality of sepsis. The most common cytokines include members of the tumor necrosis factor (TNF), interleukin (IL), and interferon (IFN) superfamilies. The primary function of these molecules is to promote inflammation; they do this via signaling pathways that induce expression of inflammatory products. The byproducts of this stimulation form a very long list that includes nitric oxide, platelet-activating factor, and eicosanoids. These products have direct effects on the host vasculature and facilitate the transmigration of leukocytes to the site of infection. TNFα and IL-6 are both noted for inducing the expression of procoagulant proteins such as TF.

Cross-talk between inflammation and coagulation

Inflammation and coagulation play an important role during Ebola virus pathogenesis. Both constitute two host defense systems with complementary roles in eliminating invading pathogens, limiting tissue damage, and restoring homeostasis.

There is cross-talk between these two systems, whereby inflammation leads not only to activation of coagulation, but coagulation also considerably affects inflammation. Some of the physiological processes involved in inflammation and The most important mechanisms by which coagulation proteases influence inflammation is by binding to protease activated receptors (PARs), localized in the vasculature on endothelial cells, mononuclear cells, platelets, fibroblasts and smooth muscle cells [START_REF] Coughlin | Thrombin signalling and protease-activated receptors[END_REF]. In addition, physiological anticoagulant pathways can influence inflammatory activity. Indeed, activated protein C has been found to inhibit endotoxin-induced production of TNF-a, IL-1b, IL-6 and IL-8 by cultured monocytes/macrophages [START_REF] Yuksel | Activated protein C inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production by inhibiting activation of both nuclear factorkappa B and activator protein-1 in human monocytes[END_REF]. It has also been shown that activated protein C abrogates inflammatory activity and improves organ function and survival during sepsis in baboons (Taylor 1994).

It has also been shown that activated platelets play an important role in inflammation. First, platelet adhesion to the subendothelial matrix supports leukocyte rolling, adhesion, and transmigration through interaction of platelet Pselectin with leukocyte P-selectin glycoprotein ligand-1. Platelets can also release various proinflammatory cytokines (such as CD40 ligand and IL-1β) and chemokines (such as RANTES and platelet factor-4), which may result in further activation of monocyte integrins and thus lead to further monocyte recruitment.

These findings indicate that natural anticoagulants can regulate inflammatory responses as well as the coagulation system by inhibiting the production of proinflammatory cytokines monocytes/macrophages.

Infection with Ebola virus leads to the production of proinflammatory cytokines that, in turn, stimulate the production of tissue factor. Activation of the coagulation system and ensuing thrombin generation are dependent on the expression of tissue factor; acute inflammation results in DIC, a systemic activation of coagulation. Conversely, activated coagulation affects inflammatory cells and endothelial cells and thereby modulates the inflammatory response.

The basic elements of clotting and inflammation include platelets, leukocytes, monocytes/macrophages, and an array of coagulation enzymes, but the essential cooperation and interactions between clotting and inflammation are essential to preserve both pathways. This reciprocal relationship is wellregulated and tightly controlled under normal health conditions but can lead to significant morbidity and mortality in disease.

DIC diagnosis

The detection of molecular markers for activation of coagulation or fibrin formation may be the most sensitive assays with which to establish a diagnosis of DIC, but unfortunately, the specificity of such tests is low (Horan & Francis, 2001).

Another problem is that a reliable test for quantifying soluble fibrin in plasma is not available and a wide discordance been shown among various assays (McCarron et al, 1999). The dynamics of DIC can also be judged by measuring activation markers that are released upon the conversion of the coagulation factor zymogen to an active protease, such as prothrombin activation fragment (Bauer & Rosenberg, 1987;Boisclair et al, 1990). Indeed, these markers are notably elevated in patients with DIC but again specificity is a problem. Other tests that can be used are: plasma thrombin clotting time, fibrinogen levels, assessment of antithrombin III -thrombin complex, fibrin degradation products (FDP) in serum, etc... [START_REF] Levi | Current understanding of disseminated intravascular coagulation[END_REF]).

3.1.4.

Another player in the coagulation pathway: MBL MBL, also known as mannan/mannose binding protein, is a glycoprotein and belongs to a subfamily of proteins called the collectins because family members possess both collagenous regions and also lectin domains. MBL is synthesized in the liver and secreted into the blood where it has an important role in the innate immune system as an acute-phase reactant [START_REF] Eisen | Impact of mannose-binding lectin on susceptibility to infectious diseases[END_REF].

MBL is composed of subunits comprising three identical peptide chains of There is evidence from studies of MBL-disease associations that point to the protein having a major role in the modulation of inflammation. Some studies suggest that the MBL protein exerts a complex effect on cytokine release by monocytes [START_REF] Jack | Mannose-binding lectin regulates the inflammatory response of human professional phagocytes to Neisseria meningitidis serogroup B[END_REF]) (figure 22(iii)). When N. meningitidis organisms are incubated with increasing concentrations of MBL and then added to MBL-deficient whole blood, the release of the cytokines TNF-α, IL-1β and IL-6 from monocytes was enhanced at MBL concentrations below 4 μg/ml but reduced at higher concentrations. This suggests that both the MBL genotype and the presence or otherwise of an on-going acute phase response will help to determine the nature of such cytokine responses in any given individual. The Ebola glycoprotein is heavily glycosylated and contains both N-linked and O-linked carbohydrate structures. N-linked carbohydrates are normally terminated by mannose residues and it has been shown that DC-SIGN, which has a high affinity for high-mannose sites on N-linked glycans, binds to Ebola glycoproteins and that this interaction is inhibited by the high-mannose carbohydrate mannan (Ji, Olinger et al. 2005).

It has previously been shown that recombinant human MBL (rhMBL) binds EBOV GP1,2 lentiviral virion-like particles and wild-type-like EBOV in a specific and dose-dependent manner (Ji, Olinger et al. 2005, Michelow, Dong et al. 2010). Treatment of EBOV infected mice with supraphysiological rhMBL was also shown to have a protective effect [START_REF] Michelow | High-dose mannosebinding lectin therapy for Ebola virus infection[END_REF]). It has also been shown that the CRD domain of MBL is capable of binding Ebola virus via highly specific interaction to N-linked glycans; cyanovirin-N, a lectin that targets N-linked high mannose, was shown to compete with MBL binding to viruses.

Moreover, deglycosylation of N-linked glycans with PNGase F inhibits Ebola virus infection [START_REF] Brudner | Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane C-type lectin receptors[END_REF]).

MBL has a complex role in infections caused by Ebola virus. MBL effectively inhibits infection of human cells by Ebola virus GP-pseudotyped lentiviral virions and wild type-like Ebola virus in the presence of active complement in serum (Michelow, Dong et al. 2010[START_REF] Michelow | High-dose mannosebinding lectin therapy for Ebola virus infection[END_REF]. In contrast to this,

MBL

3.2.Direct cytotoxicity caused by the virus itself and GP protein

Humans with fatal infections have up to 10 10 copies of viral RNA per milliliter, whereas much fewer copies (10 7 copies/ml) are found in the serum of those who survive Ebola virus infection [START_REF] Zampieri | Immunopathology of highly virulent pathogens: insights from Ebola virus[END_REF]. High

2000)

It was thought that direct damage to endothelial cells by virus replication was the first determinant of vascular injury and permeability. However, as mentioned above, Ebola virus histological analysis of autopsy tissues from several of the early outbreaks did not identify vascular lesions [START_REF] Zampieri | Immunopathology of highly virulent pathogens: insights from Ebola virus[END_REF]).

Similarly, no evidence of substantial vascular lesions in non-human primates infected with Ebola virus exists [START_REF] Jaax | Lethal experimental infection of rhesus monkeys with Ebola-Zaire (Mayinga) virus by the oral and conjunctival route of exposure[END_REF].

However, several studies have shown that Ebola virus GP down-regulates a range of cell surface markers including several involved in cell adhesion (such as integrin molecules, especially β1-and α v -integrin subunits, and PECAM-1

and VCAM) and immune surveillance (like MHC-I). In fact, it has been shown that overexpression of mature GP on the plasma membrane results in the masking of cellular surface epitopes [START_REF] Reynard | Ebolavirus glycoprotein GP masks both its own epitopes and the presence of cellular surface proteins[END_REF]). The high degree of GP glycosylation (including N-and O-linked sugars) and the fact that surface GP is a trimer, explains this masking phenomena by Ebola virus GP (Figure 24).

The loss of cell adhesion as well as the specific loss of MHC-I and adhesion molecules involved in stabilizing cell-to-cell interactions is likely to negatively impact both the stimulation of an anti-Ebola virus CD8 + T-cell response and the ability of T cells to kill infected cells.

Cells not expressing Ebola virus GP

Cells expressing Ebola virus GP: masking of celular molecules

Integrins also play a critical role in the trafficking of leukocytes through the endothelium to sites of infection. Specifically, the initial tethering and rolling of leukocytes along the endothelium, followed by the arrest and final diapedesis into sites of infection, all require regulated integrin function. Infection of endothelial by cells Ebola virus, however, inhibits this antiviral state from being induced. This inhibition is at least partially due to the Ebola virus protein VP35, which acts as a potent alpha interferon antagonist. However, the downmodulation/masking of PECAM-1 and MHC-I in endothelial cells by GP alone indicates that GP also plays a major role in this process [START_REF] Simmons | Ebola virus glycoproteins induce global surface protein downmodulation and loss of cell adherence[END_REF]. Indeed, the more specific down-modulation of MHC-I in HUVEC, macrophages, several cell lines and endothelial cells may suggest that such host defense molecules are the intended target of the down-regulation.

Shielding of MHC-I and integrins suggests that Ebola virus GP may play an important role in the evasion of antiviral immunity. Masking of surface proteins may result in the inhibition of receptor-ligand interactions and signaling and thus contribute to a functional destabilization of virus-infected cells.

3.3.Overview of Ebola virus pathogenesis

In striking contrast with fatal outcome, effective control of EBOV infection is associated with balanced immune responses. A transient and well-regulated inflammatory response is observed early in the disease not only in patients that survive, but also in asymptomatic subjects. This response may help to control the infection and to induce specific immunity. An early and robust humoral response is strongly correlated with survival in symptomatic patients. In asymptomatic EBOV-infected subjects, antibodies are detected about 3 weeks after infection and in moderate amounts. In survivors and asymptomatic subjects, high antibody titres can persist for years.

The factors underlying the radically different outcomes of EBOV infection are unclear, but may include the route of infection, the size of the inoculum, the cell types that are initially infected, previously acquired immunity, heterologous immunity, and MHC status. However no difference was found in the coding sequences of viruses isolated from survivors and fatalities and different outcomes can be observed in patients infected with the same viral source [START_REF] Leroy | Ebola and Marburg haemorrhagic fever viruses: major scientific advances, but a relatively minor public health threat for Africa[END_REF].

Ebola virus induced pathophysiology is explained by a combination of factors, including uncontrolled and non-specific inflammatory responses, viral-induced immunosuppression and direct viral destruction of several cell types; collectively contributing to the collapse of the vascular system, multiorgan failure and a septic shock-like syndrome in lethal infection.

Ebola virus, a member of the Filoviridae family, causes lethal hemorrhagic fever in man and non-human primates, displaying up to 90% mortality rates. A unique feature of EBOV is that, following infection, virus glycoproteins are released from cells in soluble forms. High amounts of truncated surface GP, named shed GP, are detected in the blood of patients and experimentally infected animals.

Even if shed GP has been discovered more than 10 years ago, besides its role as a decoy factor, no other roles have been yet determined for this soluble glycoprotein. This topic is increasingly relevant in the face of the reoccurring disease threat posed by this continually remerging and highly pathogenic virus.

In this context, the objective of the work done in the scope of this thesis was to produce and to study the role of shed GP during Ebola virus pathogenesis.

Overall, we provide new insights into the biological role of shed GP during infection and its contribution to the high pathogenicity of Ebola virus.

Since surface GP is almost not cleaved by TACE when it is transiently transfected, we first generated a system where we were able to refine and optimize production of shed GP in high enough amounts to be able to to study it in BSL-2 conditions. We have identified its primary targets to include DCs and macrophages. We have also determined that shed GP has a direct, by itself, and indirect, through cytokine release, effect on cells of the immune system and we have characterised that this activation is, principally but not exclusively, achieved by binding of glycosylated shed GP to TLR4.

We were also interested in determining if monocytes, which remain a paradoxal site of replication for Ebola virus, could be numbered amongst shed GP's early targets. To do this, we have studied if shed GP could bind and allow Ebola virus infection of monocytes. Importantly, we also show that shed GP causes DCs, monocytes, macrophages and HUVECs to increase the expression of tissue factor (TF). Importantly, TF has been shown to be the primary endothelial cell response that results in alterations in both permeability and procoagulant activity. We have also revealed that shed GP activates permeability of HUVECs, both directly and indirectly though cytokine release.

Finally, we have also studied the role of shed GP in affecting infection of cells by the virus itself. We have performed competition experiments during infection to characterise the impact of the presence of shed GP in Ebola virus binding and internalisation.

Taken together the objective of this work was to delimit a new role for EBOV shed GP. Overall data provide a potential explanation as to a major contributing factor of the excessive host responses observed during EBOV infection. death. In order to address solely the potential functions of shed GP and to allow for the possible effects caused by cytotoxicity, an EBOV GP mutant lacking shedding properties was used in this study as a negative control. This mutant contains a substitution L635V (GP-LS, low shedding) that was previously shown to prevent TACE cleavage and result in increased surface GP expression (Dolnik, Volchkova et al. 2004). Of note, expression of this mutant results in similar, if not higher, cytotoxicity than that caused by wild-type GP (GP-WT) and provides culture supernatants containing the same potential extracellular factors released from GP expressing cells whilst displaying undetectable amounts of shed GP.

It is important to stress that the expression and production of shed GP, sGP and also of all control proteins used in this study was performed in cells cultured in FCS free medium (VP-SFM). This prevents putative interaction between soluble EBOV glycoproteins and sera lectins including mannose-binding lectin (MBL), which as described above has previously had been shown to bind surface GP of EBOV (Ji, Olinger et al. 2005). In this manner, 293T cells were transfected with the plasmids expressing mutated GPs, GP-WT, sGP as well as GFP as a non-relevant control. Western blot analysis of cell lysates and culture supernatants revealed a dramatic difference in efficacy of GP shedding between GP-WT and mutated GPs as expected (Figure R2 A). Of note, due to the removal of the region spanning the transmembrane anchor, after cleavage by TACE the GP 2 within shed GP shows a lower molecular mass and faster migration on SDS-polyacrylamyde gels compared to GP 2 in intracellular GP 1,2 .

While similar amounts of intracellular GP (represented by endoplasmic reticulum GP precursor -preGPer-and mature GP 1,2 ) were seen in cells expressing either GP-WT or GP-LS (compare lines 1 and 3), GP-LS did not show any detectable levels of shed GP in the medium when compared with GP-WT. Notably, relatively low amounts of intracellular GP were detected with GP-HS whilst increased levels of shed GP were detected in the medium. As Of note, our GP construction and production approach differed from that used in an earlier study investigating soluble EBOV glycoproteins. In the previous study the authors had used purified HA-tagged soluble glycoproteins produced through the addition of a stop codon immediately upstream of the transmembrane domain that allows efficient secretion of GP into the extracellular media (Wahl-Jensen, Kurz et al. 2005). However, we have observed that when GP is produced in this way the truncated GP is clearly present in a monomeric form, as evidenced by sedimentation through a sucrose gradient (Figure R2 D). In contrast, shed GP that is released through TACE cleavage is present in a trimeric form, as observed during infection (Dolnik, Volchkova et al. 2004) and as seen with our GP-HS construction.

EBOV soluble glycoproteins bind to DC and macrophages

Human monocyte-derived DCs and macrophages (MØ) as well as peripheral blood lymphocytes (PBL) containing T lymphocytes, B lymphocytes and NK cells, were incubated with the samples of culture medium containing the soluble EBOV GPs described above. To assess the ability of shed GP to bind to DCs and macrophages (MØ) flow cytometry was performed using anti-GP specific antibodies that recognize both sGP and shed GP. DCs were shown to bind both shed GP and sGP while macrophages could only bind shed GP (Figure R3 A).

As expected, incubation of the cells with GP-LS failed to evidence any GP specific binding in agreement with the undetectable levels of shed GP in samples. It appears that neither B (Figure R3 A) nor T lymphocytes or NK cells (data not shown) bind shed GP or sGP; this is in agreement with earlier data revealing an absence of surface GP binding to these cells (Marzi, Moller et al. 2007, Dube, Schornberg et al. 2010).

In light of data revealing an interaction between surface GP and MBL, we also investigated whether human sera containing MBL is capable of affecting the Culture supernatant from cells expressing GP-LS was used as an additional negative control. Prior to experiments, all culture supernatants were tested for the absence of endotoxin and showed endotoxin concentrations below 0,25 units/ml and were thus considered to be endotoxin-negative.

EBOV shed GP induces release of pro-and anti-inflammatory cytokines from DCs and MØ

Given the transcriptional activation observed above it follows that upon stimulation DCs and MØ would release a range of cytokines into the medium.

Accordingly, culture supernatants of DCs and MØ collected 24h after addition of shed GP (as detailed above) were assayed using a Multiplex cytometric bead array against a panel of cytokines previously shown to be upregulated during EBOV infection (Stroher, West et al. 2001, Baize, Leroy et al. 2002). As expected, shed GP induced the secretion of TNFα, IL6, IL10, IL12, IL8, IL1β and IL1RA in both DCs and MØ (Figure R5 A). Comparable levels of these cytokines were also observed in the medium when cells were treated with LPS.

All negative controls used in this study, including samples of medium from GP-LS-expressing cells did not show any significant release of the cytokines tested.

Incubation of the cells with the sGP-containing sample also did not result in Results III:

Role of shed GP in coagulation and vascular permeability C-type lectins bind high mannose residues of N-linked glycans in a calciumdependent manner (Michelow, Dong et al. 2010). Previous studies have shown that particles pseudotyped with Ebola and Marburg glycoproteins bound significantly to MBL (Ji, Olinger et al. 2005).

To determine whether soluble glycoproteins interact with MBL, we performed surface plasmon resonance (SPR) using the Biacore T100 system. We immobilized recombinant MBL (rMBL) on a CM5 sensorchip and tested binding of soluble glycoproteins in the presence of CaCl 2 . The data obtained showed that soluble glycoprotein shed GP specifically interacts with MBL. Only trimeric shed GP bound strongly to immobilized rMBL. Since the monomeric form is similar to trimeric shed GP it may have conserved interaction sites to MBL but differences in oligomerisation showed lower affinity to MBL (Figure R10 A).

Analytes containing sGP, considerably less glycosylated than shed GP, and mock supernatant (from GFP expressing cells) did not show any binding to MBL ligand.

In order to confirm this data, we performed an ELISA (Figure R10

Shed GP Activates MASP1

After confirming MBL binding to shed GP, we wanted to determine if this binding was capable of activating MBL associated protein MASP-1. To explore the activity of MASP-1, which is known to have a thrombin-like substrate specificity, we performed a fluorescence-based assay by using the thrombin substrate VPR-AMC, a short peptide conjugated to 7-amino-4-methyl coumarin (AMC).

Shed GP activates C4 fixation and cleavage through MASP-2

Since the above experiments indicated that soluble glycoprotein shed GP can bind to MBL and activate MASP-1, we wanted to determine if this binding could also activate MASP-2, which is directly implicated in C4 cleavage during the lectin complement pathway.

Supernatants from the recombinant viruses described above (EBOV-HS, EBOV-LS and EBOV-WT) were coated in maxisorp plates and C4 fixation was quantified by ELISA after addition of MBL (Figure R12 A). EBOV-HS supernatant, which contains high amount of shed GP compared to LS and WT variants, fixed significantly more C4 than EBOV-LS and EBOV-WT. Accordingly EBOV-WT, that contains more shed GP than EBOV-LS fixed more C4 compared to EBOV-LS. Negative control containing no supernatant but only MBL showed the lowest C4 fixation. All samples were normalised to mock controls (supernatant from non-infected cells).

In order to confirm these results, we performed a competition assay as in Figure R11 B. Briefly, mannan, VLPs-GP and supernatants from cells transiently transfected with HS, LS and sGP were incubated with MBL and the mixture was subsequently added to plates previously coated with VLP-GP.

Finally, C4 was added and its fixation and cleavage was measured by ELISA (Figure R12 B). As expected, MBL previously incubated with HS, EBOV-GP VLPs and mannan showed weaker fixation of C4 compared to incubation with LS, sGP or nothing (MBL alone, as positive control). These data show that MBL which is already bound to shed GP, EBOV-GP or mannan, does not allow MBL fixation to EBOV-GP VLPs coated in the plate and thus prevents fixation and cleavage of C4. Thus, lectin complement pathway activation could be achieved through activation of MASP-2 by shed GP.

Shed GP induces expression of tissue factor in monocytes,

DCs, macrophages and HUVEC

TF is the primary initiator of blood coagulation. Modulation of TF expression plays an important role in regulation of endothelial thrombogenicity and vascular tone. Expression of tissue factor (TF) is known to be induced during EBOV infection (Geisbert, Young et al. 2003).

In order to test if soluble glycoproteins are able to induce the expression of tissue factor in EBOV primary targets (monocytes, DCs and macrophages) and also in human endothelial cells (HUVECs), these cells were exposed for 24 hours to LPS and supernatants from transiently transfected cells containing:

Mock (in grey), sGP, LS, HS and, as detailed above, HS previously incubated with MBL (HS+MBL) (all in white). Cells were then collected and TF expression was analysed by flow cytometry (Figure R13 A). Expression of tissue factor was strongly induced in monocytes by HS and LPS but not by LS or sGP. TF activation by HS was completely abrogated when this supernatant was previously incubated with MBL (HS+MBL). Expression of TF was also induced by LPS and HS in macrophages and HUVEC but not in DCs. LS, sGP and HS+MBL did not induce TF expression. We then wanted to determine if soluble glycoproteins could activate coagulation by increasing soluble levels of tissue factor (sTF) and decreasing soluble thrombomodulin (sThM), which has anticoagulant properties. Supernatants from HUVEC, treated as in the previous experiment, were collected and their content of sTF and sThM was analysed by Multiplex ELISA. In the case of sTF both TNF-α and HS incubation led to an increase in soluble levels of TF (Figure R13 B). Controls containing Mock, sGP, LS and HS+MBL did not affect sTF release. Surprisingly, in the case of sThM, supernatants from transiently transfected cells containing shed GP (HS), LS, or HS+MBL did not induce the release of anticoagulant biomarker thrombomoduline (ThM) when compared to positive control, TNF-α.

Supernatant containing sGP seemed to slighty decrease sThM in medium when compared with negative controls.

and thrombomodulin (sThM). The mean± sd of triplicates from one representative experiment from three independent experiments is shown.

ICAM-1 and VCAM expression on HUVEC cells

It is well known that upon stimulation, endothelial cells express cell adhesion molecules such as ICAM-1 and VCAM-1, which are known to be important mediators in cellular migration and infiltration and in inducing TF expression in monocytes and macrophages [START_REF] Celi | P-selectin induces the expression of tissue factor on monocytes[END_REF][START_REF] Mcgilvray | VLA-4 integrin cross-linking on human monocytic THP-1 cells induces tissue factor expression by a mechanism involving mitogen-activated protein kinase[END_REF]. Therefore, we evaluated the expression of ICAM-1 and VCAM-1 on the vascular endothelium after treatment with EBOV soluble glycopoteins.

Modulation of endothelial monolayer permeability by EBOV shed GP

Vascular dysregulation and instability are thought to be crucial symptoms during EBOV infection [START_REF] Ryabchikova | Animal pathology of filoviral infections[END_REF][START_REF] Ryabchikova | Animal pathology of filoviral infections[END_REF], Zaki and Goldsmith 1999, Feldmann and Geisbert 2011). While endothelial cells are susceptible EBOV targets in vitro (Yang, Duckers et al. 2000[START_REF] Simmons | DC-SIGN and DC-SIGNR bind ebola glycoproteins and enhance infection of macrophages and endothelial cells[END_REF], Wahl-Jensen, Afanasieva et al. 2005) they are considered to be rather late virus targets in vivo [START_REF] Baskerville | Ultrastructural pathology of experimental Ebola haemorrhagic fever virus infection[END_REF], Geisbert, Young et al. 2003[START_REF] Hensley | The contribution of the endothelium to the development of coagulation disorders that characterize Ebola hemorrhagic fever in primates[END_REF] suggesting an indirect effect of EBOV infection on this cells. A dysregulated inflammatory host response to EBOV infection is believed to facilitate increased permeability of endothelial barriers that would eventually cause haemorrhaging [START_REF] Royall | Tumor necrosis factor and interleukin 1 alpha increase vascular endothelial permeability[END_REF], Geisbert, Young et al. 2003, Geisbert, Young et al. 2003, Wahl-Jensen, Afanasieva et al. 2005). In this regard proinflammatory cytokines, in particular TNFα, released from immune cells activated by shed GP could affect endothelial barrier integrity.

To answer this question, an in vitro monolayer cell permeability assay was performed using HUVECs. HUVEC monolayers grown in transwell plates on semi-permeable membranes were treated for 22h with samples containing soluble glycoproteins or culture supernatants from macrophages previously stimulated for 24h with either HS or sGP and respective controls. Following treatment, FITC-dextran was added to the apical part of semi-permeable membranes in cell culture plate and the permeability of the monolayer induced directly by soluble glycoproteins (Figure R15 A Somewhat surprisingly given the absence of detectable activation in previous assays, a moderate increase in permeability was observed when culture medium from GP-LS-expressing cells was used. Nevertheless, the effect observed by HS is significantly superior when compared to LS. hours post rVSV-GP infection, replication and therefore, the amount of virus in the supernatant, was lower when cells were pretreated with HS supernatant than with LS and mock controls.

