Malika Ouldali

Karine Moncoq

Agnès De

La Croix

La Valette

Ana A Arteni

Jean-Michel Betton

Jean Lepault
email: jean.lepault@orange.fr

Agnès De La Croix De La Valette

Jean- Michel Betton

Study of membrane deformations induced by Hepatitis C protein NS4B and its terminal amphipathic peptides

Keywords: HCV-NS4B induced membrane disorder, NS4B peptides induced membrane perforation, one NS4B peptide induced membrane fusion, Cryo-EM of membranes disordered

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INTRODUCTION

All positive-stranded RNA (+RNA) viruses modify host cytoplasmic membrane complexes, and these altered structures appear important for viral genome replication (reviewed in [START_REF] Miller | Modification of intracellular membrane structures for virus replication[END_REF])although the mechanism(s) of membrane perturbation remains poorly understood. Some DNA and double-stranded RNA viruses (poxviruses, reoviruses respectively) also present a membrane-associated replication complex. To better characterize the membrane deformations induced by viruses, we studied a known active membrane protein, NS4B of the hepatitis C virus (HCV).

HCV is a major cause of chronic liver disease, with approximately 3 % of the world population infected [START_REF] Lavanchy | The global burden of hepatitis C[END_REF]. Discovered more than twenty years ago [START_REF] Choo | Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome[END_REF], HCV is an enveloped +RNA virus belonging to the genus Hepacivirus of the Flaviviridae family. The HCV genome (ca 9.6 kb) consists of a single open reading frame (ORF) flanked by 5' and 3' untranslated regions (UTRs). The ORF encodes a polyprotein that is processed by cellular and viral proteases to generate the structural (core, E1 and E2) and non-structural (NS) proteins. NS proteins are involved both in viral particle maturation/secretion (NS1 or p7, and NS2) and viral genome replication (NS3, NS4A, NS4B, NS5A and NS5B) (reviewed in [START_REF] Moradpour | Hepatitis C virus proteins: from structure to function[END_REF]). Formation of the HCV replication complex (RC) is associated with large cytoplasmic membrane rearrangements, anchoring and protecting the RC to facilitate efficient replication [START_REF] Gosert | Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons[END_REF]. These altered membranes, believed to be derived from the endoplasmic reticulum (ER), are constituted of small vesicles embedded in a membranous matrix of circular or undulating membranes, constituting a rather compact structure referred to as the membranous web (MW) [START_REF] Egger | Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex[END_REF]. Similar rearrangements have been described in the livers of HCV-infected patients [START_REF] Pfeifer | Experimental non-A, non-B hepatitis: four types of cytoplasmic alteration in hepatocytes of infected chimpanzees[END_REF].

The NS proteins have been immunolocalized in the MW [START_REF] Egger | Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex[END_REF] revealing the formation of intense cytoplasmic membrane-associated foci (MAF) where NS proteins are also localized [START_REF] Lundin | Topology of the membrane-associated hepatitis C virus protein NS4B[END_REF].

Despite the considerable amount of information available on the viral proteins involved in RC and MW (reviewed in [START_REF] Moradpour | Hepatitis C virus proteins: from structure to function[END_REF][START_REF] Moradpour | Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration taht harbors the viral replication complex[END_REF][START_REF] Tang | Cellular and molecular biology of HCV infection and hepatitis[END_REF][START_REF] Alvisi | Hepatitis C virus and host cell lipids: An intimate connection[END_REF]), the mechanism of their formation is not yet fully understood.

It is likely that cellular factors act in this process -indeed, a growing number of host factors have been identified as components of the MW, which are also required for HCV replication (see [START_REF] Salloum | Treating hepatitis C infection by targeting the host[END_REF] for a review).