Infection of Vero cells with recombinant Ebola virus expressing GFP during infection (rEBOV-GFP) showed similar results as rVSV-GP experiments. Vero cells were incubated with mock supernatant (in grey) or supernatants (in white) from recombinant viruses releasing high, low or normal amounts of shed GP into the media (named EBOV-HS, EBOV-LS and EBOV-WT respectively). We could observe that infection by rEBOV-GFP virus was clearly reduced when cells had previously been incubated with HS supernatant (Figure R16 C). Cells incubated with LS and WT did not show any reduction when compared to cells that were not treated with any supernatant (in grey).

Shed GP targets the immune system

The molecular mechanisms behind the pathogenesis of Ebola virus are yet not completely understood, however it is known that the pathogenic potential of EBOV is enhanced through several factors and both viral gene products and dysregulation of host proteins seem to be detrimental to the host's chances of survival.

Ebola virus infection is characterized by immune suppression and a systemic inflammatory response that causes impairment of the vascular, coagulation and immune systems in addition to lymphopenia. These are all hallmarks of pathogens that subvert both innate and adaptative immune responses [START_REF] Feldmann | Ebola haemorrhagic fever[END_REF]). An impaired and ineffective host response leads to high concentrations of virus and proinflammatory mediators in the late stages of disease, which play important detrimental roles in the pathogenesis of haemorrhaging and shock. It has been shown that Ebola virus-infected cells produce and release high amounts of viral soluble glycoproteins into the blood vessels in both humans and animals (Feldmann, Volchkov et al. 1999, Volchkova, Klenk et al. 1999); once released, these proteins are free to interact with host factors and cells. However, the precise role of these soluble glycoproteins during viral pathogenesis still remains unknown.

Studying the interaction of these soluble proteins with host cells and their role in pathogenesis is complicated due to the difficulty in producing these soluble proteins in large quantities equivalent to those observed during Ebola virus infection. In the case of shed GP, the efficiency of shedding in cells transiently expressing GP from transfected plasmids is much lower compared to virus infection, probably due to the low activity of TACE or another potential unidentified cellular process in uninfected cells. Moreover, the high glycosylation and postranslational modifications of these soluble proteins hinders the use of other commonly used systems such as bacteria or yeast, which are more efficient. For these reasons we sought to generate several constructs with different shedding properties that allowed us to study this protein: High shedding (HS) and Low shedding (LS) constructs, which respectively induce more or less secretion of shed GP into the media compared to GP wild-type. The fact that we have produced our proteins in serum-free medium means, on one side that different serum lectins didn't interact with our soluble glycoproteins and on the other side, we could generate a negative control for the heavily glycosylated shed GP through the subsequent addition of sera lectin MBL.

One earlier study addressed some of the same issues that we did, in that they used purified, secreted HA-tagged glycoproteins in order to study activation of macrophages (Wahl-Jensen, Kurz et al. 2005). That study differed however, in that (i) the insertion of a HA tag and stop codon before the transmembrane domain allows efficient secretion into the extracellular media. However, we have observed that when GP is produced in this way, the truncated GP is clearly present in a monomeric form, as evidenced by sedimentation through a sucrose gradient (Figure 1D); in contrast, in our construct shed GP is released through TACE cleavage and is present in a trimeric form, as also observed during infection. In addition, (ii) in the previous study, macrophage culture and experiments were done in the presence of human sera which is known to contain lectins, including MBL amongst others, that can interact with glycoproteins. These differences could explain that, in their case, no role was found for secreted glycoproteins until now.

Depending on the cytokine profile observed its effect can be either protective or damaging. Filovirus-induced expression of such mediators results in an immunological imbalance that partly contributes to the progression of disease.

The balance in cytokine release is influenced by the route, the titer and the strain of the incoming virus as well as factors specific to the individual host immune response. In this respect, it should be expected that cytokine profiles could differ between individuals and EBOV strains. It has previously been

shown that there is a link between clinical status and inflammatory responses (Baize, Leroy et al. 2002); in fact, human blood samples from survivors and nonsurvivors obtained during the five outbreaks that occurred between 1996 and 2003 in Gabon and Republic of Congo were studied and it was observed that fatal outcome is associated with exacerbated innate immune responses and with global suppression of adaptive immunity [START_REF] Wauquier | Human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis[END_REF]. This exaggerated innate immune reaction is characterized by a "cytokine storm," with aberrant secretion of numerous proinflammatory cytokines, Several studies on infected human and nonhuman primates have demonstrated the interaction of Ebola virus with different cells of the immune system [19,20].

This corresponds well with the data obtained during our study were we observed that cultured primary DCs and macrophages were activated upon treatment with Ebola virus shed GP soluble glycoprotein, resulting in an increase of a number of mediators including pro-and anti-inflammatory cytokines, namely TNFα, IL6, IL10, IL12, IL8, IL1β and IL1RA. This has also been observed in previous studies using VLPs on DCs [START_REF] Bosio | Ebola and Marburg virus-like particles activate human myeloid dendritic cells[END_REF]) and macrophages (Wahl-Jensen, Kurz et al. 2011). This observation leads to the conclusion that neither the whole virus nor viral replication are prerequisites for activation of primary target cells during Ebola virus infection.

As observed during our study the secreted form of the viral glycoprotein spike, soluble glycoprotein shed GP, is sufficient to activate DCs and macrophages and thus, probably together with other viral factors, be partially responsible for the high pathogenicity of Ebola virus. Here we compare for the first time activation of primary targets DCs and macrophages by soluble glycoproteins.

Notably, we also observe that macrophage activation and release of cytokines is considerably higher when compared to DCs.

Since susceptibility of monocytes to infection is controversial [START_REF] Feldmann | Filovirus-induced endothelial leakage triggered by infected monocytes/macrophages[END_REF] (Martinez, Johnson et al. 2013). In agreement, we show that delay in permissiveness may be explained by the fact that treatment of monocytes with shed GP differentiates them and thus, they become permissive to infection. Therefore monocyte differentiation appears to be a requirement for EBOV entry and this differentiation could be due to shed GP. This would suggest that soluble shed GP could target non-infected monocytes and rend them permissive for infection, thus considerably increasing EBOV cellular targets in vivo. Further experiments should be done in order to determine expression of cell surface markers and compare them with macrophages and DCs to fully characterise the maturation phenotype associated with permissive EBOV infection.

Since lymphocyte apoptosis is observed during EBOV infection, we thought that shed GP could be the cause of this cell death; however, shed GP was shown not to bind to lymphocyte. The simple release of proinflammatory cytokines could indirectly induce lymphocytes apoptosis but further research should be done in this field to test this hypothesis. Notwithstanding, the fact that shed GP can clearly bind to DCs and macrophages raises an interesting hypothesis; it is known that TIM-4 is restrictively expressed on the surface of DCs and macrophages and that it is the ligand of TIM-1, which is principally expressed on activated T lymphocytes, and that this delivers a costimulatory signal necessary for T cell proliferation [START_REF] Meyers | TIM-4 is the ligand for TIM-1, and the TIM-1-TIM-4 interaction regulates T cell proliferation[END_REF].. This data for activation and cytokine release. Given this common pathology, we wondered whether EBOV soluble glycoproteins might affect TLR4 as LPS does during septic shock. Indeed, when presented in VLPs EBOV GP has been shown to interact with TLR4, leading to the induction of proinflammatory cytokines as well as SOCS1 in both a human monocytic cell line (THP-1) and 293T cells expressing a functional TLR4/MD2 receptor [START_REF] Okumura | Interaction between Ebola virus glycoprotein and host toll-like receptor 4 leads to induction of proinflammatory cytokines and SOCS1[END_REF]). Thus, in the case of EBOV both the virus particles themselves and importantly shed GP as a soluble mediator would be able to activate TLR4 on target cells, making it distinct from a number of other viruses whose surface glycoproteins have been shown to act as ligands for TLR4 recognition, including respiratory syncytial virus, mouse mammary tumor virus and human CMV [START_REF] Haynes | Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus[END_REF][START_REF] Burzyn | Toll-like receptor 4dependent activation of dendritic cells by a retrovirus[END_REF][START_REF] Tal | Association between common Toll-like receptor 4 mutations and severe respiratory syncytial virus disease[END_REF][START_REF] Xie | Toll-like receptor 4 expression and function of respiratory syncytial virus-infected airway epithelial cells[END_REF]. In this way activation of non-infected immune cells through soluble shed GP would contribute to the systemic inflammatory syndrome and high pathogenicity seen with EBOV infection.

In agreement with this idea, we show that activation of DCs and macrophages by Ebola virus soluble glycoproteins is principally triggered through TLR4 Indeed, in the case of EBOV both the virus particles themselves and importantly shed GP, as a soluble mediator, would be able to activate TLR4 on target cells, making it distinct from other viruses. In this way activation of non-infected immune cells through soluble shed GP would be a major cause of the systemic inflammatory syndrome seen with EBOV infection and would considerably amplify the host's immune response, thus contributing to the high pathogenicity of Ebola virus.

Likewise, it has also been shown that MBL is able to bind to TLR4 and suppress lipopolysaccharide-induced inflammatory cytokine secretion (Wang, Chen et al. 2011, Wang, Zhang et al. 2011) and microbial [START_REF] Wang | Mannan-binding lectin inhibits Candida albicans-induced cellular responses in PMA-activated THP-1 cells through Toll-like receptor 2 and Toll-like receptor 4[END_REF]) and virus infectivity [START_REF] Reading | A serum mannose-binding lectin mediates complement-dependent lysis of influenza virus-infected cells[END_REF], Saifuddin, Hart et al. 2000) by different mechanisms. Moreover MBL has also been reported to bind the membrane-anchored receptor CD14, which facilitates the interaction between LPS and TLR4 [START_REF] Sano | Surfactant proteins A and D bind CD14 by different mechanisms[END_REF][START_REF] Chiba | Rat mannose-binding protein a binds CD14[END_REF]). As we can see in our study, blocking of TLR4 receptor with MBL or anti-TLR4 antibodies considerably reduces binding and activation of proinflammatory cytokines by shed GP in DCs and macrophages, a fact that is probably explained by the fact that MBL can block shed GP binding to TLR4 by both directly blocking TLR4 receptor and by binding to shed GP and thus preventing shed GP binding to TLR4.

It has been shown that O-glycosylations are specifically recognized by TLR-4 receptor and that this recognition induces release of proinflammatory cytokines [START_REF] Netea | Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors[END_REF]. Indeed, as discussed above, surface glycoproteins from a number of other viruses have been shown to act as ligands for TLR4 recognition [START_REF] Haynes | Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus[END_REF][START_REF] Burzyn | Toll-like receptor 4dependent activation of dendritic cells by a retrovirus[END_REF][START_REF] Tal | Association between common Toll-like receptor 4 mutations and severe respiratory syncytial virus disease[END_REF][START_REF] Xie | Toll-like receptor 4 expression and function of respiratory syncytial virus-infected airway epithelial cells[END_REF]. In agreement with this, we have observed that deglycosylation of shed GP resulted in the loss of its capacity to activate TNFα release in DCs and macrophages. This means that removal of Nand O-glycosylation of shed GP probably prevents the attachment of shed GP through TLR4 in DCs and macrophages. We speculate that shed GP activation of TLR4 could have a positive effect on viral replication by directly activating non-infected macrophages and dendritic cells, promoting subsequent infection and allowing an initial round of viral replication or feedback of TLR4-induced cytokines, which may promote virus replication.

Further studies are required however in order to determine if shed GP receptors correspond to some of the attachment factors already described for EBOV i.e.

C-type lectin membrane associated receptors, such as DC-SIGN and L-SIGN, or other attachment factors on the cell surface as βintegrins (Takada, Watanabe et al. 2000[START_REF] Schornberg | Alpha5beta1-integrin controls ebolavirus entry by regulating endosomal cathepsins[END_REF], Axl [START_REF] Shimojima | Tyro3 familymediated cell entry of Ebola and Marburg viruses[END_REF][START_REF] Shimojima | The mechanism of Axlmediated Ebola virus infection[END_REF][START_REF] Kondratowicz | T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus[END_REF] or T-cell immunoglobulin mucin domain-1 (TIM-1) [START_REF] Kondratowicz | T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus[END_REF]. A previous study has shown that carbohydrate-specific signalling through DC-SIGN involves cooperation with TLR4 [START_REF] Gringhuis | Carbohydrate-specific signaling through the DC-SIGN signalosome tailors immunity to Mycobacterium tuberculosis, HIV-1 and Helicobacter pylori[END_REF]).

Even if our data suggests that TLR4 is the principal attachment receptor for shed GP, it does not exclude the fact that other attachment factors could participate in shed GP binding and that it is possible that a cross-talk exists Data concerning the role of MBL during virus infection is controversial. In the presence of complement, MBL has been shown to be able to neutralize VSV pseudotyped with Ebola virus glycoprotein (Ji, Olinger et al. 2005). Likewise, MBL has also been shown to have a protective effect during Ebola virus infection in vivo where mice whose recombinant MBL serum concentration was increased survived otherwise fatal Ebola virus infection and became immune to virus rechallenge [START_REF] Michelow | High-dose mannosebinding lectin therapy for Ebola virus infection[END_REF]. Paradoxically, another study has recently shown that, in the absence of complement, MBL enhances infection of Ebola virus and other N-glycosylated viruses such as West-Nile virion-like particles and to a lesser degree, Nipah and Hendra viruses [START_REF] Brudner | Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane C-type lectin receptors[END_REF]). However, it should be noted that this last study was done using considerably supraphysiological concentrations of recombinant MBL (at least 5 times higher than physiological levels). Another limitation was that lentiviral pseudotyped viruses were used for mechanistic experiments. Ebola virus length ranges from 600 to 1400 nm, and peak infectivity is associated with 805-nm particles [START_REF] Aleksandrowicz | Ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis[END_REF]; Therefore, pseudotyped systems have limitations because the morphology of the virions differs significantly from that of real Ebola virions (spherical and smaller for retrovirus or VSVpseudotyped virions versus filamentous for authentic Ebola virus [START_REF] Bhattacharyya | Ebola virus uses clathrin-mediated endocytosis as an entry pathway[END_REF]). Hence, for all of these reasons, the observed interactions between HIV-EBOV-GP and host attachment factors during this study may be to some extent artificial.

In summary, the first part of this study has produced several novel findings. First we have identified primary targets of EBOV soluble glycoproteins; second, we have observed that shed GP is able to induce pro-and anti-inflammatory cytokine synthesis and secretion in DCs and macrophages, principally through TLR4 receptor, besides inducing their phenotypic maturation. Comparison of these two primary targets simultaneously under the same experimental conditions was never done before. Second, we have demonstrated the importance of shed GP glycosylation on the immunogenicity of the protein and that its activity can be blocked completely by the presence of the sera lectin MBL. Third, we have shown that both shed GP but also cytokines released from shed GP treated DCs and macrophages induce the disruption of endothelial permeability. Altogether we provide information to suggest that soluble glycoprotein shed GP plays an important role during EBOV pathogenicity by contributing to the exacerbated activation of inflammation which besides the dysregulation of the immune response, increases endothelial permeability.

Shed GP impairs coagulation and permeability pathways

Mannose-binding lectin (MBL) is known to have intrinsic antimicrobial activity and to be also implicated in the initialisation of coagulation and complement systems [START_REF] Matsushita | Structural and functional overview of the lectin complement pathway: its molecular basis and physiological implication[END_REF]. Mannose-Binding lectin is a C-type lectin that recognizes hexose sugars including mannose, glucose, fucose, and N-acetylglucosamine on the surface of glycosylated pathogens. Several viruses, such as influenza virus, human immunodeficiency virus, herpes simplex virus type 2, severe acute respiratory syndrome coronovirus (SARS-CoV), Ebola virus, and Marburg virus have been shown to be recognized by MBL through Nlinked high-mannose carbohydrate structures on their respective glycoproteins (Hartshorn, Sastry et al. 1993[START_REF] Anders | Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virusinfected patients[END_REF][START_REF] Reading | A serum mannose-binding lectin mediates complement-dependent lysis of influenza virus-infected cells[END_REF], Saifuddin, Hart et al. 2000, Gadjeva, Paludan et al. 2004[START_REF] Michelow | High-dose mannosebinding lectin therapy for Ebola virus infection[END_REF]. This process involves attaching a high-mannose core to the amide nitrogen of asparagines followed by the adornment and the reorganisation of viral oligosaccharides in the endoplasmic reticulum and Golgi apparatus (Breitling and Aebi 2013). Importantly, surface glycoprotein structures are not only critical for virus stability, cell tropism, immune evasion and host cell invasion but they are characteristic of a broad group of viruses in which N-linked glycosylation contributes to viral virulence [START_REF] Vigerust | Virus glycosylation: role in virulence and immune interactions[END_REF]. Ebola virus surface glycoprotein is heavily glysosylated and, like Marburg virus, contains both N-linked and O-linked carbohydrate structures [START_REF] Feldmann | Glycosylation and oligomerization of the spike protein of Marburg virus[END_REF][START_REF] Geyer | Carbohydrate structure of Marburg virus glycoprotein[END_REF], Feldmann, Nichol et al. 1994). Since MBL has been shown to bind to both Ebola virus and Marburg virus glycoproteins (Ji, Olinger et al. 2005), due to the structural similarity between EBOV surface glycoprotein GP and soluble glycoprotein shed GP released during EBOV infection, it is easy to imagine that surface GP and shed GP might share some functions. Our results show that, as has been demonstrated with EBOV surface glycoprotein (Ji, Olinger et al. 2005), shed GP is able to specifically bind MBL and, importantly, the trimer structure of the protein is essential for this interaction. Moreover, we have shown that binding of shed GP to MBL activates at least two MBL-associated serine proteases (MASPs): MASP-1 and MASP-2, which have previously been shown to be implicated in coagulation and complement processes respectively [START_REF] Matsushita | Structural and functional overview of the lectin complement pathway: its molecular basis and physiological implication[END_REF]. Our data significantly (Takahashi 2011, Takahashi, Chang et al. 2011). Several clinical studies have associated MBL deficiency with increased susceptibility to infection [START_REF] Sumiya | Molecular basis of opsonic defect in immunodeficient children[END_REF], Gadjeva, Paludan et al. 2004[START_REF] Shi | Mannose-binding lectin-deficient mice are susceptible to infection with Staphylococcus aureus[END_REF], Moller-Kristensen, Ip et al. 2006[START_REF] Takahashi | The mannose-binding lectin: a prototypic pattern recognition molecule[END_REF]. Importantly, we have also shown that the degree of activation of MASP-1 by shed GP is dependent on the amount of MBL in sera of patients and therefore, that a correlation exists between MBL content in blood and its activation, meaning coagulation activation, by EBOV shed GP. It is interesting to speculate that these differences in MBL levels in blood could explain the wide range of individual susceptibility and severity in the symptoms observed in EBOV infected patients.

Interestingly, it has been observed that during DIC and organ failure, both phenomena observed during EBOV infection, there is a deficiency in MBL (Takahashi, Chang et al. 2011). Since the liver is the primary organ that synthesizes MBL (Uemura, Saka et al. 2002), and it is damaged during EBOV infection, this may have repercussions on MBL production. Damage to the liver, combined with massive viremia, leads to disseminated intravascular coagulopathy (DIC), further organ failure and systemic inflammation. We hypothesize that MBL ultimately does not neutralize Ebola virus during infection because MBL is depleted when it binds to shed GP; and also, that liver injury and impairement observed during EBOV infection may reduce MBL synthesis at later stages of infection. Moreover, the fact that MBL participates in coagulation processes, may, on one side, contribute to generate DIC syndrome and therefore thrombi and organ failure and, on the other side, this implication of MBL may reduce its availability for other processes, resulting in decrease of its opsonophagocytic activity and thus contributing to viral egress.

Recently a study has shown that murine MBL has an anti-inflammatory function by interfering with the interaction between LPS and TLR4 on mast cells [START_REF] Ma | Mouse mannose-binding lectin-A and ficolin-A inhibit lipopolysaccharide-mediated pro-inflammatory responses on mast cells[END_REF]. In agreement with this, our work supports further testing of supraphysiologic MBL therapy against Ebola virus infection which could be a potential therapeutic agent against Ebola virus and may offer a novel antiviral approach. However, since MBL is activated by shed GP, treatment should be done with recombinant MBL, or synthetic MBL derivatives, not containing MASP-1 in order to avoid the impairment of coagulation. Although MASP-2 should be included in order to keep the opsophagocytic functions of MBL. A treatment with MBL combined with anti-TLR4 antibodies and anti-coagulation modulators would prevent an exacerbated release of pro-and anti-inflammatory cytokines that could allow an early and well-regulated inflammatory response, which would prepare the host to control viral replication and coagulation impairment and induce specific immunity.

It has been shown that endothelial cell function is impaired during Ebola virus infection in humans (Zaki and Goldsmith 1999) and non-human primate models [START_REF] Ryabchikova | Animal pathology of filoviral infections[END_REF][START_REF] Ryabchikova | Animal pathology of filoviral infections[END_REF].

Although endothelial cells are considered secondary targets, vascular dysregulation and instability are thought to be crucial symptoms during Ebola virus infection. However, proinflammatory cytokines such as TNFα activate the endothelium, increasing expression of cell adhesion molecules and affecting endothelial barrier integrity [START_REF] Royall | Tumor necrosis factor and interleukin 1 alpha increase vascular endothelial permeability[END_REF]. In vitro studies have demonstrated that Ebola virus-like particles expressing the transmembrane glycoprotein GP1,2 at the surface were able to activate endothelial cells and decrease the barrier function. In contrast sGP seem to have a protective role in endothelial barrier integrity when cells were treated with TNF-α (Wahl-Jensen, Afanasieva et al. 2005). Our data shows that shed GP itself but also supernatants resulting from shed GP activation of macrophages are able to increased vascular permeability. Endothelial cells are susceptible EBOV targets in vitro (Yang, Duckers et al. 2000, Wahl-Jensen, Afanasieva et al. 2005), but are considered to be rather late virus targets in vivo (Geisbert, Young et al. 2003, Hensley andGeisbert 2005) suggesting an indirect effect of EBOV infection on these cells, but suggesting a preponderant role for shed GP. These results also demonstrate that cross-talk between inflammation and endothelial permeability may direct immune responses in the vascular endothelium to recruit and activate immune cells.

It has been suggested that there is a relationship between the procoagulant and permeability pathways; in fact, endothelial cell (EC) expression of TF and extrinsic clotting factors are critical in augmenting capillarity leakage (Friedl, Puhlmann et al. 2002). It has been shown that treatment of EC with anti-TF antibodies blocks permeability , thus implicating an essential role for EC surface expression of TF and the extrinsic clotting cascade in increasing capillarity leakage (Friedl, Puhlmann et al. 2002). The importance of TF in triggering the coagulation abnormalities that typify EBOV infection is supported by a study where rhesus monkeys treated with nematode anticoagulant protein c2 (rNAPc2), a potent inhibitor of tissue factor-initiated blood coagulation, significantly improved survival. Moreover, survival was associated with reduced activation of coagulation and proinflammatory response (Geisbert, Hensley et al. 2003). Our data suggests that shed GP is not only involved in the coagulation cascade, but would also play an important role in endothelial permeability. We have been able to detect increased pro-coagulant and propermeability marker tissue factor on the surface of HUVEC cells but also on the surface of immune cells such as monocytes and macrophages, data that correlates with previous studies that showed that EBOV infected monocytes/macrophages can trigger endothelial leakage [START_REF] Feldmann | Filovirus-induced endothelial leakage triggered by infected monocytes/macrophages[END_REF].

We have previously seen that soluble glycoproteins are able to induce cytokine production. In this study we also showed that soluble glycoproteins can also be involved in the last steps of leukocyte-endothelial adhesion. Soluble glycoprotein shed GP is able to induce the surface expression of ICAM and VCAM on the surface of endothelial cells, thus contributing to high-affinity adhesive interaction between leukocytes and endothelium but also inducing this phenomena in further cells throughout the bloodstream.

Importantly, we have also been able to show that pro-permeability markers such as ICAM-1, VCAM-1 and E-selectin are also released in a soluble form. The release of adhesion molecules in a soluble form may simply arise as a consequence of inflammation or may have immune modulating properties by having inhibitory effects on neutrophil and monocyte extravasation. The soluble character of these molecules allows them to disseminate easily by traveling through the bloodstream, increasing their potential cellular targets and affecting permeability integrity. We have also studied the presence of pro-coagluant markers released from HUVEC in a soluble form, including tissue factor, also implicated in the permeability process as explained above. There are two forms of soluble TF: one anchored to membrane of microvesicles released from cells expressing TF on the surface (active form), and soluble TF that is not associated to membranes and generated by alternative splicing, asTF (inactive form). Soluble tissue factor (asTF) was thought to have a procoagulant role; however, it has recently been shown that the procoagulant activity of asTF is very low (Bogdanov, Balasubramanian et al. 2003, Eisenreich and[START_REF] Eisenreich | Regulation and differential role of the tissue factor isoforms in cardiovascular biology[END_REF] and membrane anchored TF -on cells or on vesicles -, rather than asTF, was the main source of procoagulant TF activity [START_REF] Yu | Shedding of tissue factor (TF)-containing microparticles rather than alternatively spliced TF is the main source of TF activity released from human cancer cells[END_REF], whereas asTF has recently been linked more closely to proangiogenic processes and cell proliferation (van den Berg, van den Hengel et al. 2009[START_REF] Eisenreich | Regulation of pro-angiogenic tissue factor expression in hypoxiainduced human lung cancer cells[END_REF]. We have observed that shed GP also contributes to the induction of coagulation through sTF. Surprisingly, the soluble form of thrombomodulin, a protein expressed on the surface of endothelial cells and that has an anticoagulant role, was not shown to be affected by shed GP or other supernatants compared to TNFα, which as expected, decreased its release into the medium.

surfaces but is also released in a soluble form into the medium and (iii) shed GP seems to play an important role in the induction of adhesion molecule expression on the surface of human endothelial cells thus potentially contributing to the recruitment of leukocytes as possible new cellular targets.

Therefore, even if shed GP is not the single factor affecting coagulopathy and vascular leakage, it seems reasonable to speculate that it could contribute in an important way to their impairment.

We have also determined another important role for shed GP. Current results suggest that shed GP, due to its almost identical structure and glycosylation pattern compared to viral surface GP, is capable to bind to similar receptors and help the virus to not re-infect the same cells, or neighbouring cells in close proximity, and thus spread to cells further afield. We thus hypothesize that shed GP is involved in the establishment of viremia. Our results provide a key for shed GP shares receptors with surface GP present on virions, there is a competition established between them that will prevent some virus particles from binding to neighboring cells, which already have some of the receptors coupled to shed GP. This will contribute to virus spreading and dissemination into the bloodstream thus targeting more distant cells.

Shed GP as a key factor in EBOV pathogenicity

Ebola virus GP has been very studied due to its importance in viral entry and its potential as a target for vaccine development. Moreover, it has been shown to have a very important role in Ebola virus pathogenesis. First of all, surface GP is responsible for targeting cells that are important for EBOV pathogenesis, affecting them in several ways. It is capable not only to lead to immunosupression by downregulating important molecules on cell surfaces which are important for lymphocyte binding and antigen presentation [START_REF] Simmons | Ebola virus glycoproteins induce global surface protein downmodulation and loss of cell adherence[END_REF][START_REF] Sullivan | Ebola virus glycoprotein toxicity is mediated by a dynamin-dependent protein-trafficking pathway[END_REF], Reynard, Borowiak et al. 2009), but surface GP has also been shown to inflict direct cytotoxicity in vascular cells by causing cell rounding and detachment, followed by cell death (Yang, Duckers et al. 2000). Other cell lines, such as 293T and macrophages have shown to be affected by this phenomenon [START_REF] Simmons | Ebola virus glycoproteins induce global surface protein downmodulation and loss of cell adherence[END_REF], Alazard-Dany, Volchkova et al. 2006). These effects are mediated by the mucin-domain of GP, which is heavily glycosylated. However, it is important to take into account that infection can be influenced by multiple mechanisms such as pattern recognition, cytokine production and other factors such as adherence to host cells and growth rate and that the different glycosylation patterns observed in the different strains of EBOV could imply a difference in pathogenicity. One study has shown that GP is a major pathogenicity factor (Groseth, Marzi et al. 2012), as when they exchanged surface glycoproteins of Zaire EBOV and Reston EBOV (more and less pathogenic strains respectively) they observed that recombinant RestonGP-Zaire EBOV was less lethal for mice when compared to wild type Zaire EBOV. However, ZaireGP-Reston EBOV did not render the virus more lethal when compared with wild type Reston EBOV meaning that GP, even if it does contribute to filovirus virulence, is not the only pathogenic factor of the virus and other viral elements are needed for the acquisition of full virulence.

Our data demonstrates several common characteristics shared between surface GP and shed GP. Even if, differently from surface GP, shed GP does not produce cell rounding and death, it is able to target similar cells to surface GP including monocytes, macrophages, DCs and endothelial cells and activate them to produce pro-and antiinflammantory cytokines and vascular markers of endothelial permeability. This indicates that in vivo shed GP must surely contribute to EBOV pathogenicity. It is important to stress that shed GP, differently from surface GP, would target cells that are still non-infected. This is a very important phenomenon because (i) it allows the virus to affect the immune system without the need for infection, (ii) it allows the recruitment of new possible targets such as neutrophiles, monocytes, DCs... and (iii) that shed GP can easily spread through the bloodstream reaching farther and different targets (other immune cells in other places in the body, other organs such as kidney, liver...). Therefore, shed GP probably triggers inflammation processes that on one side allow the recruitment of new possible targets and on the other side induces migration of already infected cells to new areas, spreading infection.

Activation of the innate immune system is initiated by soluble pattern recognition molecules, which are expressed either bound to the extracellular matrix on innate immune cells or circulating in the blood as soluble molecules.

One such soluble pattern recognition molecule is MBL, which is mostly (>95%) synthesized in the liver and secreted to circulate in the blood (Uemura, Saka et al. 2002[START_REF] Takahashi | The mannose-binding lectin: a prototypic pattern recognition molecule[END_REF]). The innate immune response generates signals to activate appropriate effector molecules and cells of the adaptive immune system. MBL, as a pattern recognition molecule of the innate immune system, provides a surveillance system both locally and systemically. In this manner MBL can contribute both to immunity from pathogens as well as maintenance of tissue integrity and homeostasis. It has been recognized that there is significant interaction among the regulatory and effector systems of inflammation, complement activation, coagulation and vascular permeability [START_REF] Dugina | Receptors of the PAR family as a link between blood coagulation and inflammation[END_REF], Levi, Keller et al. 2003, Takahashi 2011) Since shed GP seems to contribute to the virulence of ZEBOV, its administration could result in an effective therapeutic strategy that may induce an appropriate innate and adaptative immune response before infection. A proper inflammatory response combined with the production of antibodies against EBOV may result in a prophylactic treatment of EBOV infection.

However, since it is also able to activate endothelial leakage and to impair the coagulation pathway, its production and administration should still be studied and optimized in the guinea pig model that, after the NHP model, most accurately reflects coagulation impairment during EBOV infection.

Beyond shed GP's application as a treatment at controlled levels and prior to contact with the virus, the immunogenicity of shed GP in human DCs and macrophages described herein also points toward its role during Ebola virus high pathogenicity and also provides a better understanding about the mechanism responsible for the interaction with different components of the host immune system during Ebola virus infection.

Material and methods

Cell lines and plasmids

Both We have previously demonstrated that the substitutions L635V and D637V

dramatically alter the efficacy of EBOV GP shedding in a transient expression system (Dolnik, Volchkova et al. 2004). In this study to increase the efficiency of GP shedding in a transient expression system we introduced the substitutions D637A and Q638V using site-directed mutagenesis of a plasmid encoding wildtype GP. The resulting plasmid was designated phCMVGP-HS. Plasmid expressing GP with a substitution L635V (phCMVGP-LS) was described earlier (Dolnik, Volchkova et al. 2004). Plasmid expressing transmembrane-anchor truncated mutant GP ΔTM was generated by introducing a stop codon at position 651 using site-directed mutagenesis of a plasmid encoding wild-type GP.

Viruses

Recombinant EBOV-HS (with mutation D637L) and EBOV-LS (with mutation L635V) and EBOV-WT (with no mutations) were used in order to produce supernatants containing respectively high, low and wild type quantities of shed GP in supernatants. For production, Vero E6 were grown for 24h to a confluence of ~40% in 150 cm 2 flasks; cells were then infected with 1:500 dilution of these recombinant viruses and after 5 days in culture, supernatants were collected, centrifugated 10 minutes at 1000 rpm and then ultracentrifuged with a 20% sucrose cushion at 25000 rpm for 50 minutes at 4°C using SW32

rotor. The clarified supernatants were aliquoted and stored at -80°C. Viruses were then titrated using TCID 50 technique.

Transient expression of EBOV GP

293T cells were grown for 24h to a confluence of ~60%. Transfection of cells with recombinant plasmids was performed using TurboFect reagent (Thermo).

Briefly, 20 μg of respective plasmids (HS, LS, GP ΔTM, sGP and GFP) were added to 500 μl of DMEM with no serum in a ratio 1:4 (plasmid:turbofect); the mixture was incubated for 20 minutes at room temperature and added to 150 cm 2 flasks containing 60% confluent 293T cells in 15 ml of VP-SFM medium (Gibco). Three hours later 25 ml of VP-SFM were added in each flask. During GP expression the cells were kept in VP-SFM medium containing no serum.

Culture supernatants were harvested 36h post-transfection, clarified by centrifugation at 1000 rpm for 10 min at 4°C and then by centrifugation at 28,000 rpm at 4°C for 2 h using a SW28 rotor with a Beckman Optima L-70K ultracentrifuge. The clarified supernatants were concentrated using

Concentrator tubes (Pierce) with a molecular weight cut off of 20,000 MWCO.

All samples were aliquoted and stored at -80°C.

Culture of primary human monocyte-derived-DCs and macrophages

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by centrifugation onto a lymphocyte separation medium (Ficoll) cushion; briefly, 16 Leucosep tubes (Dutscher) were filled with 15ml of Ficoll and centrifuged at 1700 rpm for 1 minute. Then 31 ml of blood were added and Leucosep tubes were centrifuged for 30 minutes at 1700rpm with no acceleration nor brakes. PBMC rings where then collected in RPMI-1640 (Life Technologies) supplemented with 200 mM L-glutamine (Life Technologies), 10 mM HEPES buffer (Life Technologies), 10% de-complemented FCS and 8 μg/ml of Gentamicine (Life Technologies) (complete RPMI). Tubes containing PBMCs in complete RPMI were washed by centrifugation three times at 1600 rpm for ten minutes. After each centrifugation supernatant was replaced by 40 ml of fresh complete RPMI medium. Then PBMCs were enriched by centrifugation through a Percoll density gradient. Briefly, 22,5 ml of Percoll, 2,5 ml of PBS 10x and 25ml of PBS 1x with CaCl2 were mixed and 6ml were deposited in 8 x 15ml tubes. Carefully, 3 ml containing 3x10 7 cells/ml were loaded on the 6ml of Percoll. Tubes were then centrifuged for 20 minutes at 2000rpm with no acceleration nor brakes. The recovered monocyte fraction, contained not in the pellet but in the ring between medium and percoll, was washed three times as explained before (1600rpm, 10 minutes) and were depleted of cell contaminants by positive selection (Miltenyi Biotec), ensuring purification rates of ≥95%. Briefly, the cell pellets recovered after washing centrifugations were resuspended in 80μl of Equilibration Buffer (0,5% Bovine Serum Albumin, 2mM EDTA and PBS) and 20μl of CD14 MicroBeads (Miltenyi Biotec) per 10 7 total cells. Mixture was incubated at 4°C for 15 minutes and then was centrifuged at 300xg for 10 minutes and resuspended in 3ml of Equilibrium Buffer. Columns with additional filters (to remove cell clumps) were then placed on the magnetic field and were rinsed with 3ml of Equilibrium buffer. Cell suspension was then applied to the column. Unlabeled cells were collected and three washings steps were performed by adding 3ml of Equilibrium buffer.

Columns were then removed and 5ml of Equilibrium Buffer were added and placed on a collection tube. Magnetically labelled cells were flushed out by pushing the plunger into the column and the eluted fraction was resuspended in complete RPMI. Cells were then washed three times as before (1600rpm, 10 minutes) and resuspended in RPMI-1640. Immature DCs and monocyte-derived macrophages were obtained upon culturing of cells in complete RPMI-1640 for 5 days either with 40 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) or 50 ng/ml of interleukin-4 (IL-4) and 40 ng/ml of macrophage colony-stimulating factor (M-CSF), respectively.

Endotoxin test

The Limulus amebocyte lysate test (LAL Chromogenic Endotoxin Quantitation Kit, Pierce) was used to determine endotoxin levels in culture supernatants used in this study. The Endotoxin Standard vial containing 26EU of lyophilized endotoxin was reconstituted by adding 1mL of room temperature endotoxin-free water to make Endotoxin Standard Stock. Then, from this initial tube, standard stock solutions were made at 1, 0,5, 0,25 and 0,1 EU/ml. Then the microplate was warmed for 10 minutes at 37ºC. With the microplate maintained at 37ºC, 50 μl of each standard or unknown sample replicate were added and incubated for 5 minutes at 37ºC. Then, 50 μl of LAL were added to each well, gently shaken and incubated at 37ºC for 10 minutes. After exactly 10 minutes, 100 μl of substrate solution was added to each well and incubated for 6 minutes at 37ºC.

After that, 50 μl of Stop Reagent (25% acetic acid) were added, maintaining the same pipetting order as at the beginning. The absorbance was then measured at 405-410nm on Tecan 200. All values were less or equal to those of VP-SFM medium (<0.25 endotoxin units per ml).

Sedimentation analysis

The oligomeric structures of EBOV shed GP and transmembrane-anchor truncated mutant GP 1,2Δ were compared using a sedimentation assay. 293T In all assays except deglycosylation experiments, shed GP was used at a final concentration of 2 µg/ml. In deglycosylation experiments shed GP was used at a concentration of 0.5 µg/ml.

Real time RT-PCR quantitative analysis

RNAs from monocyte-derived DCs and macrophages treated with EBOV GPs were isolated using Total RNA Isolation kit (Machery Nagel). Briefly, 300 µl of RLT buffer were used to lyse cells contained in each well (5x10 5 cells) and purified RNA was eluted in 100 µl of water. Then cDNAs were generated using

Cytokine release

Culture supernatants from monocyte-derived DCs or macrophages treated with 2 µg of soluble EBOV GPs for 24 hours were assayed for the presence of TNFα, IL-6, IL-8, IL12p40, IL1β, IL10 and IL1-RA using Multiplex cytometric bead array (Bio-Rad) in Luminex MAGPIX. The assay was performed according to the manufacturer's instructions. First, each lyophilized cytokine standard (1 and 2) were reconstituted by adding 500 µl of RPMI not containing serum, then were vortexed for 5 seconds and incubated on ice for 30 minutes. During this time, frozen samples at -80ºC were slowly thawed in ice. After 30 minutes of incubation, eight serial dilutions (S1-S8) were made by adding 72 µl of standard 1 and 72 µl of standard 2 in S1 and then, four-fold serial dilution were made from S2 to S8 by adding 50 µl in 150 µl of RPMI with no serum in each tube.

Magnetic beads specific for each cytokine were then vortexed and 575 µl of each (TNF-α, IL-6, IL-8, IL12p40, IL1β, IL10 and IL1-RA) were added to 1725 µl of assay buffer (provided with the kit). Then, 50 µl of beads were added per well and 50 µl samples and standards were also added in triplicate. Incubation of the plate, previously covered with sealing tape and aluminium, was carried out in the dark on a shaker at 300 rpm for 1 hour at room temperature. Then wells were washed with 100 µl of washing buffer (provided in the kit) using a magnet to keep beads attached during washing and 300 µl of each specific antibody against beads were added to 900 µl of Bio-Plex detection antibody diluent (provided in the kit). Incubation was done as before: shaking, in the dark for 1 hour. Finally, after washing with 100 µl of washing buffer, 60 µl of streptavidin-PE (provided in the kit) were added to 5940 µl of assay buffer and 50 µl of this mixture was added per well and incubated for 10 minutes at room temperature.

Then wells were washed with 100 µl of washing buffer and add 125 µl of assay buffer per well. Plate was then read using Luminex MAGPIX.

Expression of co-stimulatory molecules

Both monocyte-derived DCs and macrophages treated with 2 µg soluble EBOV GPs for 48 hours were placed in conic-bottom 96-well plates and plates were centrifuged at 2000 rpm for 2 min. Cells were then washed in cold PBS supplemented with 2% fetal bovine serum and pre-blocked at 4°C for 30 min in FACS buffer (PBS containing 2% FBS, 2% normal rat serum, 2% normal hamster serum, 2% normal mouse serum and 2% human FcR blocking solution, Milteny Biotec). Cells were then washed with PBS and cell-surface staining was performed using 1 µl of the following antibodies: FITC-conjugated anti-CD80, PE-conjugated anti-CD40 (Beckman Coulter) and APC-conjugated anti-CD83, Peridinin Chlorophyll Protein Complex Vio 700 (PerCP-Vio700)-conjugated anti-CD86 (Miltenyi Biotec). After incubation with antibodies for 1 hour at 4°C, cells were washed three times with PBS, fixed with 1% PFA solution and analyzed using a Becton Dickinson LSRII flow cytometer (BD) and FlowJo software. A minimum of 10,000 events were collected and analyzed for each sample.

Deglycosylation assay

For GP deglycosylation, supernatants containing EBOV GPs were treated for 48 hours using EDEGLY kit (Sigma) containing PNGase F, α-( 2

EBOV replication in human monocytes

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors as explained before. After 24 hours, supernatant was removed by centrifugation at 1500 for 5 minutes. Then, 400 µl of supernatants from recombinant viruses EBOV-HS, EBOV-LS and EBOV-WT were added together with 600 µl of RPMI medium with no serum for 2 hours at 37ºC. After that, 1 ml containing of RPMI 5% of FBS was added per well (to a final concentration of 2'5% of serum). Afetr 48 hours at 37ºC, monocytes were centrifuged three times at 1500 rpm for 5 minutes and supernatants were removed and replaced by RPMI with no serum. Monocytes were then infected with recombinant EBOV expressing GFP (rEBOV-GFP) at a MOI of 1 for 2 hours at 37ºC. Monocytes were then washed and incubated with RPMI medium containing 2,5% of serum for 48 hours at 37ºC and fluorescence was then analyzed by flow cytometry.

Surface Plasmon Resonance/Fluid-phase interaction between MBL and

Ebola soluble glycoproteins

Real time surface plasmon resonance spectroscopy was performed using a Biacore T100 biosensor. Briefly, recombinant MBL, kindly provided by Jens Christian Jensenius, was covalently bound to CM5 biosensorchip as ligand according to the manufacturer's instructions. Briefly, the sensorchip was activated by injection of 80 μl of NHS/EDC (Amine Coupling Kit, GE Healthcare) at 10μl/min for 7 minutes. Then, 200 μg/ml of rhMBL in 120 μl of Acetate then washed three times as before and were developed using 100 µl of TMB according to manufacturer's instructions; reaction was stopped with 100 µl of 10% phosphoric acid in water and absorbance was read at 405 nm.

Activation of MASP-1

Flat-bottomed maxisorp polystyrene tissue culture plates (Nunc) were coated in triplicate for 1 hour at room temperature with 2ug per well of α-GP antibody in TBS supplemented with 10mM CaCl 2 . Wells were washed with 40mM HEPES, 2M NaCl, 10mM CaCl 2 pH 7,4 (buffer 1) and incubated by shaking overnight at 4°C with 100 μl of supernatants and mannan (1 mg/ml) in mock supernatant containing 5mM CaCl 2 . Mannan was used as a representative ligand of MBL.

Wells were washed with buffer 1 three times. Human serum containing 30 μg/ml of recombinant MBL was 1:1 diluted in buffer 1 and 100 μl were added in the coated plate for 1 hour in ice. After consecutive washing twice with 20 mM HEPES, 1 M NaCl, 5mM CaCl2, 0,1% Tween-20 pH 7,4 (buffer 2) and three times with 20 mM HEPES, 140mM NaCl, 5mM CaCl2, 0,1% Tween-20 pH 7,4

(buffer 3), 100 μl of 0,2mM VPR-AMC (R&D systems) in 20 mM HEPES, 5mM

CaCl2 pH 8,5 (buffer 4) was added. The samples were excited at 355nm and emission read at 460nm every 60s for 1h using a TECAN 200 plate reader.

Experiments in BSL-4 conditions were done similarly but soluble glycoproteins were obtained from recombinant Ebola virus infected cells where virus had been clarified by ultracentrifugation as explained above. Supernatants came from EBOV-HS, EBOV-LS and EBOV-WT viruses.

The competition assay was done similarly but samples containing 2 µg of soluble glycoproteins were previously incubated with human serum containing 150 µg of MBL.

Activation of MASP-2/measurement of lectin complement pathway activity

MBL-bound-MASP-2 activity was evaluated by C4 fixation assay. Flat-bottomed maxisorp polystyrene tissue culture plates (Nunc) were coated in triplicate overnight at 4°C with 200 ul of supernatants and mannan (1 mg/ml) in mock supernatant containing 5mM CaCl 2 . MBL-MASP complexes in buffer 1 were bound to mannan-coated wells as for MASP-1 assay above. Wells were then blocked with 20% gelatin in warm TBS buffer for 2 hours at 37°C and then were washed with TBS containing 0,05% Tween 20 and 10 mM CaCl2. Human serum containing 25 ug/ml of MBL was 1:1 diluted in buffer 3 and incubated for 1 hour at 37°C. Following two washes with buffer 2 and three with buffer 3, 0,1 μg/well of purified C4 (Hycult biotech) in buffer 3 was added to each well and incubated for 1 hour at 37°C. Wells were then washed with buffer 3 and goat α-C4 (Abcam) was added to 3μg/ml in buffer 3 for 1 hour at 37°C. After washing three times with buffer 3, secondary antibody α-goat HRP was added to 1:2000 in buffer 3 and incubated 30 minutes at 37°C. Wells were then washed three times with buffer 3 and developed using TMB according to manufacturer's instructions; reaction was stopped with 10% phosphoric acid in water and absorbance was read at 405 nm. Experiments in BSL-4 conditions were done similarly but soluble glycoproteins were obtained from Ebola virus recombinant infected cells where virus had been clarified by ultracentrifugation as explained before. Supernatants came from EBOV-HS, EBOV-LS and EBOV-WT viruses.

Competition assay was done similarly but samples containing 2 µg of soluble glycoproteins were previously incubated with human serum containing 150 µg of MBL.

Induction and detection of Tissue factor in monocytes, macrophages, DCs and HUVEC

Dendritic cells, macrophages and HUVEC were plated at 1x10 6 cells/ml in 6well plates. Medium in cells was removed and 600 μl of RPMI (for immune cells)

or 600 μl of EGM-2 medium (for HUVEC) with no serum but supplemented as explained before was added. 400 μl of soluble glycoproteins (shed GP, sGP, Low shedding, shed GP+MBL) and positive control TNFα (20ng/ml) was added per well and incubated for 24 hours. Cells were then trypsinized, centrifuged at 1200 rpm and resuspended in PBS in conic-bottom 96-well plates. Cells were then antibody blocked at 4°C for 30 min in FACS buffer (PBS containing 2% FBS, 2% normal rat serum, 2% normal hamster serum, 2% normal mouse serum and 2% human FcR blocking solution (Milteny Biotec). All mAbs were purchased from BD Pharmingen except where noted. Cells were then incubated with α-tissue factor for 1 hour at 4°C, washed twice with PBS and incubated with Alexa 488 for 30min at 4°C. After incubation with antibodies, cells were washed and fixed with 1% PFA solution. Cells were analyzed using a Becton

Dickinson LSRII flow cytometer (BD) and FlowJo software.

Detection of soluble coagulation and permeability marker release in culture medium

Supernatants containing soluble glycoproteins and their respective controls were assayed in MAGPIX in order to determine soluble levels of tissue factor, thrombomodulin, ICAM-1, VCAM-1 and E-selectin using Multiplex cytometric bead array Milliplex kits Human Cardiovascular Disease Panel 2 and 4 (Millipore). Assay was performed according to the manufacturer's instructions. Assay Buffer, 100 μl, was added into each well of the plate and incubated at room temperature for 10 minutes. After its removal, 25 μl of Assay Buffer and 25 μl of culture medium (EGM-2) were added per well; then, 25 μl of standards, Controls, background and samples were added into the appropriate wells.

Twenty-five microliters of sonicated Mixed Beads were added to each well and incubated on a plate shaker overnight at 4°C. The content of the plate was then removed and wells were washed 3 times with wash buffer, using the magnet so not to lose beads during washing. Next, 50 μl of Detection Antibodies (provided in the kit) were added to each well and incubated with agitation on a plate shaker for 1 hour at room temperature (20-25°C). Without washing, 50 μl

Streptavidin-Phycoerythrin were added to each well containing the 50 μl of Detection Antibodies and the plate was incubated for 30 minutes at room temperature. Well contents were then removed, washed as explained before and 100 μl of Drive Fluid was added to all wells. Beads were resuspended on a plate shaker for 5 minutes and the plate was run on a MAGPIX machine.

Plaque size assay

Confluent Vero cells in 12 well plates were incubated with supernatants containing HS, sGP and LS from transiently transfected cells for 1 hour at 37ºC. Cells were then washed three times with room temperature PBS containing Ca +2 and Mg +2 and infected with recombinant VSV pseudotyped with Ebola virus GP (rVSV-GP) or wild-type VSV for 1 hour at 37ºC. Cells were then washed three times with room temperature PBS containing Ca +2 and Mg +2 and medium was replaced by 3 mililiters of 2,5% medium containing soluble glycoproteins and avicell (final concentration 0,6%). Plaque size was analysed by counting plaques pixels with AxioVision software.

Binding and replication of rVSV GP-R18 in the presence of shed GP

VSV-containing cell culture supernatants were stained with 1 μg/ml R18 for 1 h on a shaker at RT and then centrifuged (10 min, 3000×g at 4 °C). Supernatants were diluted in up to 3 ml of PBS containing Ca +2 and Mg +2 , loaded onto a 1 ml 20% sucrose cushion and ultra-centrifuged for 1 h at 50000 rpm at 6 °C (Himac CS 150GX ultra-centrifuge with an S80473 rotor). Pellets were washed with PBS containing Ca +2 and Mg +2 and re-ultracentrifuged. Pellets were finally resuspended in 200 μL PBS containing Ca +2 and Mg +2 . Vero cells were treated with HS or LS supernatant from transiently transfected cells for 1 hour at 4ºC, washed with PBS Ca Mg three times and R18-labeled rVSV GP were added and binding was determined by flow cytometry.

For time course studies of rVSV-GP replication in the presence of HS or LS and mock as controls, Vero cells were incubated with HS, LS and mock (from GFP expressing cells) supernatants as explained before, and were then infected with rVSV-GP. Supernatants and cells were collected at 4, 8 and 12 hours post rVSV-GP infection and the presence of M protein of the virus was analysed by Western blot.

In BSL-4 conditions, Vero cells were incubated with mock supernatant or Geisbert (2007). "Recombinant human activated protein C for the postexposure treatment of Ebola hemorrhagic fever." J Infect Dis 196 Suppl 2: S390-399.

 Hoenen, T., A. [START_REF] Groseth | The Ebola virus glycoprotein contributes to but is not sufficient for virulence in vivo[END_REF]  White, J. M., S. E. Delos, M. Brecher and K. [START_REF] Dube | Cell adhesion promotes Ebola virus envelope glycoprotein-mediated binding and infection[END_REF].

"

Glycosylation in EBOV

Glycosylation process

Glycosylated proteins (glycoproteins) are found in almost all living organisms including eukaryotes, eubacteria and archae.