The presence and accumulation of NS proteins upon their synthesis in the ER is the first step in formation of the RC. In particular, it has been shown that MW or MAF specifically appear upon over-expression of NS4B [START_REF] Egger | Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex[END_REF][START_REF] Lundin | Topology of the membrane-associated hepatitis C virus protein NS4B[END_REF]. This suggests that NS4B is a possible triggering factor of MW formation. NS4B is a 27 kDa integral membrane protein [START_REF] Hügle | The hepatitis C virus non integral protein 4B is an integral endoplasmic reticulum membrane protein[END_REF] composed of three domains: the cytosolic N-and C-termini, and a central transmembrane domain comprising 4 to 5 helices [START_REF] Lundin | Topology of the membrane-associated hepatitis C virus protein NS4B[END_REF]. Each cytosolic domain contains 2 amphipathic helices, the N-terminal AH1 and AH2 [START_REF] Elazar | An N-terminal amphipathic helix in hepatitis C virus (HCV) NS4B mediates membrane association, correct localization of replication complex proteins, and HCV RNA replication[END_REF][START_REF] Gouttenoire | An amphipathic alpha-helix at the C terminus of hepatitis C virus nonstructural protein 4B mediates membrane association[END_REF], and the C-terminal H1 and H2 [START_REF] Gouttenoire | Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B[END_REF]. Studies on isolated peptides corresponding to these amphipathic helices revealed their ability to interact with and perturb model and cellular membranes [START_REF] Cho | Identification of a class of HCV inhibitors directed against the nonstructural protein NS4B[END_REF][START_REF] Guillen | A membranotropic region in the Cterminal domain of hepatitis C virus protein NS4B Interacyion with membranes[END_REF][START_REF] Palomares-Jerez | membrane interaction of segment H1 (NS4BH1) from hepatitis C virus non-structural protein 4B[END_REF][START_REF] Palomares-Jerez | Nterminal AH2 segment of protein NS4B from hepatitis c virus. Binding to and interaction with model biomembranes[END_REF][START_REF] Palomares-Jerez | Interactions with membranes of the full C-teminal domain of protein NS4B from Hepatitis C virus[END_REF]. Both N-and C terminal domains, as well as an homooligomerization of NS4B, are all thought to be linked to MW formation [START_REF] Lundin | Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein[END_REF][START_REF] Yu | Palmitoylation and polymerization of hepatitis C virus NS4B protein[END_REF][START_REF] Gouttenoire | Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory[END_REF]. In addition, NS4B has been shown to bind nucleotides [START_REF] Einav | A nucleotide binding motif in hepatitis C virus (HCV) NS4B mediates HCV RNA replication[END_REF], to hydrolyze tri-nucleotides [START_REF] Thompson | Biochemical characterization of recombinant hepatitis C virus nonstructural protein 4B: evidence for ATP/GTP hydrolysis and adenylate kinase activity[END_REF] and to bind RNA [START_REF] Einav | Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis[END_REF]. As a consequence of its apparent role in the replication process, NS4B has become a potential antiviral target (reviewed in [START_REF] Dvory-Sobol | The future of HCV theraoy: NS4N as antiviral target[END_REF]).

Data on NS4B-induced MW are only available in either a cellular context, or an artificial one involving isolated amphipathic peptides and liposomes [START_REF] Cho | Identification of a class of HCV inhibitors directed against the nonstructural protein NS4B[END_REF][START_REF] Paul | NS4B self-interaction through conserved C-terminal elements is required for the establishment of functional hepatitis C virus replication complexes[END_REF]. However, the precise intrinsic effect of NS4B on membranes remains to be determined. Our study aimed to determine the effect of NS4B on model membranes (liposomes). We produced large amounts of full-length NS4B in a cell-free expression system [START_REF] Fogeron | Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus[END_REF]. The purified protein was reconstituted into model membranes and further characterized by X-ray diffraction and cryoelectron microscopy (CryoEM). Our data demonstrate that NS4B destabilizes and fragments membrane vesicles. Peptides formed by the amphipathic helices present at the terminal extremities of NS4B are shown to perforate membranes and induce their fusion -in particular, they induce liposome hemi-fusion-like events. In conclusion, this study provides the first insights into the molecular mechanism of NS4B, and its involvement in cellular membrane reorganization and the formation of MW during HCV infection. Although numerous proteins participate in RC formation, membrane destabilization can be induced in the presence of hydrophobic domains or peptides only. The effects of NS4B on biological membranes can be generalized to all membrane-destabilizing proteins that contain amphipathic domains, whether in a viral or cellular context.

RESULTS

Protein expression and purification

Over-expression of the HCV NS4B gene (genotype 1a H77C) was lethal to E. coli (data not shown). The membrane protein was therefore produced using a cell-free expression system, in the presence of detergent to provide an hydrophobic environment. NS4B constructs were designed in fusion with a His-tag at either the N-or C-terminus (His-NS4B, NS4B-His respectively). Their expression was tested in small-scale batch mode in the presence of 1% Brij35, a detergent commonly used in cell-free expression. Although the two constructs of NS4B yielded a single band at the expected size (Figure SI 1A), only NS4B-His was detected by Western blot analysis using anti His-tag antibodies (Figure SI 1B). This suggests that the His-tag in His-NS4B is either hidden by the protein conformation or degraded, hampering correct antibody recognition -and also, potentially, its binding to the Ni-NTA column.

Therefore, the C-terminally-tagged construct NS4B-His was preferred for large-scale production -referred to henceforth as NS4B. Brij35 was exchanged for DDM and the protein was used within a few days.