Approximately half of all proteins typically expressed in a cell undergo glycosylated, which entails the covalent addition of sugar moieties to specific amino acids. Lipids and proteoglycans can also be glycosylated. Glycosylation takes place in the endoplasmic reticulum (ER) and Golgi apparatus [START_REF] Wormald | Conformational studies of oligosaccharides and glycopeptides: complementarity of NMR, X-ray crystallography, and molecular modelling[END_REF].

Protein glycosylation has multiple functions in the cell. In the ER, glycosylation is used to monitor the status of protein folding, acting as a quality control mechanism to ensure that only properly folded proteins are trafficked to the Golgi. Sugars on soluble proteins can be bound by specific receptors in the trans Golgi network to facilitate their delivery to the correct destination. These sugars can also act as ligands for receptors on the cell surface to mediate cell attachment or stimulate signal transduction pathways. Because they can be very large and bulky, oligosaccharides can affect protein-protein interactions by either facilitating or preventing proteins from binding to cognate interaction domains. Because they are hydrophilic, they can also alter the solubility of a protein [START_REF] Wormald | Conformational studies of oligosaccharides and glycopeptides: complementarity of NMR, X-ray crystallography, and molecular modelling[END_REF].

Glycoprotein diversity

Glycosylation

Glycosylation types in EBOV N-Glycosylation

Glycosylation is often characterized as a post-translational modification. While this is true with other types of glycosylation, N-glycosylation often occurs cotranslationally, in that the glycan is attached to the nascent protein as it is being translated and transported into the ER. The "N" in the name of this type of glycosylation denotes that the glycans are covalently bound to the carboxamido nitrogen on asparagine (Asn or N) residues.

Because the ER is the site of translation and processing of most membranebound and secreted proteins, it is not surprising that most of these are N-linked glycoproteins. Besides being the most common type of glycosylation (90% of glycoproteins are N-glycosylated), N-linked glycoproteins also have large and often extensively branched glycans that undergo multiple processing steps after being bound to proteins [START_REF] Ellgaard | Quality control in the endoplasmic reticulum[END_REF]Helenius 2003, Trombetta 2003).

O-Glycosylation

While Here we show that shed GP released from virus-infected cells binds and activates noninfected DCs and macrophages causing the massive release of pro-and anti-

Introduction

Ebola virus (EBOV) is the causative agent of a haemorrhagic fever in humans responsible for sporadic outbreaks in several African countries including the current epidemic in Guinea, Liberia and Sierra Leone [1,2] and for which there is currently no vaccine or approved treatment [3]. Fatal EBOV infection involves massive disseminated viral replication and host immune disregulation with uncontrolled cytokine secretion (for review, see [4]) and a disseminated intravascular coagulation syndrome resembling septic shock [5,6].

EBOV is a member of the Mononegavirales order Filoviridae family. Its genome consists of seven genes and encodes seven structural and at least one nonstructural protein [7][8][9]. Due to RNA editing phenomenon, transcription of the GP gene results in the synthesis of several GP gene specific mRNAs coding for viral glycoproteins including non-structural sGP and surface virion GP [10,11]. Both glycoproteins are synthesized as a precursor molecule that is proteolytically cleaved by the cellular protease furin during intracellular processing [12]. sGP forms dimers [10,13] whereas the cleaved carboxy-terminal fragment, termed delta peptide, is a monomer [14]. Viral surface spikes are formed as a trimer of GP 1,2 , made up of two subunits: GP 1 and GP 2 linked by a disulfide bond [15,16]. GP 1 is known to mediate virus attachment to host cells whereas GP 2 is involved in membrane fusion [11,17].

GP 1,2 is a type I glycoprotein containing multiple N-and O-linked glycans [7,18]. The majority of O-glycans are grouped in a region termed mucin-like domain [18][19][20].

A unique feature of EBOV is that following infection, virus encoded glycoproteins are released from cells in soluble forms. High amounts of sGP and truncated surface GP are detected in the blood of patients and experimentally infected animals [11,21]. Release of sGP is explained by its secretion via the classical secretory pathway due to the lack of a transmembrane anchor [11,22]. Cleavage of surface GP by the cellular metalloprotease TACE (TNFα-converting enzyme), a member of the ADAM (a disintegrin and metalloproteinase) proteinase family, is responsible for GP shedding [21]. In this manner soluble shed GP resembles virion GP 1,2 , and has been shown to bind and sequester virus-neutralizing antibodies directed against surface GP [21]. Aside from this antibody-neutralizing activity the role of shed GP in virus replication and pathogenicity has not yet been clearly defined.

Following EBOV infection, primary virus replication occurs in dendritic cells (DCs) and macrophages and is then followed by massive replication in hepatocytes and splenocytes [23][24][25][26]. It has recently been revealed that monocytes are also involved in virus replication albeit in an entry-delayed and differentiation-dependent fashion [27].

Massive release of cytokines, chemokines and vasoactive substances follows the course of infection thus promoting the development of inflammatory disorders and playing an important role in EBOV pathogenesis [24][25][26]. Overall, excessive inflammatory responses to infection contribute to massive infiltration of virus target cells to sites of infection but also result in the observed septic shock-like syndrome. Remarkably, little cytokine secretion is detected following EBOV infection of DCs which can be explained by their impaired maturation upon infection [26,[28][29][30]. Monocytes also appear to secrete inflammatory cytokines when infected, but at lower levels [27]. Based on in vitro data macrophages are the only cells that produce cytokines at relatively high levels following EBOV infection [26,28]. Using EBOV VLPs it has been demonstrated that cytokine and chemokine secretion from macrophages and DCs is likely to be dependent on GP [19,30,31] whereas the question remains as to how these host responses are triggered during infection.

In this study we identify the cellular targets for EBOV shed GP and answer the question if soluble EBOV glycoproteins could trigger the release of cytokines from noninfected immune cells. We show that shed GP but not sGP activates human-derived DCs and macrophages and induces the secretion of pro-and anti-inflammatory cytokines. We demonstrate that this activation can be abolished by anti-TLR4

antibodies or by addition of sera lectin MBL. Moreover, we show that the glycosylation pattern of shed GP is important for the activation of cytokine release. We also demonstrate the ability of shed GP to affect endothelial cell permeability both directly and indirectly through cytokine release. Our results indicate that shed GP plays an important role in viral pathogenicity.

Results and Discussion

Production and characterization of recombinant EBOV soluble glycoproteins.

During EBOV infection significant amounts of soluble glycoproteins, including shed GP and secreted sGP, are released from virus-infected cells [21]. The role that these proteins might play in virus replication and pathogenicity has not yet been clearly defined. In effect, EBOV shed GP is structurally identical to virion surface GP except for the lack of its carboxy-terminal part consisting of 13 amino acids upstream of the membrane anchor, the anchor itself and a short cytoplasmic tail [21,32]. Consequently, it was of interest to investigate whether shed GP and potentially sGP, that shares 295 amino-terminal amino acids with surface GP, can interact with and affect the function of DCs and macrophages that constitute an important and primary line of immune defense but that are also the major primary targets for viral infection in vivo. While transient expression of sGP from plasmid provides sufficient amounts of the protein secreted into the medium, the low efficiency of GP shedding seen with transient expression systems has hampered production of large enough quantities of this protein equivalent to those observed during EBOV infection. Of note, TACE, an enzyme responsible for GP shedding, is activated during EBOV infection [33] and the efficiency of shedding from virus-infected cells appears to be significantly higher. On the other hand the extent of glycosylation and post-translational modifications of EBOV GP prevents the use of more efficient expression systems such as bacteria or baculovirus. To surmount these difficulties we substituted two amino acids immediately surrounding the TACE cleavage site at positions D637A and Q638V and thus expected to increase efficiency of GP shedding as this newly generated sequence resembles that previously shown to be recognized by TACE for efficient release of TNFα [34]. The plasmid expressing this mutant was designated phCMVGP-HS and GP expressed from this plasmid as GP-HS (high shedding). Shed GP expressed by this mutant contains a single amino acid substitution at the carboxy-terminal end of shed GP. According to structural predictions it is highly unlikely that this single amino acid change would alter the structure or function of shed GP. Another difficulty faced in the production of recombinant GP of EBOV results from its strong cytotoxic effects [20,35,36] causing cell rounding, loss of cell attachment, and possibly the release of cellular factors due to eventual cell death. In order to address solely the potential functions of shed GP and to allow for possible effects caused by cytotoxicity, an EBOV GP mutant lacking shedding properties was used in this study as a negative control. This mutant contains a substitution L635V (GP-LS, low shedding) that was previously shown to prevent TACE cleavage and result in increased surface GP expression [21]. Of note, expression of this mutant is expected to result in similar, if not higher, cytotoxicity than that caused by wild-type GP (GP-WT)

and would provide culture supernatants containing the same potential extracellular factors released from GP expressing cells whilst displaying undetectable amounts of shed GP.

In this manner, 293T cells were transfected with the plasmids expressing mutated GPs, GP-WT and sGP as well as GFP as a non-relevant control. Western blot analysis of cell lysates and culture supernatants revealed as expected, an important difference in efficacy of GP shedding between GP-WT and mutated GPs (Figure 1A).

Of note, due to the removal of the region spanning the transmembrane anchor, after cleavage by TACE the GP 2 within shed GP showed a lower molecular mass and faster migration on SDS-polyacrylamyde gels compared to GP 2 in intracellular GP 1,2 (compare lines 1 and 7; lines 2 and 8). While similar amounts of intracellular GP (represented by endoplasmic reticulum GP precursor -preGPer and mature GP 1,2 ) were seen in cells expressing either GP-WT or GP-LS (compare lines 1 and 3), GP-LS did not show detectable levels of shed GP in the medium when compared with GP-WT. Notably, relatively low amounts of intracellular GP were detected with GP-HS whilst increased levels of shed GP were detected in the medium. As expected EBOV sGP is efficiently secreted from 293T cells transfected with corresponding plasmid (Figure 1A, lane 10).

Using Vivaspin concentrators the volume of culture supernatants was reduced by 10 fold to increase the concentration of shed GP in supernatant samples. Of note, no significant differences were detected in total amount of proteins between samples (2 mg/ml), as also illustrated in Figure 1B by Coomassie staining. The presence of shed GP in concentrated samples from GP-HS expressing cells and its absence in samples from GP-LS expressing cells was confirmed by Western Blot analysis (Figure 1B, left panel, line 3).

Using serial dilutions of a commercially available recombinant baculovirusexpressed EBOV GP lacking the transmembrane domain we were able to estimate the amounts of shed GP and sGP in concentrated supernatants by western blot analysis and quantification software. The standard curve was linear over the range used for quantification and the GP-HS sample was found to contain ~1 µg/ml and sGP ~18 µg/ml (Figure S1 A and B). In subsequent experiments with immune cells shed GP (GP-HS) was typically used at a concentration of ~0.4 µg/ml and sGP at a concentration of ~7.2 µg/ml.

To avoid using the produced recombinant shed GP in subsequent experiments at concentrations superior to those observed with infection the quantity of shed GP in concentrated samples was compared with that present in the blood of experimentally infected animals. For this purpose groups of guinea pigs were inoculated intraperitoneally with 500 infectious units (TCID 50 ) of guinea pig-adapted recombinant EBOV [37]. Infected guinea pigs were monitored for clinical manifestations and showed an increase in temperature starting by day 3 post-infection. When animals reached the ethical end point of infection they were euthanized. Blood was collected at different intervals post infection and the sera were assayed for the presence of shed GP in comparison with recombinant-produced shed GP. As demonstrated in Figure 1C and 1D the amounts of shed GP present in the blood of animals significantly exceed those used in our study. Quantification of shed GP in the sera based on amounts of truncated GP 2 reveals approximately 85 times higher concentrations of shed GP (34 µg/ml) than those used in this study. Furthermore the analysis of sera samples collected at different intervals post infection showed that the amounts of shed GP in the blood of animals significantly increased during the course of infection (Figure 1D), which is likely to be explained by both virus-induced activation of TACE [34] and also by spreading of infection that increases the number of virus infected cells.

Of note, our GP construction and production approach differed from that used in an earlier study investigating role of soluble EBOV glycoproteins [32]. In the previous study to address the role of shed GP the authors had used purified amino-terminal HAtagged soluble glycoprotein produced through the addition of a stop codon immediately after the TACE cleavage site that allows efficient secretion of this truncated GP into the extracellular media. However, we have observed that when GP is produced through truncation of its transmembrane anchor it does not appear to wholly represent shed GP.

Whilst truncated forms of GP had been previously used for crystal structure analysis of surface GP, and were principally shown to form trimers [18], the stability of such truncated oligomeric structures is suspected to be significantly lower in cell culture conditions. Indeed, as evidenced by our sedimentation analysis the transmembraneanchor truncated GP mutant is mostly a monomer in solution (Figure S2). In contrast shed GP produced in our study is a trimer as was also observed during EBOV infection [21] and is in agreement with the trimeric structure of surface GP. Of note the process of shedding includes proper GP oligomerization, maturation, and transport to the cell surface where the mature GP spikes are cleaved by cellular TACE and shed from the cells.

In addition, in the previous study the soluble recombinant glycoproteins were produced in conditions containing human sera [32] which are known to contain sera lectins, including human MBL [38][39][40]. Indeed, MBL is a C-type lectin capable of acting as a pattern recognition molecule for pathogens by recognizing heavily glycosylated proteins, particularly those containing a high-mannose type of glycosylation such as that seen with EBOV surface GP [41][42][43][44][45]. Importantly, MBL has recently been shown capable of binding EBOV surface GP [42,46,47]. The presence of such sera lectins during production of soluble GP could hamper the investigation of the interaction of this protein with its cellular targets and therefore the expression and production of shed GP, sGP and also of all control proteins used in this study was performed in cells cultured in FCS free medium (VP-SFM, Gibco).

EBOV soluble glycoproteins bind to DC and macrophages

Next, human monocyte-derived DCs and macrophages (Mø) as well as peripheral blood lymphocytes (PBL) were incubated with the samples of culture medium containing the soluble EBOV GPs described above. To assess the ability of shed GP to bind to DCs, Mø or specific fractions of lymphocytes, flow cytometry was performed using anti-GP specific antibodies. DCs were shown to bind both shed GP and sGP while Mø could only bind shed GP (Figures 2A andS3A). As expected, incubation of the cells with GP-LS failed to evidence any GP specific binding in agreement with undetectable levels of shed GP in samples. It appears that neither B nor T lymphocytes (Figures 2A andS3A) bind shed GP or sGP in agreement with earlier data revealing an absence of surface GP binding to these cells [48,49].

In light of data revealing an interaction between surface GP and MBL [42,46],

we also investigated whether MBL present in human sera is capable of affecting the binding of shed GP to cells. Of note, normal human plasma contains MBL concentrations ranging from 10 to 5000 ng/ml [START_REF] Babovic-Vuksanovic | Mannose-binding lectin (MBL) deficiency. Variant alleles in a midwestern population of the United States[END_REF][START_REF] Garred | Mannose-binding lectin and its genetic variants[END_REF]. Using commercially available human sera (Statens Serum Institute, Denmark) we demonstrate that pre-incubation of shed GP with such sera (final MBL concentration of 150 ng/ml) blocked shed GP binding to both DCs and Mø (Figures 2A andS3A).

Previously it was demonstrated that surface GP of EBOV interacts with toll-like receptor 4 (TLR4) [START_REF] Okumura | Interaction between Ebola virus glycoprotein and host toll-like receptor 4 leads to induction of proinflammatory cytokines and SOCS1[END_REF], a cellular surface receptor that is known to recognize highly glycosylated molecules containing O-linked mannosyl residues such as those present on GP. It was thus of interest to investigate if shed GP could also mediate its activity through TLR4. To address this question DCs and Mø were pre-treated with mouse anti-TLR4 antibody (HTA125) and then incubated with shed GP. As a control, the cells were first incubated with mouse isotype control antibodies and then with shed GP.

Binding of shed GP to both DCs and Mø was dramatically reduced upon treatment of the cells with anti-TLR4 antibody whereas no effect on GP binding was observed when control antibodies were used (Figures 2B andS3B). These results suggest that shed GP binding to DCs and Mø is likely to be explained by its interaction with TLR4. In this regard an absence of shed GP binding to PBLs is in agreement with little to no surface TLR4 expression on lymphocytes from healthy donors [START_REF] Ganley-Leal | Differential regulation of TLR4 expression in human B cells and monocytes[END_REF][START_REF] Muzio | Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells[END_REF][START_REF] Zarember | Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines[END_REF].

It is worthy to note that MBL can also bind TLR4 [START_REF] Shimizu | Mannose binding lectin and lung collectins interact with Toll-like receptor 4 and MD-2 by different mechanisms[END_REF][START_REF] Wang | Mannan-binding lectin directly interacts with Toll-like receptor 4 and suppresses lipopolysaccharide-induced inflammatory cytokine secretion from THP-1 cells[END_REF][START_REF] Ma | Mouse mannose-binding lectin-A and ficolin-A inhibit lipopolysaccharide-mediated pro-inflammatory responses on mast cells[END_REF]. In this regard the MBL-mediated reduction in shed GP binding to the cells could be explained either by the interference of MBL in shed GP binding to TLR4 and/or by sequestering shed GP via direct interaction with MBL. In an attempt to clarify this question we performed experiments where cells were pre-treated with sera containing MBL with subsequent washing and incubation with shed GP. As demonstrated in Figures 2C andS3C, shed GP binding to the cells was also blocked under these conditions, in this case most likely via interaction of TLR4 with MBL. In agreement, pre-incubation of cells with MBL deficient sera did not affect shed GP binding.

Thus far data obtained clearly indicate an interaction between shed GP and DCs and Mø. This is contrary to a previous publication in which no evidence of an interaction was found between shed GP and immune cells [32]. In this regard, the avoidance of sera lectins during GP production appears to be of vital importance as we demonstrate that the addition of human sera containing MBL abrogates binding of shed GP to target cells in vitro. Of note, as demonstrated in Figure 1C significantly higher amounts of shed GP are present in the blood of EBOV-infected guinea pigs than in our experimental conditions. Furthermore the continuous and massive release of shed GP during infection (Figure 1D) most likely would reduce or counteract the inhibitory effect of sera lectins in vivo even further.

Importantly, the data obtained in this study suggest that TLR4 is likely to be the key cellular partner for binding of shed GP to DCs and Mø. As TLR4 is abundantly expressed on macrophages and DCs [START_REF] Muzio | Toll-like receptors: a growing family of immune receptors that are differentially expressed and regulated by different leukocytes[END_REF][START_REF] Zhang | Molecular mechanisms of NF-kappaB activation induced by bacterial lipopolysaccharide through Toll-like receptors[END_REF][START_REF] Brightbill | Toll-like receptors: molecular mechanisms of the mammalian immune response[END_REF] our findings led us to investigate further the role that such an interaction might play, in terms of activation of these cells and the induction of the release of pro-inflammatory cytokines that is known to be a characteristic of EBOV pathogenesis [START_REF] Baize | Inflammatory responses in Ebola virus-infected patients[END_REF][START_REF] Hensley | Proinflammatory response during Ebola virus infection of primate models: possible involvement of the tumor necrosis factor receptor superfamily[END_REF].

EBOV shed GP induces upregulation of cytokine transcription in both DCs and

Mø

To address whether shed GP binding to immune cells results in their activation, monocyte-derived DCs and Mø were incubated with samples of culture supernatants containing the soluble EBOV glycoproteins detailed above and expression of TNFα, IL6, IL10 and IL12p40 mRNA was measured by real-time PCR at 4, 8, 12 and 24h post-treatment. In controls, cells were treated either with culture supernatants from cells expressing GFP (Mock) or LPS (500 pg/ml). Culture supernatants from cells expressing GP-LS were used as an additional negative control. Prior to experiments, all culture supernatants were tested for the absence of endotoxin and showed endotoxin concentrations below 0.25 units/ml and were thus considered to be endotoxin-negative.

In a sharp contrast to all negative controls used in this experiment, treatment of DCs and Mø with either shed GP or LPS results in transcriptional activation of a number of analysed genes (Figure 3A andB). Upregulation of TNFα and IL6 occurred as early as 4h post treatment whereas IL10 and IL12p40 were activated only 8h and 12h post treatment, respectively. The increase in mRNA levels appears to be higher in Mø than in DCs and when compared to LPS, cell activation caused by shed GP appears to be more durable. Treatment of the cells with sGP did not reveal any significant effects on cytokine mRNA synthesis. In agreement with experiments concerning shed GP binding, pre-treatment of both DCs and Mø with anti-TLR4 antibody significantly reduced shed GP-induced activation of TNFα mRNA synthesis (Figure 3C). A similar or even complete neutralizing effect of anti-TLR4 antibody was observed in the case of LPS treatment, in agreement with previous publications demonstrating that LPS acts on cells via TLR4 [START_REF] Wang | Mannan-binding lectin directly interacts with Toll-like receptor 4 and suppresses lipopolysaccharide-induced inflammatory cytokine secretion from THP-1 cells[END_REF][START_REF] Beutler | Toll-like receptors: how they work and what they do[END_REF]. Furthermore, as shown in Figure 3D, pre-treatment of shed GP with human sera containing MBL results in the abrogation of shed GP-dependent TNFα activation. Importantly experiments with dose-dependent inhibition of shed GP activity by MBL-containing sera clearly support the conclusion that the inhibitory effect of sera lectins will be countered by the continuously growing amounts of shed GP in vivo (Figure 3D). A two-fold decrease in MBL amount led to a significant reduction of the MBL inhibitory effect on the activation of both DCs and macrophages by shed GP. No blocking effect was observed after pre-treatment of shed GP with MBL-deficient sera.

As expected, both human sera used did not themselves activate DCs or Mø.

EBOV shed GP induces release of pro-and anti-inflammatory cytokines from DCs and Mø

Given the transcriptional activation observed above it follows that upon stimulation DCs and Mø would release a range of cytokines into the medium.

Accordingly, culture supernatants of DCs and Mø collected 24h after addition of shed GP (as detailed above) were assayed using a Multiplex cytometric bead array (Bio-Rad) against a panel of cytokines previously shown to be upregulated during EBOV infection [START_REF] Baize | Inflammatory responses in Ebola virus-infected patients[END_REF][START_REF] Stroher | Infection and activation of monocytes by Marburg and Ebola viruses[END_REF]. As expected, shed GP induced the secretion of TNFα, IL6, IL10, IL12, IL8, IL1β and IL1RA in both DCs and Mø (Figure 4A). Comparable levels of these cytokines were also observed in the medium when cells were treated with LPS. All negative controls used in this study including samples of medium from GP-LS expressing cells did not show any significant release of the cytokines. Incubation of the cells with the sGP-containing sample also did not result in detectable cytokine secretion.

Expectedly, pre-treatment of shed GP-containing samples with MBL resulted in a complete block in cytokine release that correlates well with the absence of activation of cells, as demonstrated in figure 3A and3B. In accordance with the results presented above, pre-treatment of cells with an anti-TLR4 antibody considerably reduced the release of TNFα, IL6, IL10, IL12, IL8, IL1β and IL1RA (Figure 4B). Again a similar or even stronger neutralizing effect of anti-TLR4 antibody was observed in the case of LPS treatment. Importantly, shed GP produced in 293T cells cultured with 5% FCS also failed to show activation of immune cells (Figure S4) supporting the importance of using sera-free medium for shed GP production.

In a separate experiment we determined that shed GP activity is dosedependent. Several dilutions of shed GP from GP-HS expressing cells as well as from wild-type GP-expressing cells were used to treat immune cells and culture supernatants from DCs and Mø collected 24h after addition of shed GP were assayed (as detailed above) using a Multiplex cytometric bead array (Bio-Rad) against a panel of cytokines.

As shown in figure 4C the activity of shed GP released from wt GP-and GP-HS expressing cells is comparable when the concentration of the proteins is adjusted. This also demonstrates that whilst increasing GP shedding efficacy the single amino acid exchange at the carboxy-terminal end of GP-HS does not affect the protein's activity in comparison to the wild-type sequence.

As activation of DCs and Mø is usually associated with an increase in expression of phenotypic activation markers such as the co-stimulatory molecules CD86, CD80, CD83 and CD40, we investigated whether the binding of shed GP or sGP to these immune cells could induce the expression of these surface molecules. As demonstrated in figure 5 A and B and figure S5 A and B, exposure to shed GP but not to sGP induced the expression of co-stimulatory molecules on both DCs and Mø. Similar expression of surface markers was observed upon treatment of the cells with LPS. Samples of culture medium from GP-LS expressing cells, used here as a negative control, did not reveal any significant effect on immune cells. Pre-treatment of cells with MBL prevents cell activation as demonstrated by the absence of any increase in co-stimulatory molecule expression.

Glycosylation of shed GP is vital to activate DCs and Mø

Since anti-TLR4 antibodies were capable of blocking shed GP binding and also of preventing immune cell activation we speculated that GP glycosylation might serve as a pathogen associated molecular pattern (PAMP) recognized by TLR4. Indeed, the glycosylation pattern of EBOV GP is complex and composed of mature and immature N-glycans but also a dense cluster of O-glycosylated serine and threonine residues referred to as mucin-like domain [18,19]. In order to verify this idea shed GP was Taken together these results suggest that the particular glycosylation pattern of shed GP is responsible for its functions as an inducer of immune cell activation and that this function is primarily mediated via shed GP binding to TLR4 present on DCs and Mø acting as a pathogen pattern recognition receptor. This would appear consistent with the hypothesis that shed GP is acting in a manner somewhat similar to that seen for LPS in its ability to cause systemic inflammation.

Modulation of endothelial permeability by EBOV shed GP

Vascular dysregulation and instability are thought to be crucial symptoms during EBOV infection [24,[START_REF] Ryabchikova | Animal pathology of filoviral infections[END_REF]. While endothelial cells are susceptible EBOV targets in vitro [35,[START_REF] Wahl-Jensen | Effects of Ebola virus glycoproteins on endothelial cell activation and barrier function[END_REF], they are considered to be rather late virus targets in vivo [START_REF] Geisbert | Pathogenesis of Ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells[END_REF][START_REF] Hensley | The contribution of the endothelium to the development of coagulation disorders that characterize Ebola hemorrhagic fever in primates[END_REF] suggesting an indirect effect of EBOV infection on this cells. A dysregulated inflammatory host response to EBOV infection is believed to facilitate increased permeability of endothelial barriers that would eventually cause haemorrhaging [START_REF] Wahl-Jensen | Effects of Ebola virus glycoproteins on endothelial cell activation and barrier function[END_REF][START_REF] Royall | Tumor necrosis factor and interleukin 1 alpha increase vascular endothelial permeability[END_REF]. In this regard proinflammatory cytokines, in particular TNFα, released from immune cells activated by shed GP could affect endothelial barrier integrity [START_REF] Geisbert | Pathogenesis of Ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells[END_REF][START_REF] Royall | Tumor necrosis factor and interleukin 1 alpha increase vascular endothelial permeability[END_REF]. Furthermore, as we demonstrated that shed GP is capable of activating immune cells for release of different cytokines, we also sought to determine if shed GP could directly affect endothelial cell permeability. To answer these questions, an in vitro monolayer cell permeability assay was performed using HUVECs. Firstly, HUVEC monolayers grown on semi-permeable membranes in cell culture plate inserts were treated for 22h with samples containing soluble EBOV glycoproteins. Following treatment FITC-dextran was added to the apical part of the insert and the permeability of the monolayer (Figure 7A) was assessed by measuring fluorescence in the basal compartment. As controls, cells were treated with samples of culture medium from GFP-(Mock), sGP-or GP-LS-expressing cells and also with LPS (as above), MBL-containing sera, MBL-deficient sera and TNFα (10ng/ml). The integrity of HUVEC monolayers was verified with a fluorometer both before and after 22h of incubation with supernatants containing soluble glycoproteins.

HUVEC monolayers were also photographed under a light microscope (Figure 7C). As seen in Figure 7A and 7C, samples containing GP-HS, LPS and TNFα significantly increased the permeability of the HUVEC monolayer when compared with negative controls and sGP. As expected, culture supernatants containing GP-HS in the presence of MBL-containing sera were significantly less capable of inducing an increase in permeability of the HUVEC monolayer compared to MBL-deficient sera. Treatment with human sera alone did not show any significant effect on permeability either with or deficient for MBL. Secondly, we also assayed whether the levels of soluble modulators released from macrophages treated with samples of shed GP and all controls (as above)

were sufficient to contribute to the decreased barrier function of the endothelium. As seen in figure 7B and D, the supernatants from shed GP-treated macrophages significantly increased the permeability of the HUVEC monolayer when compared to samples of culture medium from GFP-(Mock) or sGP-expressing cells, in a manner comparable with that seen with LPS-treated macrophages or when TNFα was used as a control. Somewhat surprisingly given the absence of detectable activation in previous assays, a moderate increase in permeability was observed when culture medium from GP-LS-expressing cells was used, probably explained either by residual amounts of shed GP present in GP-LS samples used to treat macrophages or by the presence of another undetermined factor released during cell treatment with medium upon GP expression. Culture supernatants from macrophages treated with shed GP in the presence of MBL containing sera were significantly less capable of inducing an increase in permeability of the HUVEC monolayer compared to MBL-deficient sera. Treatment with human sera alone did not show any significant effect on permeability either with or deficient for MBL. The ability of shed GP to affect endothelial cells function both directly and indirectly highlights the interplay between shed GP, systemic cytokine release and increased vascular permeability.

Taken together the data obtained support a role for EBOV shed GP in the creation of excessive and dysregulated host inflammatory responses and an increased vascular permeability. Based on the results presented here we postulate that once released from the surface of infected cells shed GP could be responsible for the induction and release from non-infected macrophages and DCs of a battery of pro-and anti-inflammatory cytokines. This is especially interesting as elevated levels of pro-and anti-inflammatory cytokines have been shown to be present in fatal EBOV infections in man [START_REF] Baize | Inflammatory responses in Ebola virus-infected patients[END_REF][START_REF] Wauquier | Human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis[END_REF]. Importantly, a clear link has been demonstrated between clinical status and inflammatory responses in that fatal outcome of infection is often associated with aberrant innate immune responses [START_REF] Wauquier | Human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis[END_REF][START_REF] Cilloniz | Functional genomics reveals the induction of inflammatory response and metalloproteinase gene expression during lethal Ebola virus infection[END_REF].

This study provides a potential explanation as to the cause of these disregulated host responses, since it has been demonstrated that neither the whole virus nor viral replication in immune cells could explain an excessive activation of immune cells for release of these cytokines [30,[START_REF] Baize | Inflammatory responses in Ebola virus-infected patients[END_REF][START_REF] Wauquier | Human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis[END_REF][START_REF] Groseth | The Ebola virus glycoprotein contributes to but is not sufficient for virulence in vivo[END_REF]. We now show that cultured primary DCs and macrophages are activated upon treatment with EBOV shed GP, resulting in release of several mediators including pro-and anti-inflammatory cytokines such as TNFα, IL6, IL10, IL12, IL8, IL1β and IL1RA independently of virus replication in these cells.

The apparent similarity in the response of immune cells to shed GP and LPS highlights a likely common triggering of TLR4 and shared downstream pathway for activation and cytokine release. Intriguingly, a common feature of both LPS as a bacterial component and EBOV infection is their ability to trigger a similar physiological syndrome typified by elevated cytokine release, which for EBOV infection closely resembles the septic shock seen with bacterial infection. Indeed, when presented in VLPs EBOV GP has been shown to interact with TLR4, leading to the induction of proinflammatory cytokines as well as SOCS1 in both a human monocytic cell line (THP-1) and 293T cells expressing a functional TLR4/MD2 receptor [START_REF] Okumura | Interaction between Ebola virus glycoprotein and host toll-like receptor 4 leads to induction of proinflammatory cytokines and SOCS1[END_REF]. Furthermore, a recent study by Lubaki et al. [30] provided clear evidence that an unrelated viral vector carrying EBOV GP could effectively cause maturation of dendritic cells. However, during EBOV replication in dendritic cells interferon antagonism mediated by virus-encoded proteins VP35 and VP24 strongly reduces both the expression of maturation markers and secretion of cytokines and chemokines from these cells. On the contrary, EBOV shed GP as a soluble mediator is able to activate non-infected immune cells, making it distinct from a number of other viruses whose surface glycoproteins have been shown to act as ligands for TLR4 recognition but that either do not shed their surface glycoproteins into the extracellular environment or do not spread systemically and thus only cause local inflammatory disorders [START_REF] Xie | Toll-like receptor 4 expression and function of respiratory syncytial virus-infected airway epithelial cells[END_REF][START_REF] Tal | Association between common Toll-like receptor 4 mutations and severe respiratory syncytial virus disease[END_REF][START_REF] Haynes | Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus[END_REF][START_REF] Burzyn | Toll-like receptor 4dependent activation of dendritic cells by a retrovirus[END_REF][START_REF] Bukreyev | The secreted form of respiratory syncytial virus G glycoprotein helps the virus evade antibody-mediated restriction of replication by acting as an antigen decoy and through effects on Fc receptor-bearing leukocytes[END_REF].

In conclusion, in the present study we have identified the primary targets of EBOV soluble glycoprotein shed GP. In our model, EBOV infected cells release both virions and shed GP into extracellular environment. Shed GP, most likely via interaction with TLR4 on the surface of macrophages and DCs induces activation of these cells and the release of pro-and anti-inflammatory cytokines. The soluble nature of both shed GP and cytokines enables them to trigger new, remote targets such as noninfected APCs, which will then amplify inflammation by recruiting and/or activating other cell types including lymphocytes, endothelial cells and other cell types including hepatocytes, leading to the impairment of cell functions and organ failure (Figure 8).

Our results strongly suggest that the target cell range for EBOV infection is considerably broader than previously imagined. In effect, activation of non-infected immune cells by shed GP could play an important role in the establishment of systemic inflammation during infection. Furthermore the data obtained with shed GP provide a potential link between systemic inflammation and increased endothelial permeability that together contribute to the disseminated intravascular syndrome seen with lethal EBOV infection. Overall, our data contribute to a better understanding of the way EBOV might provoke the excessive cytokine storm that appears to be detrimental to survival of infection and provide new insights with which to develop therapeutic strategies to combat this newly defined role for shed GP in high viral pathogenicity. In this regard, it is intriguing to speculate that treatment with anti-TLR4 antibodies could be used to reduce the inflammatory reaction caused by shed GP in a similar way than has been demonstrated for the dramatically successful treatment of septic shock in mice after lethal LPS challenge [START_REF] Daubeuf | TLR4/MD-2 monoclonal antibody therapy affords protection in experimental models of septic shock[END_REF]. Similarly, it is conceivable that neutralizing antibodies targeting shed GP could also help to alleviate the systemic shock-like syndrome seen with EBOV infection.

Materials and methods

Virus

The work with infectious EBOV belonging to Filoviridae family, ebolavirus genus, species Zaire Ebolavirus was performed at INSERM "Laboratoire Jean Mérieux" within biosafety level 4 containment at Lyon, France. The virus was propagated on Vero E6 cells. Culture supernatants were collected, clarified, frozen and kept at -80°C before virus titration by TCID 50 [START_REF] Moe | Plaque assay for Ebola virus[END_REF] The virus was generated using a reverse genetics system previously described in [START_REF] Volchkov | Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity[END_REF]. Amino acid substitutions M71I, L147P, T187I

were introduced into the VP24 gene to increase the pathogenicity in recombinant EBOVs for guinea pigs [37,[START_REF] Mateo | VP24 is a molecular determinant of Ebola virus virulence in guinea pigs[END_REF]. Mutations introduced into the virus were confirmed by sequencing of complete genomic RNA isolated from virus stocks.

Cell lines and plasmids

293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, PAA laboratories) supplemented with 10% fetal calf serum (PerbioHyclone). Human umbilical vein endothelial cells (HUVECs) (Lonza) were maintained at 37ºC with 5% CO2 in EGM-2 Media (Lonza) enriched with EGM-2 Single Quots containing FBS, hydrocortisone, hFGF-B, VEGF, R3-IGF-1, ascorbic acid, hEGF, GA-1000 and heparin. Cells were cultured for 3-4 days with EGM-2 medium and were passaged for no more than 5 generations for the experiments described.

We have previously demonstrated that the substitutions L635V and D637V dramatically alter the efficacy of EBOV GP shedding in a transient expression system [21]. In this study to increase the efficiency of GP shedding in a transient expression system we introduced the substitutions D637A and Q638V using site-directed mutagenesis of a plasmid encoding wild-type GP. The resulting plasmid was designated phCMVGP-HS.

Plasmid expressing GP with a substitution L635V (phCMVGP-LS) was described earlier [21]. Plasmid expressing transmembrane-anchor truncated mutant GP 1,2Δ was generated by introducing a stop codon at position 651 using site-directed mutagenesis of a plasmid encoding wild-type GP.

Transient expression of EBOV GP

293T cells were grown for 24h to a confluence of ~60%. Transfection of cells with recombinant plasmids was performed using TurboFect reagent (Thermo). During GP expression the cells were kept in VP-SFM medium (Gibco) containing no serum.

Culture supernatants were harvested 36h posttransfection, clarified by centrifugation at 1000 rpm for 10 min at 4°C and then by centrifugation at 28,000 rpm at 4°C for 2 h using a SW28 rotor with a Beckman Optima L-70K ultracentrifuge. The clarified supernatants were concentrated 10 fold using Concentrator tubes (Pierce) with a molecular weight cut off of 20000. All samples were aliquoted and stored at -80°C.

Culture of primary human monocyte-derived-DCs and macrophages

Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by centrifugation onto a lymphocyte separation medium (Ficoll) cushion

Endotoxin test

The Limulus amebocyte lysate test (LAL Chromogenic Endotoxin Quantitation Kit, Pierce) was used to determine endotoxin levels in culture supernatants used in this study. The values were less or equal to those of VP-SFM medium (<0.25 endotoxin units per ml).

Sedimentation analysis

The oligomeric structures of EBOV shed GP and transmembrane-anchor truncated mutant GP 1,2Δ were compared using a sedimentation assay. 293T cells were transfected with plasmids phCMVGP-HS or phCMVGP 1,2Δ and culture medium was collected 36h post-transfection. Medium was clarified by low-speed centrifugation and subjected to ultracentrifugation through a linear 5-25% (w/w) sucrose gradient prepared on co-IP buffer (1% NP40, 0.4% deoxycholate, 5 mM EDTA, 100 mM NaCl, 20 mM Tris-HCl,

Deglycosylation assay

For GP deglycosylation, supernatants containing EBOV GPs were treated using EDEGLY kit (Sigma) for 48h according to the manufacturer's instructions. Shed GP mixed with deglycosylation enzymes pretreated at 80ºC for 30 min were used as a control. Monocyte-derived DCs and macrophages were treated with deglycosylated shed GP samples for 24 hours and culture supernatants were assayed for the presence of TNFα using a Multiplex cytometric bead array (Bio-Rad) in Luminex MAGPIX.

HUVEC permeabilization assay

HUVEC were grown in collagen-coated inserts of permeability assay chambers (Millipore) for 72h until confluence. The media was replaced with 600 μl of fresh EBM

Animal experiments

Guinea pigs, strain Hartley (3 week-old females), were infected intraperitoneally with 500 TCID 50 of recombinant guinea-pig adapted EBOV as previously described [START_REF] Mateo | VP24 is a molecular determinant of Ebola virus virulence in guinea pigs[END_REF].

Mock-infected controls were inoculated with DMEM. Animals were monitored for clinical manifestations and were euthanized when they reached an ethical end-point. Samples were also analyzed by Western blot with appropriate antibodies (left panel).

(C). Guinea pigs (strain Hartley) were infected intraperitoneally with 500 TCID50/animal of guinea pig-adapted EBOV [START_REF] Mateo | VP24 is a molecular determinant of Ebola virus virulence in guinea pigs[END_REF]. Serial dilutions of sera collected at des patients ou du matériel contaminé par le virus Ebola (5,24). Cette observation est a rapproché du fait que la plupart des mammifères non primates, infectés expérimentalement développent également une infection asymptomatique (40) ainsi il existe probablement des facteurs génétiques, immunitaires restrictifs à l'infection.

Une nécrose multifocale est observé dans les tissus lymphoïdes, celle-ci ne résulte pas directement de l'infection par le virus, les lymphocytes n'étant pas infectable mais provient d'un emballement du système immunitaire induisant l'apoptose massive des lymphocytes (3)(4)(5)26). La stimulation des monocytes par des endotoxines, du TNF, de l'IL-1β ou encore par MCP-1, induit l'expression à leurs surfaces du TF (7,10). La libération de TF actif dans la circulation contribuent directement à l'élaboration de thrombose (9). En effet, le TF s'associe au facteur VIIa pour former un complexe protéolytique activant le facteur X, celui-ci étant l'acteur direct du clivage de la prothrombine en thrombine (Figure 3). De plus, le complexe TF-VIIa est un élément de signalisation connu notamment pour induire dans les cellules un afflux de Ca2+ et pour être pro-inflammatoire (induction d'IL6 et IL8) (33). La génération de dépôt de fibrine dû à l'action de la thrombine sur le fibrinogène résulte dans la formation de multiples thrombi et dans la consommation des facteurs de coagulation. Cette activation de la voie du TF conduit également à la consommation des plaquettes. Ainsi différentes protéines recombinantes (rNAPC2 et APC/Xigris) inhibitrices de la voie du TF ont montré leurs activités dans le traitement de l'infection à virus Ebola chez le singe (12,17). Le rNAPC2, pour Recombinant et semblent varier selon l'espèce de virus Ebola (3,23,28,44). De plus les données de concentrations de cytokines sériques représentent la somme des sécrétions par différents types cellulaires et dans différents compartiments, ceci pouvant expliquer partiellement les différences entre les observations in vitro et in vivo. Le TNFα est fortement secrété par les macrophages infectés in vitro ( 16) et sa sécrétion est inhibée dans les cellules dendritiques dérivées de monocytes (19). Il est intéressant de noter qu'il existe plusieurs publications montrant in vitro le rôle de la glycoprotéine de surface (sous forme de VLP) dans l'induction de la sécrétion de TNFα par les cellules dendritiques (6,47). Il constitue de plus un facteur essentiel de stimulation des cellules endothéliales, engendrant l'expression du TF à leur surface. De plus l'IL6 et l'IL1 ont montré leurs actions dans l'induction de la coagulation (20,39). Une telle libération de cytokines devrait également libérer le facteur d'agrégation plaquettaire et des eicosanoides (thromboxane). La libération massive de cytokines inflammatoires et l'atteinte hépatique engendrent à leur tour la libération massive d'oxyde nitrique (NO), en effet lors de l'épidémie à Sudan ebolavirus de 2000, il a pu être mesurée à partir de 8 jours après le début des symptômes, des taux de plus de 150µM de NO (38). L'oxyde nitrique a pour principal effet le relâchement de la tunique de muscle lisse des vaisseaux sanguins, une libération importante comme celle observée en phase aiguë a pour effet une vasodilatation massive associée à une chute de la pression artérielle et une détresse cardiaque (des symptômes observés en phase aiguë de l'infection). Cependant l'oxyde nitrique est aussi connu pour son rôle protecteur de l'endothélium, sa présence en quantité importante pourrait aussi aider à préserver l'intégrité de l'endothélium et expliquerait l'absence d'altération vasculaire dans les infections à filovirus (41).

Cellules endothéliales

Les cellules endothéliales constituent la barrière entre le sang et les tissues, elles

Figure 1 . 27 Figure 2 . 28 Figure 3 . 30 Figure 4 . 37 Figure 5 . 51 Figure 6 . 55 Figure 7 . 56 Figure 8 . 62 Figure 9 . 68 Figure 10 . 70 Figure 11 . 74 Figure 12 . 75 Figure 13 . 78 Figure 14 .Figure 15 .Figure 16 .Figure 17 .Figure 18 .Figure 19 .Figure 20 .Figure 21 .Figure 22 .Figure 23 .Figure 24 .Figure R1 .Figure R6 .Figure R9 .Figure R11 .Figure R13 .Figure R14 .Figure R15 .Figure R16 .Figure 25 .Figure 26 .Figure 27 .Figure 28 .Figure 29 .Figure 30 .

 12722833043755165575686296810701174127513781415161718192021222324R1R6R9R11R13R14R15R16252627282930 Figure 1. Geographic distribution of filovirus outbreaks ...................................27

Figure 1 .

 1 Figure 1.-Geographic distribution of filovirus outbreaks. (Prior to 2014)

Figure 2 .

 2 Figure 2.-Discovery of an ebolavirus-like filovirus in europe. Adapted from (Negredo, Palacios et al. 2011).

Figure 4 .

 4 Figure 4.-Phases of EBOV hemorrhagic fever (EHF)

  nucleoprotein (NP), virion protein 35 (VP35), VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L) (Figure 5A). Intergenic regions contain initiation and termination signals. Leader and Trailer are noncoding regions which are very important during transcription, replication and encapsidation. The central core of the virion is composed of the ribonucleoprotein complex (RNP), which consists of the genomic RNA molecule encapsulated by NP and linked to the inner matrix proteins VP30 and VP35 and the L polymerase. This complex is involved in transcription and replication. The other three proteins, GP, VP24 and VP40 are membrane-associated. The matrix proteins VP40 and VP24 are linked to the ribonucleoprotein complex at the inner surface of the lipid bilayer of the viral envelope, which is derived from the host cell. VP40 and VP24 are involved in viral nucleocapsid formation, viral budding or assembly, and host range determination. The viral envelope contains only the glycoprotein GP, organized as trimeric spikes (Figure 5B and C).

Figure 5 .

 5 Figure 5.-Ebola virus morphology and organisation.-(A) genome organisation of Ebola virus; (B) Protein organisation in viral particles and (C) Transversal cut of a virion; Spikes are indicated with green arrows.

Figure 6 .

 6 Figure 6. Ebola virus glycoprotein synthesis and conformation. (A)Processing of EBOV glycoproteins. The EBOV genome contains seven genes but nine proteins are produced due to editing of the GP gene. The GP gene primary transcript encodes for a secreted GP (pre-sGP). Furin cleavage of pre-sGP produces mature sGP and a secreted Δ-peptide. Transcriptional stuttering results in the production of the envelope-attached GP and a small, secreted GP (ssGP). GP is cleaved by furin to form a disulfide-linked GP1 (green)-GP2 (yellow) heterodimer, which then assembles as trimers on the virus surface.Cleavage at the membrane-proximal external region by the tumor necrosis factor-a converting enzyme (TACE) releases shed GP. (B) Molecular representation of EBOV GP subunits are shown in green (GP1) and yellow (GP2). Complex-type N-linked glycans are modelled onto the EBOV GP surface as red spheres revealing a heavy glycan layer that buries much of the GP

Figure 8 .

 8 Figure 8.-TACE cleavage site selectivity.-A peptide library was used to determine preferences at the positions upstream and downstream of the cleavage site. Data were normalized such that a value of 1 (black) corresponds to the average quantity per amino acid in a given sequencing cycle and would

Figure 9 .

 9 Figure 9.-Illustration of Ebola virus infection cycle. Filoviruses enter the cell by receptor-mediated endocytosis or macropinocytosis (1). After fusion of the viral and cellular membrane (2), the nucleocapsid is released into the cytoplasm and serves as a template for transcription (3) and replication (4). The replicated RNA is encapsidated by the nucleocapsid proteins. The newly synthesized nucleocapsids and viral proteins are transported to the sites of viral release (5),

Figure 10 .

 10 Figure 10.-Ebola virus infection and spreading. (a) Ebola virus infects subjects through contact with body fluids or secretions from an infected patient. Entry can occur through abrasions in the skin, accidental needle sticks,contact with infected bushmeat or across mucosal surfaces. Early targets for replication are monocytes, macrophages and dendritic cells which, through the lymphatic

Figure 11 .

 11 Figure 11.-IFN pathway.-IFN type I binds receptor IFNAR thus activating Jak-STAT signaling cascade and upregulating IFN-stimulated genes.

Figure 12 .

 12 Figure 12.-Type I IFN induction, signaling and action. During the IFN independent pathway, Protein Kinase R (PKR) and helicases RIG-1 and MDA-5 can recognize dsARN synthesized during viral replication but also the 5'triphosphate extremities of some viral genomes (Yoneyama, Kikuchi et al. 2004). The recognition of viral components in the cellular cytoplasm by these intracellular sensors leads to activation of the transcription factors NF-κB, IRF-3 and AP-1. The cooperative action of these factors is required for full activation of the IFN-β promoter. A second signaling pathway involves endosomal TLR-3 and TRIF.

(Figure 13 .

 13 Figure 13.-Function of Ebola virus-encoded IFN-antagonist proteins. (A) EBOV VP35 inhibits IFN-α/β production by interacting with the IRF-3 kinases IKK-ɛ and TBK-1. This prevents efficient phosphorylation of IRF-3, blocking activation of the IFN-β promoter. (B) EBOV VP24 prevents nuclear accumulation of activated, tyrosine-phosphorylated STAT1 through an interaction with NPI-1 family karyopherin-α proteins, which otherwise import dimerized STAT1 into the nucleus.

  against a lethal infection by passive transfer of virus-specific polyclonal antibodies, obtained from vaccinated monkeys. Treatments were delayed 48 h, with additional treatments on days 4 and 8 post-exposure; the delayed treatments protected EBOV-challenged NHPs (Dye, Herbert et al. 2012). These studies clearly demonstrate that post-exposure antibody treatments can protect NHPs (probably depending on the antibody quality, quantity and the moment of administration) and open avenues for filovirus therapies for human use.

Figure 14 .

 14 Figure 14.-Tissue Factor coagulation pathway Tissue factor expression is enhanced leading to increased production of prothrombin that is converted to thrombin, and that in turn generates fibrin from fibrinogen thus generating clotting.

Figure 16 .

 16 Figure 16.-Disseminated Intravascular Coagulation. (A) Deposition of fibrin leads to clotting (B) Clotting implies consumption of coagulation factors thus inducing abnormal bleeding. Both will contribute to the development of multiorgan failure.

Figure 17 .

 17 Figure 17.-Schematic representation of pathogeneic pathways in disseminated intravascular coagulation. During systemic inflammatory response syndromes, both perturbed endothelial cell function and activated mononuclear cells may produce proinflammatory cytokines that mediate coagulation activation. Activation of coagulation is initiated by tissue factor expression on activated macrophages, monocytes and endothelial cells. In addition, downregulation of physiological anticoagulant mechanisms (like Protein C) and impairment of fibrinolysis (by inhibition of Tissue Factor Pathway



  The ability to elicit responses in leukocytes, platelets and endothelial cells  Cleavage of proteins outside the coagulation pathway, such as complement components C3 and C5.  Stimulate the production and secretion of inflammatory cytokines principally through NF-κB signaling pathways. It activates a proinflammatory response through its interaction with specific receptors known as protease-activated receptors or PARs, which are expressed on ECs, mononuclear cells, platelets, fibroblasts, and smooth muscle cells. IL-6 production enhanced by thrombin leads to TF expression through a positive feedback loop.  Enhancement of monocytes or monocyte-derived macrophages adhesion and increase of their production of pro-inflammatory cytokines with downregulation of anti-inflammatory cytokines.  Stimulation of endothelial cell production of platelet-activating factor (PAF), whose role is to attract neutrophils and contribute to their adhesion.

3. 1 . 3 . 2 . 2 .

 1322 Impairment of anticoagulant mechanisms Activation of coagulation is regulated by four major anticoagulant factors: antithrombin (AT), thrombomodulin (TM), the activated protein C (PCA) and tissue factor pathway inhibitor (TFPI). An impaired function of various natural regulating pathways of coagulation activation may amplify thrombin generation and contribute to fibrin formation (Okajima 2001). The dysregulation of these anticoagulants are detailed below: Antithrombin (AT)'s role in coagulation involves the inhibition of several coagulation modulators including factors Xa, IXa, XIa, and thrombin. AT's antiinflammatory actions include:  Inhibition of leukocyte activation (rolling and adhesion)  Binding of AT to heparin sulfate proteoglycan (HSPG) on the endothelial surface and blockade of the expression of pro-inflammatory cytokines.