Visualization of the effects of NS4B on model membranes by cryo-electron microscopy (cryoEM)

Insertion of detergent-isolated membrane proteins in their native medium relies on detergent removal of a ternary aqueous solution containing three solutes: lipid, protein and detergent.

Detergent removal can be achieved by either dialysis or BioBeads treatment (see [START_REF] Richard | Reconstitution of CF0F1 into liposomes using a new reconstitution procedure[END_REF]). Judged by the formation of liposomes in the absence of protein, both methods worked well (Figure SI 2). The resultant liposomes are, however, formed more rapidly with Biobeads (hours versus days), and displayed lower polydispersity (Figure SI 2). All experiments were therefore performed using BioBeads. The effects of NS4B on the structure of model membranes were analyzed at different lipid-to-protein molar ratios (LPR).

Membrane-reconstituted samples with or without the protein were first analyzed by conventional negative staining (Figure SI 2 and 3), and then by cryoEM in order to preserve the structure of aqueous lipidic phases [START_REF] Lepault | Cryo-electron microscopy of artificial biological membranes[END_REF] (Figure 1). Pure lipid samples contained liposomes that were mostly multilamellar when observed by cryoEM (Figure 1A). In the presence of NS4B at molar lipid-to-protein ratios (LPR) of 500 or lower, NS4B affected the morphology of the liposomes in a concentration-dependent manner (Figure 1 B, C andD). The lower the LPR, the more heterogeneous and disordered the membranes appeared (see Figure 1B and1C). For LPR lower than 50, no liposomes were present at all -instead, small objects were observed consistent with the presence of lipid micelles (Figure 1D). While the exact structure of these objects is unknown, lipid micelles surrounding protein molecules represent a reasonable hypothesis. Figure 2 shows enlarged areas of figure 1. In the absence of NS4B, the membranes are parallel and their doublelayered structure is clearly visible (Figure 2A). For increasing ratios of NS4B, membranes become more locally curved and fewer double-layered structures are observed (Figures 2B and2C). Due to the low molecular weight of the protein (27kDa), the micelles containing NS4B exhibit low contrast (figure 2D). These observations suggest that NS4B possesses the ability to disorganize membranes by imposing local constraints that alter membrane curvature. Similar results were obtained with liposomes of different composition (soybean phosphatidyl-choline, synthetic lecithine such as dimerystoyl-phosphatidyl-choline (DMPC); data not shown). Further experiments were carried out at LPR between 100 and 300, corresponding to the region of major membrane alterations.

Overall effects of NS4B on lipid phases by various biophysical studies

DMPC has been extensively characterized [START_REF] Tardieu | Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases[END_REF] and constitutes a simple and appropriate system to assess the effect of a protein on the structure of membranes. At a water concentration above 20 % (weight / total weight), DMPC presents three temperaturedependant phases: (i) Below 14 °C, the aliphatic chains are stiff, arranged on a quasihexagonal lattice and tilted with respect to the normal to the lattice plan. This phase is generally referred as Lβ' [START_REF] Luzzati | X-ray diffraction studies of lipid-water system[END_REF]). (ii) At 14-24 °C, the aliphatic chains have the same conformation as for Lβ', but the surface of the membrane is rippled, forming the Pβ' phase [START_REF] Tardieu | Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases[END_REF]. (iii) Above 24°C, the membrane flattens and the chains disorder, forming the Lα phase [START_REF] Luzzati | X-ray diffraction studies of lipid-water system[END_REF]. These three lamellar structures are observable in hydrated DMPC (30 % water) by their characteristic X-ray diffraction patterns at the appropriate temperatures (Figure 3 A-C). In particular, the complex, sharp reflection located around 4.2 Å, observed at 20 °C (Fig 3B ), is replaced by a broad reflection centered around 4.6 Å when the temperature is increased above was incorporated in the hydrated DMPC phase at an LPR of 300, both the low and high angle reflections broadened so that the phase transitions were difficult to detect (Figure 3 D).

Similar patterns were obtained in the 10-30 °C temperature range (data not shown), demonstrating that NS4B greatly affects the structure of DMPC membranes and anneals the phase transitions of aqueous DMPC lamellae.

Membrane perforation and fusion activities of terminal peptides

To further investigate the molecular mechanism of NS4B-induced membrane disorganization, we investigated the effects of consensual N-and C-terminal amphipathic helices (AH1, AH2 and H2) on liposomes. The synthesized peptides are referred to as pepAH1, pepAH2 and pepH2. Due to its rather small length, the properties of H1 were not investigated. The effects of the different peptides were analyzed using a fluorescent test on artificial membranes.