  Inhibition of leukocyte adhesion to activated endothelium.  Acceleration of the activation of the plasma carboxipeptidase thrombinactivatable fibrinolysis inhibitor (TAFI), an important inhibitor of fibrinolysis.  TM is considered anti-inflammatory through its interference with complement activation and the neutralization of LPS. Serum specimens from the Sudan Ebola virus outbreak in 2000 in Uganda were analysed for several biomarkers and elevated levels of thrombomodulin were associated with mortality and haemorrhaging (McElroy, Erickson et al. 2014). Activated protein C (PCA) inhibits, through proteolytic cleavage, coagulation factors Va and VIIIa, providing critical negative feedback regulation of thrombin generation. In addition, PCA possesses cytoprotective properties including antiapoptotic mechanisms (inhibits endothelial cells apoptosis), endothelial barrier stabilization, and the inhibition of inflammation. In addition, binding of activated protein C to the protein C receptor has been shown to inhibit endotoxin-induced tissue factor expression on mononuclear cells.

2 .

 2 A secondary fibrinolytic response to fibrin deposition results from the activation of the plasminogen activator (PA) from endothelium. There are two types of PA: tissue-type plasminogen activator (t-PA) and urokinasetype plasminogen activator (u-PA), found in storage sites in vascular endothelial cells. t-PA is principally synthisized by endothelial cells in normal blood vessels and u-PA is produced and secreted by a variety of cell types including monocytes, macrophages, trophoblasts and epithelial cells. The plasmin formed after PA action can generate fibrin degradation products (FDP) which, as for fragments X and Y, have a direct antithrombin action. Other FDP fragments such as D (D-Dimers) and E interfere with fibrin formation by inhibiting its polymerisation. Both fibrin deposition in tissues, and the rapid increase in D-dimer plasma levels are hallmark features of Ebola hemorrhagic fever in cynomologus macaques and humans. Indeed, samples obtained from Ebola virus infected macaques showed the development of fibrin-degradation products (D-dimers)

Figure 18 .

 18 Figure 18.-Cross-talk between the inflammation and coagulation systems. Infection of monocytes/macrophages will lead to the expression of tissue factor, directly due to infection or indirectly through cytokine release as consequence of Ebola virus infection. The inflammatory process would then induce the coagulation pathway. Several coagulation factors induce the release of proinflammatory cytokines, thus creating a cross-talk between coagulation and inflammation.

  coagulation that are regulated by several coagulation factors and cytokines are represented in Figure 19. Coagulation and fibrin deposition can be activated due to inflammation and are essential in containing both the invading entity and the consequent inflammatory response to a limited zone. On the other side, coagulation can also modulate the inflammatory response; coagulation factors including thrombin and anticoagulant proteins, such as activated protein C, activate mononuclear and endothelial cells, which in turn affect cytokine production and inflammatory cell apoptosis. Coagulation activation yields proteases that interact with the coagulation protein zymogens, but also with specific cell receptors to induce signalling pathways that mediate inflammatory responses. Coagulation proteins, such as Xa, thrombin and fibrin, can activate endothelial cells, eliciting the synthesis of pro-inflammatory cytokines and growth factors (van der Poll, de Jonge et al. 2001).

Figure 19 .

 19 Figure 19.-Effects of physiological anticoagulant systems and fibrinolysis on coagulation and inflammation. Thrombomodulin plays a central role as an endothelial receptor and activator of protein C and TAFI respectively and has direct effects on endothelium. Activated protein C and TAFI have effects on coagulation/fibrinolysis as well as in inflammation. TFPI is the endothelial cell associated inhibitor of tissue factor but also has direct effects on cytokine production by mononuclear cells. Antithrombin binds to glycosaminglicans, which causes a more efficient inhibition of activated coagulation proteases and also affects inflammation. Release of plasminogen activators (PA) from endothelial cells will induce local fibrinolysis; the complex of u-PA and u-PAR plays an important role in inflammatory cell recruitment and migration. Plus (+) and minus (-) signs indicate stimulatory and inhibitory effects respectively. Adapted from (Williams 2007).

  32kDa. Each chain is characterised by a lectin or carbohydrate-recognition domain (CRD), a hydrophobic neck region, a collagenous region and a cysteine-rich-N-terminal region. The collagenous regions of three such chains interact to give a classical triple helix. This can then generate disulphide-bridge links at the N-terminal extremity thus forming higher order structures. MBL circulates in the serum mainly as a hexameric molecule (6 trimeric subunits with 18 CRD) (figure20).∼ MBL has at least four distinct functions: (i) the promotion of (complementindependent) opsonophagocytosis; (ii) the activation of complement; (iii) the modulation of inflammation; and (iv) the promotion of apoptosis (figure22).Firstly, it acts directly as an opsonin, promoting phagocytosis of foreign material to which it has bound (figure22(i)). The second mechanism through which MBL functions is by triggering the lectin pathway of complement activation via MBL associated serine proteases (MASPs). Complement activation leads to opsonisation of the material and cell lysis. MASPs are homologues of the complement C1 serine proteases C1r and C1s. Three MASPs have been identified to date; MASP-1, MASP-2 and MASP-3 and also a small 19 kDa protein lacking a serine protease domain named MAp19. Upon MBL binding to targets, MASP-2 cleaves the complement proteins C4 and C2, resulting in the formation of a C3 convertase, C4b2a. C3 is cleaved by C4b2a, generating the fragments C3a and C3b (figure 21). C3b is further cleaved to form the iC3b opsonin. Activation of C3 also leads to cell lysis through the formation of the membrane attack complex (MAC) (figure 22(ii)).

Figure 21 .

 21 Figure 21.-MBL binding to certain patterns activates MASPs and subsequently activates the lectin complement pathway. (A) MASP-2 fixes and cleaves C4, leading to the formation of C4a and C4b. Following cleavage of C4, C4b still binds to MASP-2. (B) Free C2 then binds to C4b and is also cleaved by MASP-2 to produce C2a and C2b. (C) C4b and C2b combine on the surface of the pathogen to form C3-convertase (D), while C4a and C2a act as chemoattractants. Adapted from[START_REF] Wallis | Molecular interactions between MASP-2, C4, and C2 and their activation fragments leading to complement activation via the lectin pathway[END_REF]

Figure 22 .

 22 Figure 22.-Role of MBL during infection. MBL, which is found in a soluble form in blood, has several functions during infection: (i) promotion of opsonisation/phagocytosis, (ii) activation of complement, (iii) induction of inflammatory cytokines release and (iv) initiation of uptake of apoptotic cells.

  seems to have a paradoxical function depending on the levels of complement. It has been shown that rhMBL and native MBL enhanced pseudotyped and wild type-like Ebola virus infection of human cells when complement components were reduced or removed. It has been speculated that MBL would concentrate virus at the surface of the target cell (Brudner, Karpel et al. 2013) According to the currently accepted paradigm, MBL deficiency predisposes children and immunocompromised individuals to diseases whereas normal or high levels of MBL are protective. Therefore, due to MBL single nucleotide polymorphisms (SNPs), different individuals containing different levels of MBL may respond differently to EBOV infection. Taken together, these data suggest that MBL and the lectin complement pathway can influence the course of EBOV infection.

  rates of viral replication lead to lysis and necrosis in cells of many organs, including the liver, spleen, kidneys and gonads. Much of the observed necrosis is virally induced, as there is little immune infiltration within infected tissues, and extraordinary numbers of viral particles are present in the necrotic debris. In addition, microscopic examination of infected human tissues shows a correlation between tissue damage and the presence of viral antigens, nucleic acid and sites of viral replication. This observation indicates that direct viral damage of tissues and organs may lead to organ failure and shock. However, despite widespread viral replication, the observed damage to endothelia, liver and other organs is not sufficiently severe to cause terminal shock and death and, as mentioned before, a pathogenic role of the host response contributes to the high pathogenicity of Ebola virus[START_REF] Zampieri | Immunopathology of highly virulent pathogens: insights from Ebola virus[END_REF].Part of the cytotoxicity of Ebola virus can be explained by viral glycoprotein of surface GP. In fact, several studies in vitro have shown that Ebola virus surface glycoprotein GP, through its highly glycosylated mucin domain, can induce the rounding and detachment of infected cells (Figure23)(Yang, Duckers et al. 2000).

Figure 23 .

 23 Figure 23.-Induction of cytopathic effects by Ebola virus glycoproteinsMorphology of 293T cells 24 hours after transfection with plasmids expressing Ebola (subtype Zaire) GP, sGP or a 'no-insert' control.(Yang, Duckers et al.

Figure 24 .

 24 Figure 24.-EBOV GP masks cellular surface proteins. Surface markers such as MHC-I and β1-integrins are masked by Ebola GP expression in these cells due to the highly level of glycosylation of Ebola virus GP.

  Moreover, since impairment of coagulation and vascular disruption are both typical phenomena caused by Ebola virus infection, we sought to study the possible role of shed GP in their disruption. Particularly, we demonstrate that EBOV soluble protein shed GP binds to Mannose Binding Lectin (MBL) and activates the MBL-associated proteases MASP-1 and MASP-2, implicated in the coagulation pathway.

  of EBOV soluble glycoproteinsAs discussed above, EBOV shed GP is structurally identical to virion surface GP except for the lack of its carboxy-terminal part consisting of 13 amino acids upstream of the membrane anchor, the anchor itself and a short cytoplasmic tail. Consequently, it was of interest to investigate whether shed GP and potentially sGP, that shares 365 amino-terminal amino acids with surface GP, can interact with and affect the function of DCs and macrophages, that are the major primary targets for viral infection in vivo. While transient expression of sGP from plasmid provides sufficient amounts of the protein secreted into the medium, the low efficiency of GP shedding seen with transient expression systems has hampered production of large quantities of shed GP protein equivalent to those observed during EBOV infection. To surmount these difficulties we modified the TACE cleavage site by substituting amino acids at positions D637A and Q638V and thus increased efficiency of GP shedding. This newly generated sequence resembles that previously shown to be recognized by TACE for efficient release of TNFα[START_REF] Caescu | Active-site determinants of substrate recognition by the metalloproteinases TACE and ADAM10[END_REF]. The plasmid expressing this mutant was designated phCMVGP-HS and GP expressed from this plasmid as GP-HS (high shedding).As already mentioned, during EBOV infection significant amounts of soluble glycoproteins, including shed GP and secreted sGP, are released from virusinfected cells. As demonstrated in FigureR1the amounts of shed GP present in the blood of animals significantly exceed those used in our study.Quantification of shed GP in the sera of infected animals (500 TCID50/animal of guinea pig-adapted EBOV), based on amounts of truncated GP 2 , reveals approximately 85 times higher concentrations of shed GP (34 µg/ml) than those used in this study.

Figure R1 .

 R1 Figure R1. Production of recombinant EBOV shed GP and analysis of shed GP release in the blood of infected guinea pigs. (A) Guinea pigs (strain Hartley) were infected intraperitoneally with 500 TCID50/animal of guinea pig-adapted EBOV (Mateo, Carbonnelle et al. 2011). Serial dilutions of sera collected at day 6 post-infection (5µl, dilutions 1/4, 1/8, 1/16 & 1/32, lanes 2-5), supernatant from EBOV-infected Vero E6 cells (20µl, EBOV Vero, lane 1) and a sample of concentrated recombinant shed GP (5µl, HS, lane 6) were analyzed by Western blot using anti-GP2 antibodies. Estimation of shed GP amounts in the sera of animals in comparison with shed GP from GP-HS expressing cells was performed using ImageQuantTL software (GE Lifesciences). Sera of animals contained approximately 85 times higher amounts of shed GP than those used in experiments with immune cells.

Figure

  Figure R2. Shed GP production and characterisation. (A) Western Blot analysis of culture medium from 293T cells expressing different recombinant EBOV GP constructs. 293T cells were transfected with wild type GP (WT), shed GP (HS), low shedding GP (LS), sGP and GFP constructs and 36h p.t. cells

  Figure R3. DCs and MØ are primary targets of shed GP. (A) Human monocyte-derived dendritic cells (DCs), monocyte-derived macrophages (MØ)

  In a sharp contrast to all negative controls used in this experiment, treatment of DCs and MØ with either shed GP or LPS results in transcriptional activation of a number of analysed genes (FigureR4 A and B). Upregulation of TNFα and IL6 occurred as early as 4h post treatment whereas IL10 and IL12p40 were activated only 8h and 12h post treatment, respectively. The increase in mRNA levels appears to be higher in MØ than in DCs and when compared to LPS, cell activation caused by shed GP appears to be more durable. Treatment of the cells with sGP did not reveal any significant effects on cytokine mRNA synthesis. In agreement with the above experiments concerning shed GP binding, pre-treatment of both DCs and MØ with anti-TLR4 antibody significantly reduced shed GP-induced activation of TNFα mRNA synthesis (FigureR4 C).A similar but even stronger neutralizing effect of anti-TLR4 antibody was observed in the case of LPS treatment, in agreement with previous publications demonstrating that LPS acts on cells via TLR4(Beutler 2002, Beutler 2002, Salomao, Martins et al. 2008, Wang, Chen et al. 2011). Furthermore as shown in FigureR4 D, pre-treatment of shed GP with human sera containing MBL results in the abrogation of shed GP-dependent TNFα activation in a dose dependent manner. Accordingly, no such blocking effect was observed after pre-treatment of shed GP with MBL-deficient sera. As expected, both human sera used did not themselves activate DCs or MØ.

Figure R4 .

 R4 Figure R4. Shed GP induces transcriptional activation of cytokines in DCs and MØ. DCs and MØ (5x10 5 cells) were incubated with concentrated culture supernatants as above (figure1R C,D) and with LPS. Cells were collected at 4, 8, 12 and 24h post-incubation as indicated. Expression levels of mRNA encoding TNF-α, IL-6, IL-10 IL-12p40, and GAPDH were measured by RT-Q-

  Figure R5. Shed GP induces secretion of pro-and anti-inflammatory cytokines from DCs and MØ and induces their phenotypic maturation. (A)

Figure R6 .

 R6 Figure R6. Deglycosylation of shed GP affects activation of DCs and macrophages. (A) Concentrated culture supernatants of 293T cells expressing GP-HS (HS) were treated either with inactivated deglycosylation enzymes or a mix of deglycosylases (*HS). Samples were analyzed by Western blot under non-reducing conditions, line 1 and 2, respectively and shown. Deglycosylated EBOV GP is indicated as *GP 1,2Δ . (B) DCs (in white) and MØ (in grey) were incubated for 24h with concentrated culture supernatants of 293T cells expressing GP-HS (HS), GP-LS (LS) after treatment with inactivated deglycosylation enzymes, LPS as a positive control and HS after treatment with

Figure R7 .

 R7 Figure R7.-Ebola virus shed GP binds to human monocytes. Human monocytes were incubated with isotype control antibodies (in grey) and supernatants containing soluble glycoproteins (HS and sGP) and supernatants containing low shedding (LS) or shed GP with sera containing MBL (HS+MBL + )

  B). Maxisorp plates previously coated with MBL were incubated with soluble glycoproteins from transiently transfected cells (HS, GP ΔTM , LS, sGP and mock -from GFP expressing cells-) and UV inactivated VSV-GP and VSV-G as positive and negative control respectively. As expected, VSV-GP was shown to strongly bind to rMBL. Shed GP also bound to MBL in a dose-dependent manner and, as observed with surface plasmon resonance and dot blot, GP ΔTM binding levels to rMBL were lower when compared to shed GP. Negative controls LS, sGP, mock, inactivated VSV-G and MBL with no supernatant were not shown to unspecifically bind to MBL.

Figure 6 ResponseFigure

 6 Figure 6

  Figure R11. Shed GP induces VPR-AMC cleavage through MASP-1. A) VPR-AMC cleavage was quantified after addition of mannan, VLPs pseudotyped with EBOV surface GP and supernatants containing HS, LS and sGP. VPR-AMC cleavage was measured after 60 (white), 80 (grey) and 100 (black) minutes. The mean of triplicates from one representative experiment

Figure

  Figure R12.-C4 fixation and cleavage by shed GP through MBL. Human serum containing MBL was incubated with (A) supernatants from recombinant Ebola virus and C4 fixation was measured by ELISA. The mean± sd of triplicates from one representative experiment from three independent experiments is shown. Statistically significant differences (paired-sample t test) compared to EBOV-HS are shown as follows: * -p<0.05. (B) Competition assay between MBL and EBOV soluble glycoproteins. MBL was incubated with mannan, VLP-GP and supernatants from cells expressing HS, LS and sGP; this was then added to VLP-GP coated plateq and C4 fixation and MBL cleavage by MASP-2 was detected by ELISA and is shown as arbitrary units. The mean± sd of triplicates from one representative experiment from three independent experiments is shown. Statistically significant differences (paired-sample t test) compared to GP-HS are shown as follows: * -p<0.05.

  HUVECs were cultured in the presence of EBOV soluble glycoproteins and expression of ICAM-1 and VCAM-1 on the surface of human endothelial cells was studied by flow cytometry analysis. Release of soluble forms of tissue factor, thrombomodulin, ICAM-1, VCAM-1 and E-Selectin was assayed using a Multiplex cytometric bead array (Millipore). As shown in FigureR14A, ICAM-1 and VCAM-1 were expressed on HUVECs after 24hours of incubation with supernatant containing shed GP (HS) and its level of expression was considerably increased. Supernatants containing controls Mock (in grey), LS and sGP supernatant were not shown to increase ICAM-1 and VCAM-1 levels.In agreement with previous results, when supernatant containing shed GP was incubated with MBL containing serum, no increase in expression of ICAM-1 and VCAM-1 was observed.Soluble levels of ICAM-1, VCAM-1 and E-Selectin correspond with data observed with surface expression of these molecules (FigureR14 B); whereas supernatant containing shed GP (HS) and TNF-α did induce the release of these molecules into HUVEC culture medium, supernatants containing LS, and sGP did not induce ICAM-1, VCAM-1 and E-Selectin release. Again, supernatant containing shed GP together with MBL containing serum (HS + MBL + ) lost the capacity to induce the release of these coagulation and permeability biomarkers (Figure R14 B).

)

  or indirectly through supernatants from treated macrophages (Figure R15 B) was assessed by measuring fluorescence in the basal compartment. Cells were treated with samples of culture medium containing Mock, HS, LS, sGP, LPS, MBL containing sera (MBL + ), MBL-deficient sera (MBL -) and TNFα (10ng/ml in Figure 11A and 1ng/ml in figure 11B). Integrity of HUVEC monolayers was verified with a fluorometer both before and after 22h of incubation with supernatants containing soluble glycoroteins. HUVEC monolayers treated with supernatants containing soluble glycoproteins (Figure R15 C) or supernatants from macrophages previously stimulated with soluble glycoproteins (Figure R15 D) were also photographed under a light microscope. As seen in Figure R15 A and C, supernatants containing HS, LPS and TNFα significantly increased the permeability of HUVEC monolayer when compared with controls Mock, LS, MBL + , MBL -and sGP. Moreover, culture supernatants containing HS in the presence of MBL (HS+MBL + ) were significantly less capable of inducing an increase in permeability of the HUVEC monolayer compared supernatants containing HS in the presence of MBL-deficient sera.As we confirmed that HS is capable of activating immune cells for release of different cytokines including TNFα, we sought to determine if the levels of such soluble modulators were sufficient to contribute to the decreased barrier function of the endothelium (FigureR15 B and D). As expected, supernatants from macrophages incubated with the relevent soluble factors had a greater effect in disrupting the integrity of a HUVEC monolayer when compared to direct treatment with supernatants containing soluble glycoproteins, reaching comparable effect as that seen with LPS/TNFα-treated macrophages or LPS/TNFα alone used as a controls (compare FigureR15Awith 15B).

  Figure R15. EBOV shed GP increases cell permeability of HUVEC in vitro.HUVEC cells were seeded onto the culture inserts of permeability assay chambers and incubated for 22h with concentrated culture supernatants from 293T cells expressing GFP (Mock), sGP, GP-LS (LS), GP-HS (HS) and LPS (10ng/ml). Cells were also incubated with human sera containing MBL (MBL + ),

  chemokines and growth factors that seem to be indicative of fatal outcome. At even moderately elevated concentrations these soluble mediators act at various points in the first line of defence by recruiting circulating mononuclear cells to the site of infection, increasing endothelial permeability, activating macrophage and DC cytotoxic functions, and inducing adaptive immune responses by providing co-stimulatory signals for naïve T cells. Therefore, compared to the exacerbated release of cytokines observed during Ebola virus infection, a proper and controlled inflammatory response against the virus during the early steps of infection would help to counteract Ebola virus pathogenesis.

  able to receive co-stimulatory signals through TIM-4 and could induce their apoptosis.A series of studies have shown that EBOV stimulates antigen-presenting cells to release large quantities of proinflammatory cytokines, chemokines and other mediators(Geisbert, Hensley et al. 2003, Hutchinson and Rollin 2007, Mohamadzadeh, Chen et al. 2007). In fact, virus spread rapidly causes an intense systemic inflammatory syndrome resembling septic shock[START_REF] Feldmann | Effective post-exposure treatment of Ebola infection[END_REF]. Ebola virus infection and septic shock present fairly similar innate responses. The major clinical features of septic shock are brought about through the action of cytokines, chemokines, and other mediators produced by monocytes, macrophages, and related cells as a result of the binding of lipopolysaccharide (LPS) and other bacterial components to pattern-recognition receptors, including Toll-like receptors. This produces the typical traits of an inflammatory response[START_REF] Mahanty | Protection from lethal infection is determined by innate immune responses in a mouse model of Ebola virus infection[END_REF]. As with septic shock caused by bacterial pathogens, fatal cases of Ebola virus infection are thought to involve inappropriate or maladapted host responses including an exacerbated release of proinflammatory cytokines. The triggering mechanism by which Ebola virus stimulates induction of proinflammatory cytokines remains unknown. The apparent similarity in the response of immune cells to shed GP and LPS highlights a likely common triggering of TLR4 and shared downstream pathway

Figure 26 .

 26 Figure 26. Potential effects of shed GP on the immune system during EBOV infection.-In our model, EBOV infected cells release both virions and shed GP into the bloodstream. Shed GP is able to bind TLR4 on the surface of macrophages and DCs thus inducing the release of pro-and anti-inflammatory cytokines. The soluble nature of both shed GP and cytokines enables them to trigger new, remote targets such as non-infected APCs, which will then amplify inflammation by recruiting and/or activating other cell types including; lymphocytes, that could explain the lymphopenia observed during EBOV infection; endothelial cells, which display increased permeability due to cytokines released from DCs and macrophages; and other cell types including hepatocytes, leading to the impairment of other functions and organ failure.

  between different signalling pathways or attachment factors (involving NFκB pathway and TLR4 receptor among others) that could be crucial in the induction of pathogen-specific immune responses in EBOV targets. In fact, our data shows that treatment of DCs and macrophages with antibodies against TLR4 did not completely block shed GP binding nor the release of cytokines. Shed GP is also N-glycosylated and therefore it may also bind lectins such as Mannose receptor (MR) or DC-SIGN. The fact that we can still observe shed GP binding after blocking with TLR4 antibodies may be explained by the fact that immune sensing of Ebola virus probably requires other attachment factors on the surface of the cell. Importantly, our data shows that release of proinflammatory cytokines is downregulated but not completely abrogated, a fact that could indicate that TLR4 signalling could also be modulated by cooperative recognition of glycosylation by lectins and Toll-like receptors as it has previously been shown with HIV concerning TLR-4 and DC-SIGN. It should also be considered that, blocking TLR-4 with antibodies could prevent this modulationcooperation (and even shed GP binding), suggesting the possibility that TLR4 acts in concert with other plasma membrane-associated proteins to mediate productive uptake of filoviruses.Moreover, since GP distribution on the surface of Ebola virus is not the same as soluble shed GP even if their trimeric structure and glycosylation are almost identical, this may affect the type of receptors that they engage and could explain the wide range of receptors that surface GP on the virion can bind. It is also possible that shed GP binds similar receptors but, contrarely to VLPs and virus, does not activate cells response due to differences and indeed lack of viral spike conformation. More experiments including blocking different cell receptors with antibodies, silencing them or even using KO animal models, should be done in order to clarify this.It is likely that C-type lectins increase filovirus attachment to cells rather than serving as cellular receptors that mediate internalization of the virus into endosomes[START_REF] Matsuno | C-type lectins do not act as functional receptors for filovirus entry into cells[END_REF]. MBL, as a soluble C-type lectin receptor of Ebola virus, could target the virion and facilitate its binding to the surface of a cell. Although cellular MBL receptors have not been completely identified, potential receptors include C1qRp, CD35 and CD91[START_REF] Mcgreal | Structure-function studies of the receptors for complement C1q[END_REF] Gasque 2002, Takahashi and Ezekowitz 2005). However, we hypothesize that the high amounts of shed GP produced during Ebola virus infection may trigger MBL thus preventing virus binding to MBL and therefore, promoting viral attachment to cell surfaces.

  deficiency results from genetic defects caused by single nucleotide polymorphisms (SNPs) that occur in 5-30% of the population, depending on ethnicity. Combinations of these SNPs result in reduced blood concentration and/or dysfunctional MBL proteins. As a result, human MBL blood levels vary

Figure 29 .

 29 Figure 29.-Role of shed GP on virus spreading.-During EBOV infection, virions and soluble glycoprotein shed GP are released into the medium. Since

Figure 30 .

 30 Figure 30.-Role of shed GP during EBOV pathogenesis.-In our model, EBOV-infected cells release both virions, soluble shed GP and cytokines (CK) (i). Shed GP binds to DCs and macrophages via TLR4 and activates these cells for release of pro-and anti-inflammatory cytokines (ii). The soluble nature of shed GP enables triggering of additional and remote targets which are still not infected. Release of cytokines from these cells will further amplify inflammation by recruiting and activating other APCs, lymphocytes, endothelial cells, and other cell types including hepatocytes. Overall the release of shed GP is likely to contribute to increased permeability, disseminated intravascular coagulation, disregulated inflammation and impairment of cell function and organ failure (iii, iv, v). Shed GP itself and the CKs released due to its binding to primary targets, are able to directly induce expression of tissue factor (TF) on monocytes, macrophages and endothelial cells that will initiate the coagulation pathway (iii). Shed GP and CKs can also activate MBL and MASP-1 will contribute to coagulation activation and clot formation (iii) and MASP-2 will promote C4 fixation and complement pathway activation (iv), thus preventing pathogen opsonisation by phagocytic cells. Since shed GP and CKs are both able to induce expression of adhesion molecules such as ICAM-1 and VCAM-1, they may increase leukocyte rolling and firm fixation to the endothelium; moreover shed GP itself and CKs can also induce EC permeability (v) thus probably contributing to vascular impairment observed during EBOV infection. Finally, binding of shed GP to similar receptors as surface GP could contribute to virus spreading (vi) through the bloodstream by preventing EBOV from binding neighboring cells. Altogether, our model indicates that shed GP contributes to the high pathogenicity of Ebola virus by altering essential systems to the detriment of host homeostasis and survival.

  cells were transfected with plasmids phCMVGP-HS or phCMVGP 1,2Δ and culture medium was collected 36h post-transfection. Medium was clarified by low-speed centrifugation and subjected to ultracentrifugation through a linear 5-25% (w/w) sucrose gradient prepared in co-IP buffer (1% NP40, 0.4% deoxycholate, 5 mM EDTA, 100 mM NaCl, 20 mM Tris-HCl, pH 7.5, 25 mM iodacetamide) on a Beckman Optima L-70K ultracentrifuge using a SW60 rotor for 21 h at 40 000 rpm, 10°C. Fractions were collected from the bottom to the top, proteins were separated by SDS-PAGE and analyzed by Western blot using anti-GP antibodies.Binding assay5 × 10 5 monocyte-derived DCs or macrophages per well were placed in conicbottom 96-well plates and plates were centrifuged at 2000 rpm for 2 min. The culture medium was removed and replaced by medium containing EBOV GPs (in VP-SFM medium, Gibco). Cells were incubated for 1 hour at 4°C, washed twice with ice-cold phosphate-buffered saline (PBS) and then incubated in FACS buffer (PBS containing 2% FBS, 2% normal rat serum, 2% normal hamster serum, 2% normal mouse serum and 2% human FcR blocking solution, Milteny Biotec) at 4°C for 30 min. Cells were then incubated with mouse anti-GP 1 antibody for 1 hour at 4°C, washed twice with PBS and incubated with antimouse IgG1 coupled to Alexa 488 for DCs and MØ or to Allophycocyanin (APC) for lymphocytes for 30min at 4°C. All mAbs were purchased from BD Pharmingen except those indicated otherwise. After incubation with antibodies, cells were washed and fixed with 1% PFA solution. Cells were analyzed using a Becton Dickinson LSRII flow cytometer (BD) and FlowJo software. In some experiments samples of GP were incubated with human sera containing MBL (150ng/ml) or MBL-deficient sera (Statens Serum Institute, Denmark) for 1 hour at 4°C prior to addition to immune cells. In TLR4-related experiments, cells were incubated for 10 min at room temperature with 30 μg/ml of anti-TLR4 antibodies (HTA125) or unspecific isotype antibody prior to addition of EBOV GPs.Treatment of cells with EBOV GPs5×10 5 of monocyte-derived DC or macrophages per well were placed in 48-well plates and after 5 days differentiation, media was replaced with 600 μl of fresh RPMI 1640 containing 200 mM L-glutamine, 10 mM HEPES buffer, 5 mM CaCl 2 and 400 μl of culture supernatants containing soluble EBOV glycoproteins (in VP-SFM medium) or 500 pg/ml of LPS as positive control. Cells were incubated for 4, 8, 12 and 24h at 37°C and culture supernatants were collected, clarified from cell debris by centrifugation (1600 rpm at 4°C for 10 min), aliquoted, and stored at -80°C until analysis. The cells were also used for RNA extraction and real time RT-PCR analysis. Experiments using either anti-TLR4 antibodies or sera containing MBL were performed as described above.

  iScript cDNA Synthesis kit (Bio-Rad); reaction was setup by adding 2 µl of RNA template to 8 µl of iScript reaction mix, 2 µl of iScript reverse transcriptase and 28 µl nuclease-free water. Complete reaction mix was then incubated at 5 minutes at 25ºC, then 30 minutes at 42ºC and finally 5 minutes at 85ºC. PCR products were then synthesized from cDNA samples by real-time PCRs using FastStart Universal SYBR Green Master (ROX) in triplicate following manufacturer's recommendations. Briefly, each reaction was performed in a total volume of 50 µl containing: 25 µl of FastStart Universal SYBR Green Master (ROX), 0,5 µl of forward primer and 0,5 µl of reverse primer at a final concentration of 300 µM each, 23 µl of RNase/DNAse free water and 1 µl of template cDNA. Parameters used to program STEPONE Plus machine (Roche) were 10 minutes at 95ºC in order to activate the FastStart Taq DNA Polymerase and then 40 cycles comprising: 10 seconds at 95ºC and 30 seconds at 60ºC in order to amplify PCR products. Validated RT-PCR primers specific for human TNFα (5'-CCTGCCCCAATCCCTTTATT and 5'-CCCTAAGCCCCCAATTCTCT), IL-6 (5'-TGCAATAACCACCCCTGACC and 5used to quantify mRNA levels. The real-time PCR analysis was performed on STEPONE Plus apparatus using Roche software. Relative quantification was made by normalization to GAPDH mRNA levels. All samples were also normalized to mock.

  GP mixed with deglycosylation enzymes inactivated at 80ºC for 30 min were used as a control. Monocyte-derived DCs and macrophages were treated with 100 µl of deglycosylated shed GP samples for 24 hours and culture supernatants were analysed by western blot in nonreducing conditions and assayed for the presence of TNFα using a Multiplex cytometric bead array (Bio-Rad) in Luminex MAGPIX.HUVEC permeabilization assayHUVEC were grown in collagen-coated inserts of permeability assay chambers (Millipore) for 72h until confluence. The media was replaced with 600 μl of fresh EBM (endothelial cell basal medium without phenol red, Lonza) and 400 μl of culture supernatants containing soluble EBOV glycoproteins (VP-SFM medium, Gibco) or 400 μl of culture supernatants from MØ previously stimulated with 2 µg of soluble EBOV glycoproteins for 24h. After 22 h of incubation, FITC-Dextran was added to each insert and FITC-Dextran leaking out into the bottom chamber was measured. Monolayer permeability was assessed both on the basis of fluorescence intensities measured using a Tecan fluorometer and the integrity of the HUVEC monolayer using a MacroFluo microscope.

10mM pH 4

 4 was coupled to the surface of the sensorchip by injection into one channel at 10 μL/minute flow rate to a refractive unit (RU) value of 7000. Another channel was used as a negative control. Deactivation of unreacted NHS-esters was done by injection of 80 μl of ethanolamine 1M pH 8.5 (Amine Coupling Kit, GE Healthcare) at 10μl/min for 7min. Analytes containing soluble glycoproteins in running buffer with no detergent (TBS 10 mM HEPES buffer [pH 7.4], 150 mM NaCl, 10mM CaCl2) were then were flowed onto the coated surface (10 μl/minute for a 7-minute association time), after which the channels were rinsed with the running buffer (TBS 10 mM HEPES buffer [pH 7.4], 150 mM NaCl, 10mM CaCl2, 0.05% surfactant P-20 10%) to analyze the dissociation phase. Sensorchip was regenerated by the injection of 100 μl of 2M NaCl at 10 μl/ml for 7 minutes. Affinities were determined by analysis of the association using BIAevaluation software. Measurement of shed GP binding to rMBL by ELISA Shed GP bound to rMBL was evaluated by ELISA. Briefly, flat-bottomed maxisorp polystyrene tissue culture plates (Nunc) were coated in triplicate overnight at 4°C with recombinant MBL (20 µg/well) in TBS containing 10 mM CaCl 2 . Wells were then incubated with supernatants containing VSV GP (as positive control), VSV G (as negative control) and supernatants containing 1 µg of HS, 500 ng of HS, 250 ng of HS, GP ΔTM , LS, sGP, mock (supernatant from GFP expressing cells) from transiently transfected cells. After 1 hour of incubation at room temperature, wells were blocked for 1 hour at room temperature with 3% milk in TBS buffer containing 10 mM CaCl2. Wells were then washed three times with 200 µl of TBS 0,1% Tween containing 10 mM CaCl2 and incubated with 1:20000 dilution of α-GP antibody 3357 in TBS containing 10 mM CaCl2. After consecutive washing as before, wells were incubated for 30 minutes at room temperature with 100 µl containing secondary antibody α-mouse-HRP at 1:20000 dilution in TBS 10 mM CaCl2. Wells were

First

  , antibody-bead vials were sonicated for 30 seconds on ice at 10 kHz to avoid clumping. Then, 150 μl from each antibody bead vial (ICAM-1, VCAM-1, E-selectin, sTissue Factor and sThrombomodulin) were added to the Mixing Bottle and were brought to a final volume of 3 mL with Bead Diluent (provided in the kit). Quality Control 1, Quality Control 2 and standards were each reconstituted with 250 μl of deionized water, vortexed and left to sit for 10 minutes. Working standards were prepared by adding 250 μl of Assay Buffer (provided in the kit) to each of 6 polypropylene tubes. Serial dilutions were prepared by adding 125 μl of Reconstituted Standard to the 250 μl of the next tube; after mixing, 125 μl were transferred consecutively from tube 1 to 6. A 0 pg/ml Standard (Background) was made with Assay Buffer. Sixty mililiters of Wash buffer 10x (provided in the kit) were diluted into 540 mL of deionized water.

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  supernatants from recombinant viruses releasing high, low or normal amounts of shed GP into the media (named EBOV-HS, EBOV-LS and EBOV-WT respectively) for 1 hour at 37ºC. Then cells were washed three times with PBS containing Ca +2 and Mg +2 and were infected with recombinant Ebola virus expressing GFP during infection (rEBOV-GFP). Replication was determined by flow cytometry 48 hours post infection. . Lawrence P, Escudero-Perez B. Drexler JF, Corman VM, Müller MA, Drosten C, Volchkov V., Virus Research, January 2014.  Dérégulation de l'hémostase dans l'infection à filovirus. Reynard O, Escudero-Pérez B., Volchkov V., Submitted to Médecine/sciences, presentation: Soluble proteins of Ebola virus activate dendritic cells and macrophages causing release of pro-and antiinflammatory cytokines. Escudero Pérez B., Lawrence P., Volchkov V., Presented at 15 th International Negative Strand Virus meeting, Palacio de Congresos, Granada, Spain  2013 Oral presentation: Soluble proteins of Ebola virus activate dendritic cells and macrophages causing release of pro-and antiinflammatory cytokines. Escudero Pérez B., Lawrence P., Volchkov V., Presented at 5 th European Congress of Virology, Centre de Congrès de Lyon, Lyon, France Posters  2014 Poster: Ebola virus shed GP binds and activates dendritic cells and macrophages for release of pro-and anti-inflammatory cytokines. Escudero Pérez B., Lawrence P., Volchkova V., Reynard O., Volchkov V., Presented at European meeting on Viral zoonoses 2014, Saint-Raphael, France "Complement-dependent neutralization of influenza virus by a serum mannose-binding lectin." J Gen Virol 75 ( Pt 3): 615-622.  Arnold, J. N., R. A. Dwek, P. M. Rudd and R. B. Sim (2006). "Mannan binding lectin and its interaction with immunoglobulins in health and in disease." Immunol Lett 106(2): 103-110.  Arthur, R. R. (2002). "Ebola in Africa--discoveries in the past decade." Euro Surveill 7(3): 33-36.  Ayithan, N., S. B. Bradfute, S. M. Anthony, K. S. Stuthman, J. M. Dye, S. Bavari, M. Bray and K. Ozato (2014). "Ebola virus-like particles stimulate type I interferons and proinflammatory cytokine expression through the toll-like receptor and interferon signaling pathways." J Interferon Cytokine Res 34(2): 79-89.  Babovic-Vuksanovic, D., K. Snow and R. M. Ten (1999). "Mannosebinding lectin (MBL) deficiency. Variant alleles in a midwestern population of the United States." Ann Allergy Asthma Immunol 82(2): 134-138, 141; quiz 142-133.  Bailly, V., Z. Zhang, W. Meier, R. Cate, M. Sanicola and J. V. Bonventre (2002). "Shedding of kidney injury molecule-1, a putative adhesion protein involved in renal regeneration." J Biol Chem 277(42): 39739-39748.  Bogdanov, V. Y., V. Balasubramanian, J. Hathcock, O. Vele, M. Lieb and Y. Nemerson (2003). "Alternatively spliced human tissue factor: a circulating, soluble, thrombogenic protein." Nat Med 9(4): 458-462.  Bosio, C. M., M. J. Aman, C. Grogan, R. Hogan, G. Ruthel, D. Negley, M. Mohamadzadeh, S. Bavari and A. Schmaljohn (2003). "Ebola and Marburg viruses replicate in monocyte-derived dendritic cells without inducing the production of cytokines and full maturation." J Infect Dis 188(11): 1630-1638.  Bosio, C. M., B. D. Moore, K. L. Warfield, G. Ruthel, M. Mohamadzadeh, M. J. Aman and S. Bavari (2004). "Ebola and Marburg virus-like particles activate human myeloid dendritic cells." Virology 326(2): 280-287.  Bradfute, S. B., P. E. Swanson, M. A. Smith, E. Watanabe, J. E. McDunn, R. S. Hotchkiss and S. Bavari (2010). "Mechanisms and consequences of ebolavirus-induced lymphocyte apoptosis." J Immunol 184(1): 327-335.  Bradfute, S. B., K. L. Warfield and S. Bavari (2008). "Functional CD8+ T cell responses in lethal Ebola virus infection." J Immunol 180(6): 4058-4066.  Bradfute, S. B., K. L. Warfield and M. Bray (2012). "Mouse models for filovirus infections." Viruses 4(9): 1477-1508.  Branco, L. M., J. N. Grove, L. M. Moses, A. Goba, M. Fullah, M. Momoh, R. J. Schoepp, D. G. Bausch and R. F. Garry (2010). "Shedding of soluble glycoprotein 1 detected during acute Lassa virus infection in human subjects." Virol J 7: 306.  Bray, M. and T. W. Geisbert (2005). "Ebola virus: the role of macrophages and dendritic cells in the pathogenesis of Ebola hemorrhagic fever." Int J Biochem Cell Biol 37(8): 1560-1566.  Bray, M., S. Hatfill, L. Hensley and J. W. Huggins (2001). "Haematological, biochemical and coagulation changes in mice, guineapigs and monkeys infected with a mouse-adapted variant of Ebola Zaire virus." J Comp Pathol 125(4): 243-253.  Bray, M. and S. Mahanty (2003). "Ebola hemorrhagic fever and septic shock." J Infect Dis 188(11): 1613-1617.  Breitling, J. and M. Aebi (2013). "N-linked protein glycosylation in the endoplasmic reticulum." Cold Spring Harb Perspect Biol 5(8): a013359.  Brindley, M. A., C. L. Hunt, A. S. Kondratowicz, J. Bowman, P. L. Sinn, P. B. McCray, Jr., K. Quinn, M. L. Weller, J. A. Chiorini and W. Maury (2011). "Tyrosine kinase receptor Axl enhances entry of Zaire ebolavirus without direct interactions with the viral glycoprotein." Virology 415Brudner, M., M. Karpel, C. Lear, L. Chen, L. M. Yantosca, C. Scully, A. Sarraju, A. Sokolovska, M. R. Zariffard, D. P. Eisen, B. A. Mungall, D. N. Kotton, A. Omari, I. C. Huang, M. Farzan, K. Takahashi, L. Stuart, G. L. Stahl, A. B. Ezekowitz, G. T. Spear, G. G. Olinger, E. V. Schmidt and I. C. Michelow (2013). "Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane C-type lectin receptors." PLoS One 8(4): e60838.  Bukreyev, A., P. E. Rollin, M. K. Tate, L. Yang, S. R. Zaki, W. J. Shieh, B. R. Murphy, P. L. Collins and A. Sanchez (2007). "Successful topical respiratory tract immunization of primates against Ebola virus." J Virol 81(12): 6379-6388.  Bukreyev, A., L. Yang, J. Fricke, L. Cheng, J. M. Ward, B. R. Murphy and P. L. Collins (2008). "The secreted form of respiratory syncytial virus G glycoprotein helps the virus evade antibody-mediated restriction of replication by acting as an antigen decoy and through effects on Fc receptor-bearing leukocytes." J Virol 82(24): 12191-12204.  Bukreyev, A. A., E. F. Belanov, V. M. Blinov and S. V. Netesov (1995). "Complete nucleotide sequences of Marburg virus genes 5 and 6 encoding VP30 and VP24 proteins." Biochem Mol Biol Int 35(3): 605-613.  Burzyn, D., J. C. Rassa, D. Kim, I. Nepomnaschy, S. R. Ross and I. Piazzon (2004). "Toll-like receptor 4-dependent activation of dendritic cells by a retrovirus." J Virol 78(2): 576-584.  Caescu, C. I., G. R. Jeschke and B. E. Turk (2009). "Active-site determinants of substrate recognition by the metalloproteinases TACE and ADAM10." Biochem J 424(1): 79-88.  Gupta, M., C. Spiropoulou and P. E. Rollin (2007). "Ebola virus infection of human PBMCs causes massive death of macrophages, CD4 and CD8 T cell sub-populations in vitro." Virology 364(1): 45-54.  Halfmann, P., G. Neumann and Y. Kawaoka (2011). "The Ebolavirus VP24 protein blocks phosphorylation of p38 mitogen-activated protein kinase." J Infect Dis 204 Suppl 3: S953-956.  Haller, O., G. Kochs and F. Weber (2006). "The interferon response circuit: induction and suppression by pathogenic viruses." Virology 344(1): 119-130.  Halpin, K., P. L. Young, H. E. Field and J. S. Mackenzie (2000). "Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus." J Gen Virol 81(Pt 8): 1927-1932.  Harrison, S. C. (2008). "Viral membrane fusion." Nat Struct Mol Biol 15(7): 690-698.  Hartshorn, K. L., K. Sastry, M. R. White, E. M. Anders, M. Super, R. A. Ezekowitz and A. I. Tauber (1993). "Human mannose-binding protein functions as an opsonin for influenza A viruses." J Clin Invest 91Harty, R. N., M. E. Brown, G. Wang, J. Huibregtse and F. P. Hayes (2000). "A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding." Proc Natl Acad Sci U S A 97(25): 13871-13876.  Hayman, D. T., M. Yu, G. Crameri, L. F. Wang, R. Suu-Ire, J. L. Wood and A. A. Cunningham (2012). "Ebola virus antibodies in fruit bats, Ghana, West Africa." Emerg Infect Dis 18(7): 1207-1209.  Haynes, L. M., D. D. Moore, E. A. Kurt-Jones, R. W. Finberg, L. J. Anderson and R. A. Tripp (2001). "Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus." J Virol 75(22): 10730-10737.  Hensley, L. E. and T. W. Geisbert (2005). "The contribution of the endothelium to the development of coagulation disorders that characterize Ebola hemorrhagic fever in primates." Thromb Haemost 94(2): 254-261.  Hensley, L. E., E. L. Stevens, S. B. Yan, J. B. Geisbert, W. L. Macias, T. Larsen, K. M. Daddario-DiCaprio, G. H. Cassell, P. B. Jahrling and T. W.

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  2000). "Downregulation of beta1 integrins by Ebola virus glycoprotein: implication for virus entry." Virology 278(1): 20-26.  Takahashi, K. (2011). "Mannose-binding lectin and the balance between immune protection and complication." Expert Rev Anti Infect Ther 9(12): 1179-1190.  Takahashi, K., W. C. Chang, M. Takahashi, V. Pavlov, Y. Ishida, L. La Bonte, L. Shi, T. Fujita, G. L. Stahl and E. M. Van Cott (2011). "Mannosebinding lectin and its associated proteases (MASPs) mediate coagulation and its deficiency is a risk factor in developing complications Takahashi, K. and R. A. Ezekowitz (2005). "The role of the mannosebinding lectin in innate immunity." Clin Infect Dis 41 Suppl 7: S440-444.  Takahashi, K., W. E. Ip, I. C. Michelow and R. A. Ezekowitz (2006). "The mannose-binding lectin: a prototypic pattern recognition molecule." Curr Opin Immunol 18(1): 16-23.  Tal, G., A. Mandelberg, I. Dalal, K. Cesar, E. Somekh, A. Tal, A. Oron, S. Itskovich, A. Ballin, S. Houri, A. Beigelman, O. Lider, G. Rechavi and N. Amariglio (2004). "Association between common Toll-like receptor 4 mutations and severe respiratory syncytial virus disease." J Infect Dis 189(11): 2057-2063.  Taylor, D. J., R. W. Leach and J. Bruenn (2010). "Filoviruses are ancient and integrated into mammalian genomes." BMC Evol Biol 10: 193.  Warfield, K. L. and G. G. Olinger (2011). "Protective role of cytotoxic T lymphocytes in filovirus hemorrhagic fever." J Biomed Biotechnol 2011: 984241.  Warren, T. K., K. L. Warfield, J. Wells, D. L. Swenson, K. S. Donner, S. A. Van Tongeren, N. L. Garza, L. Dong, D. V. Mourich, S. Crumley, D. K. Nichols, P. L. Iversen and S. Bavari (2010). "Advanced antisense therapies for postexposure protection against lethal filovirus infections." Nat Med 16(9): 991-994.  Wauquier, N., P. Becquart, C. Gasquet and E. M. Leroy (2009). "Immunoglobulin G in Ebola outbreak survivors, Gabon." Emerg Infect Dis 15(7): 1136-1137.  Wauquier, N., P. Becquart, C. Padilla, S. Baize and E. M. Leroy (2010). "Human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis." PLoS Negl Trop Dis 4(10).  White, B., M. Schmidt, C. Murphy, W. Livingstone, D. O'Toole, M. Lawler, L. O'Neill, D. Kelleher, H. P. Schwarz and O. P. Smith (2000). "Activated protein C inhibits lipopolysaccharide-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) and tumour necrosis factor alpha (TNF-alpha) production in the THP-1 monocytic cell line." Br J Haematol 110(1): 130-134.

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  increases the diversity of the proteome to a level unmatched by any other post-translational modification. The cell is able to facilitate this diversity, because almost every aspect of glycosylation can be modified, including: Glycosidic linkage -the site of glycan (oligosaccharide) binding  Glycan composition -the types of sugars that are linked to a particular protein  Glycan structure -branched or unbranched chains  Glycan length -short-or long-chain oligosaccharides Glycosylation is one of the most complex post-translational modification because of the large number of enzymatic steps involved. Glycosylation process includes linking monosaccharides together, transferring sugars from one substrate to another and trimming sugars from the glycan structure.Glycosylation is non-templated, and thus, all of these steps do not necessarily occur during every glycosylation event. Instead of using templates, cells rely on a host of enzymes that add or remove sugars from one molecule to another to generate the diverse glycoproteins seen in a given cell. The different mechanisms of glycosylation are highly-ordered, step-wise reactions in which individual enzyme activity is dependent upon the completion of the previous enzymatic reaction. Because enzyme activity varies by cell type and intracellular compartment, cells can synthesize glycoproteins that differ from other cells in glycan structure[START_REF] Spiro | Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds[END_REF].Types of GlycosylationGlycopeptide bonds can classified in 5 groups (N-, O-and C-linked glycosylation, glypiation and phosphoglycosylation) based on the nature of the sugar-peptide bond and the oligosaccharide attached. N-and O-glycosylation are the glycosylations present in EBOV.N-linkedGlycan bind to the amino group of asparagine in the ERO-linkedMonosaccharides bind to the hydroxyl group of serine or threonine in the ER, Golgi, cystosol and nucleus Glypiation Glycan core links a phospholipid and a proteinC-linkedMannose binds to the indole ring of tryptophanPhosphoglycosylationGlycan binds to serine via phosphodiester bond Proteins are not restricted to a particular type of glycosylation. Indeed, proteins are often glycosylated at multiple sites with different glycosidic linkages, which depends on the enzime availability to glycosylate, the amino acid sequence and the protein conformation. Glycoproteins are unique from other post-translationally modified proteins, because they are a combination of a protein portion and a glycan portion, both of which can comprise a significant proportion of the molecular weight of the molecule.

  N-glycosylation is the most common glycosidic linkage, O-glycoproteins also play a key role in cell biology. This type of glycosylation is essential in the biosynthesis of mucins, a family of heavily O-glycosylated, high-molecular weight proteins that form mucus secretions. O-glycosylation is also critical for the formation of proteoglycan core proteins that are used to make extracellular matrix components. O-glycosylation occurs post-translationally on serine and threonine side chains in the Golgi apparatus. N-glycosylation does not preclude the other from occurring, as O-glycosylation commonly occurs on glycoproteins that were Nglycosylated in the ER. Besides the different linkage, O-glycosylation also differs in the method of glycosylation. While a precursor glycan is transferred en bloc to Asn via N-glycosylation, sugars are added one-at-a-time to serine or threonine residues. O-glycosylation can also occur on oxidized forms of lysine and proline. Additionally, O-linked glycans usually have much simpler oligosaccharide structures than N-linked glycans (Gemmill and Trimble 1999, Dell and Morris 2001).GP activity is mediated through cellular toll-like receptor 4 (TLR4) and is dependent on GP glycosylation. Treatment of cells with anti-TLR4 antibody completely abolishes shed GP-induced activation of cells. We also demonstrate that shed GP activity is negated upon addition of mannose-binding sera lectin MBL, a molecule known to interact with sugar arrays present on the surface of different microorganisms.Furthermore, we highlight the ability of shed GP to affect endothelial cell function both directly and indirectly, demonstrating the interplay between shed GP, systemic cytokine release and increased vascular permeability. In conclusion, shed GP released from virus-infected cells could activate non-infected DCs and macrophages causing the massive release of pro-and anti-inflammatory cytokines and effect vascular permeability. These activities could be at the heart of the excessive and dysregulated inflammatory host reactions to infection and thus contribute to high virus pathogenicity.Author SummaryEbola virus, a member of the Filoviridae family, causes lethal hemorrhagic fever in man and primates, displaying up to 90% mortality rates. Viral infection is typified by an excessive systemic inflammatory response resembling septic shock. It also damages endothelial cells and creates difficulty in coagulation, ultimately leading to haemorrhaging, organ failure and death.A unique feature of EBOV is that following infection high amounts of truncated surface GP, named shed GP, are released from infected cells and are detected in the blood of patients and experimentally infected animals. However the role of shed GP in virus replication and pathogenicity is not yet clearly defined.

  treated under non denaturating and non-reducing conditions with a mix of N-and Oglycosidases and then used for incubation with immune cells, as detailed above. As shown in figure6Athe treatment with deglycosylation enzymes reduced the molecular weight of shed GP as expected, in comparison to shed GP treated with the same mix of deglycosidases that were previously inactivated by heating for 30 min at 80°C (Figure6A, compare lanes 1 and 2). DCs and Mø were incubated with shed GP samples or LPS alone for 12h, and then culture supernatants were analyzed by ELISA for amounts of secreted TNFα. As an additional control cells were subjected to incubation with LPS following incubation with deglycosylated shed GP. Strikingly, as shown in Figure6B, deglycosylation of shed GP considerably decreased the release of TNFα in both DCs and Mø, confirming the importance of the glycosylation pattern of shed GP for its activity. Cells incubated with deglycosylated shed GP remained viable and capable of responding to LPS stimuli ruling out a possible negative effect of deglycosydases on cells.

  and then were enriched by centrifugation of PBMCs through a Percoll density gradient.The recovered monocyte fraction was depleted of cell contaminants by positive selection (Miltenyi Biotec), ensuring purification rates of ≥95%. Immature DCs and monocyte-derived macrophages were obtained upon incubation of cells in complete RPMI-1640 (Life Technologies) supplemented with 200 mM L-glutamine (Life Technologies), 10 mM HEPES buffer (Life Technologies), 10% de-complemented FCS and 8 μg/ml of Gentamicine (Life Technologies) for 5 days either with 40 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) or 50 ng/ml of interleukin-4 (IL-4) and 40 ng/ml of macrophage colony-stimulating factor (M-CSF), respectively.

pH 7 . 5 ,

 75 25 mM iodacetamide) on Beckman Optima L-70K ultracentrifuge using a SW60 rotor for 21 h at 40 000 rpm, 10°C. Fractions were collected from the bottom to the top, proteins separated by SDS-PAGE and analyzed by Western blot using anti--derived DCs or macrophages per well were placed in conic-bottom 96-well plates and plates were centrifuged at 2000 rpm for 2 min. The culture medium was removed and replaced by medium containing EBOV GPs (in VP-SFM medium, Gibco). Cells were incubated for 1 hour at 4°C, washed twice with ice-cold phosphatebuffered saline (PBS) and then incubated in FACS buffer (PBS containing 2% FBS, 2% normal rat serum, 2% normal hamster serum, 2% normal mouse serum and 2% human FcR blocking solution, Milteny Biotec) at 4°C for 30 min. Cells were then incubated with mouse anti-GP1 antibody for 1 hour at 4°C, washed twice with PBS and incubated with anti-mouse IgG1 coupled to Alexa 488 for DCs and Mø or to Allophycocyanin (APC) for lymphocytes for 30min at 4°C. All mAbs were purchased from BD Pharmingen except those indicated otherwise. After incubation with antibodies, cells were washed and fixed with 1% PFA solution. Cells were analyzed using a Becton Dickinson LSRII flow cytometer (BD) and FlowJo software. In some experiments samples of GP were incubated with human sera containing MBL (150ng/ml) or MBLdeficient sera (Statens Serum Institute, Denmark) for 1 hour at 4°C prior to addition to immune cells. In TLR4-related experiments, cells were incubated for 10 min at room temperature with 30 μg/ml of anti-TLR4 antibodies (HTA125) or unspecific isotype antibody prior to addition of EBOV GPs. The statistical significance of the differences in median fluorescence intensity (MFI) values for each donor were evaluated by paired-

(

  endothelial cell basal medium without phenol red, Lonza) and 400 μl of culture supernatants containing soluble EBOV glycoproteins (VP-SFM medium, Gibco) or 400 μl of culture supernatants from Mø previously stimulated with soluble EBOV glycoproteins for 24h. After 22 h of incubation, FITC-Dextran was added to each insert and FITC-Dextran leaking out into the bottom chamber was measured. Monolayer permeability was assessed on the basis of both fluorescence intensities measured using a Tecan fluorometer and the integrity of the HUVEC monolayer using a MacroFluo microscope.

Figure 1 .

 1 Figure 1. Production of recombinant EBOV shed GP and analysis of shed GP

day 6

 6 post-infection (5µl, dilutions 1/4, 1/8, 1/16 & 1/32, lanes 2-5), supernatant from EBOV-infected Vero E6 cells (20µl, EBOV Vero, lane 1) and a sample of concentrated recombinant shed GP (5µl, HS, lane 6) were analyzed by Western blot using anti-GP2 antibodies. Estimation of shed GP amounts in the sera of animals in comparison with shed GP from GP-HS expressing cells was performed using ImageQuantTL software (GE Lifesciences). Sera of animals contained approximately 85 times higher amounts of shed GP than those used in experiments with immune cells. (D). Samples of sera collected at different intervals post infection from Mock (day 3 p.i.) -and EBOVinfected guinea pigs(animal numbers 192, 193, 195 & 196) as indicated, were analyzed by Western blot using anti-GP2 antibodies. The release of shed GP dramatically increased during the course of infection. Sera of infected animals contains both virion GP and shed GP as demonstrated by the presence of full-length virion-associated GP 2 and truncated GP 2 (GP 2Δ ).

Figure 2 .

 2 Figure 2. EBOV shed GP binding to DCs and macrophages.

Figure 3 .

 3 Figure 3. Shed GP induces transcriptional activation of cytokines in DCs and MØ.DCs and MØ (5 x 10 5 cells) were incubated with concentrated culture supernatants as

Figure 4 .

 4 Figure 4. Shed GP induces secretion of cytokines from DCs and MØ. (A) 5×10 5 of

Figure 5 .

 5 Figure 5. Shed GP induces the phenotypic maturation of DCs and MØ. 5×10 5 of

Figure 6 .

 6 Figure 6. Deglycosylation of shed GP affects activation of DCs and macrophages.

Figure 7 .

 7 Figure 7. EBOV Shed GP increases cell permeability of HUVEC in vitro. HUVEC

Figure 8 .

 8 Figure 8. Role of shed GP during EBOV infection. In our model, EBOV-infected

Figure S1 .

 S1 Figure S1. (A and B) Quantification of shed GP (A) and sGP (B) produced in GP-HS-

Figure

  Figure S2. (A) Schematic representation of EBOV surface GP, shed GP and a truncated

Figure S3 .

 S3 Figure S3. Quantitative data and statistical analysis of data presented in Figure2.

Figure 1 :

 1 Figure 1 : Les chauves-souris, les réservoirs présumés des filovirus : a) Hypsignathus monstrosus, b) Epomops franqueti, c) Myonycteris torquata, d) Rousettus aegyptiacus, e) Miniopterus Schreibersi

  Durant l'évolution de la maladie, les modifications hématologiques comprennent une lymphopénie, une hyperleucocytose des polynucléaires, et au niveau biochimiques une élévation des concentrations sériques d'aspartate et d'alanine aminotransférases caractéristique de l'atteinte hépatique(18). Une thrombocytopénie, associée à une augmentation des D-dimers (produit de dégradation de la fibrine) et une élévation du temps de céphaline activée sont retrouvées et sont les marqueurs du syndrome hémorragique et notamment de la coagulation intra vasculaire disséminé (CIVD). Cette CIVD est une caractéristique majeure de l'infection à filovirus.La CIVD, une origine multifactorielleLa CIVD se caractérise par la formation localisée de multiples caillots dans la circulation sanguine ce qui a pour effet de consommer les facteurs de coagulation et les plaquettes. Ce syndrome induit des thromboses localisées entraînant des défaillances fonctionnelles et simultanément une perte de la coagulation sanguine par déplétion des facteurs de coagulations. Lors des fièvres hémorragiques à filovirus, la CIVD est retrouvée chez l'homme ainsi que dans les modèles infectieux de primates non-humains cependant elle n'est pas retrouvée chez les rongeurs à l'exception du hamster(35). La CVID semble donc être dépendant de l'espèce, a contrario, la dérégulation du système immunitaire est retrouvée dans tous les modèles animaux et joue un rôle majeur dans l'absence de contrôle de la réplication virale. La CIVD trouve son origine dans plusieurs facteurs, certains étant liés directement aux virus, d'autres provenant des dommages induits et de l'emballement de la réponse inflammatoire.Le facteur tissulaire (TF)Les études sur le virus Ebola Zaïre dans un modèle primate ont montré que les anomalies de la coagulation sont initiées en tout début d'infection, lorsque les macrophages infectés expriment le facteur tissulaire (TF) à leurs surfaces(14). Cette glycoprotéine de 47kDa est un récepteur transmembranaire exprimé par les épithéliums, les muqueuses, la capsule des organes et de manière inductible par les macrophages, les monocytes et les cellules endothéliales. Le TF est exprimé dans des compartiments n'étant pas en contact avec le sang et cette protéine joue un rôle de senseur des hémorragies, en induisant la coagulation du sang lorsque celui-ci entre en contact avec ces tissus. Cette protéine est également retrouvée, de façon pathologique dans la circulation sanguine, à la surface de microparticules membranaires et ce notamment dans les cas de CIVD. Ces microparticules sont notamment libérées par les monocytes.

  nematode anticoagulant protein c2 inhibe directement l'activité du complexe TF/facteur VIIaen se fixant sur le facteur X. Le Xigris, lui, est une forme recombinante de la protéine C activée (APC). Cette protéine recombinante est indiqué dans le traitement des chocs septiques quelle qu'en soit la cause. La protéine C activée est un élément clef de la régulation de la coagulation et dans des modèles primates, il a pu être montré clairement une chute du taux d'APC au cours de l'infection(12, 14). Cependant, une récente étude a montré que pendant l'épidemie à Sudan Ebolavirus de 2000-2001, il n'y avait pas d'élévation du TF ni de corrélation entre son taux et la présence de signe hémorragique ou la létalité(28). Les différences observées entre le modèle primate et l'homme pourraient être de nature hôte spécifique ou virus spécifique (Zaïre ebolavirus dans l'étude primate).Une des caractéristiques des infections à filovirus est la libération désordonnée de cytokine pro et anti inflammatoire et notamment d'IFN de type I, de TNFα, d'IL-1, IL-2, IL-6IL, IL-8, IL-10 et d'IL1RA. Les données bibliographiques concernant le profil des cytokines sont toutefois encore incomplètes et parfois contradictoires(IFN et TNFα)

  assurent l'échange contrôlé des molécules biologiques via un ensemble de jonctions pour l'eau et les sels (voie paracellulaire) et via une voie trans-cellulaire pour les macromolécules. Il existe des données contradictoires concernant le rôle des cellules endothéliales dans les infections à filovirus. En effet celles-ci peuvent répliquer le virus et s'activer, cependant leur infection in vivo n'a été montrée chez le singe qu'à des stades très tardifs de l'infection. De plus, il n'a pas été noté post mortem chez le singe et l'homme de dommages vasculaires notables. L'activation de cette barrière naturelle par le TNFα produit par les cellules infectées devrait conduire à un remodelage de leur architecture, ceci entraînant une fuite de liquide vers l'interstitium. Etonnamment la sGP une protéine spécifique au virus Ebola, a montré un rôle protecteur contre les effets du TNFα sur ces cellules. La glycoprotéine structurale a quant à elle montré des propriétés inverses(46). De plus l'activation des cellules endothéliales induit l'expression à leurs surfaces du facteur tissulaire qui comme nous l'avons décrit précédemment, est initiateur de la coagulation. Ainsi ces cellules pourraient participer à la mise en place de la CIVD sans toutefois subir de modification majeure de leur architecture. Cependant la plupart des données disponibles sur ces cellules relèvent de modèle in vitro et des études complémentaires in vivo seraient nécessaires afin de mieux identifier leur rôle dans les fièvres hémorragiques à filovirus.Charges viralesLors de la phase aiguë de l'infection à virus Ebola on retrouve chez les patients des charges virales sanguines atteignant 10 6.5 pfu/ml. Ces particules arborent à leur surface la glycoprotéine d'enveloppe (GP). Cette protéine fortement glycosylée a montré des pouvoirs activateurs sur les cellules dendritiques et les macrophages grâce à son domaines mucinique(27). Il a de plus été suggéré qu'elle pourrait se lier au récepteur TLR4 et ainsi être l'initiatrice d'une forte réponse inflammatoire(30). Dans le modèle cobaye, la forme soluble (shedGP) et la forme alternative (sGP) de la glycoprotéine virale sont également retrouvées en forte quantité. Ces formes non structurales pourraient également servir d'activateurs du système immunitaire (communication personnelle). En effet, le récepteur TLR4 initialement découvert comme senseur du LPS permet une transduction du signal via Myd88 et Trif qui a pour conséquence la libération de cytokines inflammatoires et d'interférons de type 1. Enfin sa coiffe glycosylée constitue un excellent ligand pour les lectines sériques (communication personnelle). Certaines collectines sont des acteurs majeurs de la voie d'activation du complément et de l'activation de la coagulation via le recrutement des sérines-protéases sériques (MASP).

Table 1 .

 1 Ebola virus outbreaks in human beings ............................................26

Table 2 .

 2 Techniques to analyse and detect Ebola virus infection .....................41

Table 3 .

 3 Comparison of pathological features of different animal models of Ebola virus infection ..........................................................................................43

Table 4 .

 4 Efficacy of post-exposure treatment in animal models of Ebola virus infection .............................................................................................................46

Table 5 .

 5 Overview of Ebola virus vaccines .......................................................49

Table 6 .

 6 Correlative differences between survivors and non survivors of Ebola virus infection ………………………………………………………………………...82

Table 7 .

 7 Sequence of TACE cleavage sites in different EBOV strains ...........188

	IFL	Internal Fusion Loop
	IFN	InterFeroN

Table 1 )

 1 (Arthur 2002, Mahanty and Bray 2004, Baize,

	Pannetier et al. 2014).

. A number of sporadic outbreaks have been reported since the initial discovery of EBOV in 1976 (

Table 1 .-Ebola virus outbreaks in human beings

 1

. Adapted from (Arthur

to 21 days Until 6 months From 5 to 10 days REMISSION Phase II

	Incubation 2 Fever Phase I	Abdominal pain	Phase III
	Chills	Nausea	Delires
	Myalgia	Diarrhea	Coma
	Headache	Cough	Difuse coagulopathy
	Fatigue	Rash	Respiratory problems
		Ocular burning	Metabolic disorders
			Arthralgy
			Muscle pain
			Hepatitis
			Ocular affections
			Neurologic desorders
			Psycosis

  symptoms and disappear between 30 and 168 days after infection. IgG-specific antibodies develop between day 6 and 18 after onset and persist for many years. An IgM or rising IgG titre constitutes a strong

	presumptive diagnosis. Decreasing IgM, or increasing IgG titres (four-fold), or
	both, in successive paired serum samples are highly suggestive of a recent
	infection. ELISAs show a good measure of sensitivity and specificity and are
	able to detect all of the EBOV species. Antigen-capture diagnostic assays
	together with nested reverse-transcription (RT)-polymerase chain reaction
	(PCR) have been developed and used in previous outbreaks. ELISA is the best
	test for identifying patients because of its rapidity and robustness; it is
	Several serological techniques have been developed to diagnose EBOV
	infection. Nowadays, RT-PCR and antigen detection ELISA are the primary
	assays to diagnose an acute infection. Viral antigen and nucleic acid can be
	detected in blood from day 3 up to 7-16 days after onset of symptoms. For

antibody detection the most generally used assays are direct IgG and IgM ELISAs and IgM capture ELISA. IgM antibodies can appear as early as 2 days post onset of particularly likely to be positive in the more severely ill patients. RT-PCR is usually more sensitive but also more subject to artifacts and contamination.

Table 2 .-Techniques to analyse and detect Ebola virus infection.

 2

Table 3 .-Comparison of pathological features of different animal models of

 3

Ebola virus infection. Adapted from (Nakayama and Saijo 2013).

1.6.

The fight against Ebola virus 1.6.1.Treatments

Table 4 .-Efficacy of post-exposure treatment in animal models of Ebola virus

 4

infection. Adapted from

[START_REF] Nakayama | Animal models for Ebola and Marburg virus infections[END_REF]

Table 5 .-Overview of Ebola virus vaccines

 5

. Adapted from

(Hoenen, Groseth et al. 2012)

3.1.1.2.Ebola virus inhibits the interferon response

	mediated immune responses, which may contribute to the lack of control of viral
	replication.
	It appears that there is a difference in the cytokine profile observed according to
	Filovirus species. Different expression of TNF-α and IFN-γ has been observed
	between Ebola Zaire, Sudan and Bundibugyo during human infections,
	suggesting a different immune response depending on Ebola virus species
	which could explain the differences in the virulence and the mortality rate
	between the different Ebola virus species (Hutchinson and Rollin 2007, Gupta,
	MacNeil et al. 2012).
	The interferon (IFN) system represents a major element of the innate immune
	response against viral infection (Mordstein, Neugebauer et al. 2010). We can
	distinguish three types of IFN: IFN type I, which includes IFNα and IFNβ and
	produced by all cell types, IFN type II which is only represented by IFNγ and is
	produced by dendritic cells, lymphocytes T and Natural killer cells and type III
	IFN, which is represented by IFN-λ and is principally produced by endothelial
	cells. Type I interferons (IFNs) are recognized as an early and powerful host
	defense against virus infection. Virus infection generally results in the synthesis
	and secretion of the type I alpha/beta interferons (IFN-α/β). Once secreted, IFN-
	α/β acts in an autocrine or paracrine manner by binding the ubiquitously
	expressed IFN-α/β receptor (IFNAR) (Figure
	Inhibition of DC function with live or inactivated virus, but not with VLPs,
	indicates that the suppression of DC function and maturation is likely to be due
	to the presence of viral proteins or genomic material not present in the VLPs.
	Further studies are needed to clarify the detrimental effects of Ebola virus
	infection on other subpopulations of DCs, in particular on plasmacytoid DCs,
	which are important in antiviral interferon responses. The consequences of
	nonfunctional DCs include a diminished ability to stimulate humoral or cell-

Table 6 .-Correlative differences between survivors and non survivors of Ebola virus infection. Adapted from

 6

	CD8 + activation	No CD8 + activation
	Above-normal numbers of T cells	Below-normal numbers of T cells
	10 7 viral genome copies/ml serum	10 10 viral genome copies/ml serum
	Detectable antibodies in blood at	No detectable antibodies in blood at
	onset of symptoms	onset of symptoms
	Low Nitic Oxide (NO)	High NO

  HEK 293T cells and Vero E6 cells were routinely cultured in Dulbecco's

	modified Eagle's medium (DMEM, PAA laboratories) supplemented with 10%
	fetal calf serum (PerbioHyclone) and without antibiotics. For transient protein
	expression 293T cells were seeded into 150 cm2 flasks 24 h prior to
	transfection and were cultured in DMEM supplemented with 10% fetal calf
	serum (PerbioHyclone) and without antibiotics. Human umbilical vein
	endothelial cells (HUVECs) (Lonza) were maintained at 37ºC with 5% CO 2 in
	EGM-2 Media (Lonza) enriched with EGM-2 Single Quots containing FBS,
	hydrocortisone, hFGF-B, VEGF, R3-IGF-1, ascorbic acid, hEGF, GA-1000 and
	heparin. Cells were cultured for 3-4 days with EGM-2 medium and were
	passaged for no more than 5 generations for the experiments described.

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

Objectives

Publications

Acknowledgments

I would first like to thank Professor Viktor Volchkov for having welcomed me to his laboratory and for making my dream come true: since I was eight years old I have been dreaming of working with the Ebola virus. I would specially like to thank him for his trust, which has encouraged me to be more independent within the research framework, and for his bright ability to present and organise concepts.

Acknowledgments.

We are grateful to Thierry Walzer (CIRI, Lyon) for his advice in conducting experiments with immune cells. We would also like to thank Olivier Reynard for providing help in performing animal experiments. We thank the biosafety team members for their assistance in conducting experiments.

The most promising results so far were obtained when siRNA was used and a 100% survival rate in NHPs was achieved when animals were treated daily for 7 days and the first treatment started 30 minutes after challenge (Lodmell, Parnell et al. 2002).  Garbutt, M., R. Liebscher, V. Wahl-Jensen, S. Jones, P. Moller, R. Wagner, V. Volchkov, H. D. Klenk, H. Feldmann and U. [START_REF] Dolnik | Ectodomain shedding of the glycoprotein GP of Ebola virus[END_REF].  Levi, M., T. T. Keller, E. van Gorp and H. ten Cate (2003). "Infection and inflammation and the coagulation system." Cardiovasc Res 60(1): 26-39.  Levi, M. and H. Ten Cate (1999). "Disseminated intravascular coagulation." N Engl J Med 341(8): 586-592.

Results

Soluble EBOV glycoproteins bind to rMBL

Discussion

 Levi, M., T. van der Poll and H. R. Buller (2004).

Annexes

Annexes

Shed GP of Ebola Virus Triggers Systemic Immune Activation and

Increased Vascular Permeability. 1 Escudero-Pérez B., 1 Volchkova V.A., 1 Dolnik O., 1 Lawrence P., and 1* Volchkov V.E.

Abstract

During Ebola virus (EBOV) infection a significant amount of surface glycoprotein GP is shed from infected cells in a soluble form due to cleavage by cellular metalloprotease TACE. Shed GP and non-structural secreted glycoprotein sGP, both expressed from the same GP gene, have been detected in the blood of human patients and experimentally infected animals. In this study we demonstrate that shed GP plays a particular role during EBOV infection. In effect it binds and activates non-infected dendritic cells and macrophages inducing the secretion of pro-and anti-inflammatory cytokines (TNFα, IL1β, IL6, IL8, IL12p40, and IL1-RA, IL10). Activation of these cells by shed GP correlates with the increase in surface expression of co-stimulatory molecules CD40, CD80, CD83 and CD86. Contrary to shed GP, secreted sGP activates neither DC nor macrophages while it could bind DCs. In this study, we show that shed sample t test and GraphPad Prism 6 software (GraphPad Software, La Jolla California, USA).

Treatment of cells with EBOV GPs

5×10 5 of monocyte-derived DCs or macrophages per well were placed in 48-well plates and after 5 days differentiation, media was replaced with 600 μl of fresh RPMI 1640 containing 200 mM L-glutamine, 10 mM HEPES buffer, 5 mM CaCl2 and 400 μl of culture supernatants containing soluble EBOV glycoproteins (in VP-SFM medium).

Cells were incubated for 4, 8, 12 and 24h at 37°C and culture supernatants were collected, clarified from cell debris by centrifugation (1600 rpm at 4°C for 10 min), aliquoted, and stored at -80°C until analysis. The cells were used for real time RT-PCR analysis. Experiments using either anti-TLR4 antibodies or sera containing MBL were performed as described above in Binding assay.

Unless otherwise stated shed GP was used at a final concentration of 0.4 µg/ml and sGP at 7.2 µg/ml. In deglycosylation experiments shed GP was used at a concentration of 0.2 µg/ml.

Real time RT-PCR quantitative analysis

RNAs from monocyte-derived DCs and macrophages treated with EBOV GPs were isolated using Total RNA Isolation kit (Machery Nagel). cDNAs were generated using iScript cDNA Synthesis kit (Bio-Rad), followed by real-time PCRs using Sybr-Green-Master-Mix (Roche) in triplicate following manufacturer's recommendations.

Validated RT-PCR primers specific for human TNFα (5'-CCTGCCCCAATCCCTTTATT and 5'-CCCTAAGCCCCCAATTCTCT), IL-6 (5'-TGCAATAACCACCCCTGACC and 5'-TGCGCAGAATGAGATGAGTTG), IL10

(5'-GAGGCTACGGCGCTGTCAT and 5'-CCACGGCCTTGCTCTTGTT), IL12p40

(5'-CCAGAGCAGTGAGGTCTTAGGC and 5'-TGTGAAGCAGCAGGAGCG) and GAPDH (5'-CCATGTTCGTCATGGGTGTG and 5'-GGTGCTAAGCAGTTGGTGGTG) were used to quantify mRNA levels. The real-time PCR analysis was performed on Stepone Plus apparatus using Roche software. Relative quantification was made by normalization to GAPDH mRNA levels.

Cytokine release

Culture supernatants from monocyte-derived DCs or macrophages treated with soluble EBOV GPs for 24 hours were assayed for presence of TNF-α, IL-6, IL-8, IL12p40, IL1β, IL10 and IL1-RA using Multiplex cytometric bead array (Bio-Rad) in Luminex MAGPIX. The assay was performed according to the manufacturer's instructions.

Expression of co-stimulatory molecules

Both monocyte-derived DCs and macrophages treated with soluble EBOV GPs for 48 hours were washed in cold PBS supplemented with 2% fetal bovine serum and preblocked at 4°C for 30 min in FACS buffer. Cell-surface staining was performed using the following antibodies: FITC-conjugated anti-CD80, PE-conjugated anti-CD40 Retro-orbital sinus sampling of blood was performed after local anaesthesia at day 3, 6 and 9 and prior to euthanasia. The sera were purified using VenoSafe PET tubes (VenoSafe).

All animals were handled in strict accordance with good practices as defined by the French national charter on the ethics of animal experimentation. Animal work was approved by the regional ethical committee (CREEA), and experiments were performed in the INSERM Jean Mérieux BSL-4 laboratory in Lyon, France.