Liposomes containing high concentrations of carboxyfluorescein display reduced fluorescence intensity as a result of self-quenching. When the liposomes are disrupted the drug is released into the external medium, lowering its concentration and hence the degree of self-quenching, thereby increasing its fluorescence activity. This constitutes a simple test to investigate the membrane perforation properties of any compound. All three peptides, pepAH1, pepAH2 and pepH2, did indeed perforate liposomes (Figures 4 A andSI 4). The amplitude and rate of the fluorescence increase varied with peptide concentration. While the activity of pepH2 and pepAH2 were similar, the perforation activity of pepAH1 was five times lower. It should be noted that 1 mM pepH2 and pepAH2 exhibit an activity similar to 0.1 % Triton X-100 (v/v). None of these peptides disrupted red blood cell at physiological pH, and so the pore size could not be measured by hemolysis protection experiments.

In addition to perforation, peptides may also induce membrane fusion [START_REF] Wharton | Membrane fusion by peptide analogues of influenza virus haemagglutinin[END_REF]. We tested this possibility by fluorescence resonance energy transfer between donor and acceptor fluorescent molecules associated with lipid. When liposomes marked with both fluorescent molecules are mixed with regular liposomes, peptides inducing membrane fusion lead to a greater distance between donor and acceptor -reducing the energy transfer rate and thus increasing the yield of fluorescence emitted by the donor (and decreasing that from the acceptor). As seen in Figure B, the addition of pepH2 to a mixture of regular and fluorescent liposomes led to an increase in donor fluorescence. This increase required the presence of both regular and fluorescent liposomes -it did not appear when pepH2 was added to fluorescent liposomes alone (violet trace in Figure 4 B). The increase did not arise from adsorption phenomena induced by the peptide, but resulted from the fusion of regular and fluorescent membranes.

PepAH1 and pepAH2 did not show any fusion activity (data not shown); only pepH2 was thus further investigated.

Liposome fusion required a rather high concentration of peptide (> 1 µM). Indeed, the peptides are not soluble in water and only a small fraction can insert into membranes. Thus when NS4B-liposome samples were centrifuged through a sucrose density gradient, most of the peptides were found in the bottom fraction (Figure SI 5), while the peptides dissolved in water exhibited large aggregates as observed by dynamic light-scattering (data not shown).

The large concentration of peptides necessary to affect liposome membrane can thus be attributed to their low solubility in water and not to the existence of different destabilization mechanisms for NS4B and pepH2.

Cryo-electron microscopy of liposomes in the presence of pepH2

Liposomes were visualized by cryo-EM, as spherical objects defined by a single bilayer. The apparent diameter of the liposomes was rather constant, with values generally less than 0.5 µm (Figure 5A). When several liposomes aggregate, the membranes keep their doublelayered structure (figure 5A). Multi layered liposomes were rare. When pepH2 was added at a concentration of 25 µΜ, most liposomes aggregated (Figure 5 B). Many liposomes contained a second one -suggesting that the presence of the peptide lead to an invagination of the membrane and formation of an internal liposome (Figures 5B). PepH2 appears thus to have the capacity to transform liposome into double membrane vesicles by a mechanism that remains unknown. When the liposomes aggregated, the contrast of the interacting layers is reduced (Figure 5 B-D) and a succession of high and low densities is observed along the centre. These two observations indicate a disorganization of the lipid polar head domains of the interacting bilayers, reflecting a reorientation of their aliphatic chains as a result of peptide insertion. Figure 6 shows enlarged views of interacting liposomes in absence (A) and presence (B) of pepH2. In presence of pepH2 the contrast of the interacting leaflets reduces in some areas. In addition, the aqueous medium between the liposomes completely disappears when NS4B is present. While these structures were absent in absence of NS4B, most aggregated liposomes displayed interacting layers with a reduced contrast. These structures suggest that the external liposome membranes have locally merged. The fact that the fusion area has conserved the thickness of two membranes suggests that the external leaflets fused in a succession of local hemi-fusion events. As the extent of each fused event is unknown, we referred this structure as arising from an hemi-fusion-like event.. Although most observed liposome interactions involved hemi-fusion-like event, full fusion events cannot be excluded.

Although rare, pores were observed and displayed small diameter (Figure SI 6 A-B).

Reduced aqueous densities are observed between the interacting membranes in presence of pep H2 (C-D), in contrast to pure liposomes (A). Note that hemi-fusion appears between membranes of single-lamellar liposomes (large arrow in B) or between membranes of concentric liposomes (small arrow in B).

Figure 6: Images of the contact between liposome membranes in the absence (A) and presence (B) of peptide H2. Without pepH2, the low density arising from the external medium is clearly visible between the two bilayers (A); in the presence of pepH2, the lipid layers in contact have low contrast and the aqueous phase is no longer visible (B). The distance between the internal layers are identical, suggesting the existence of several hemi-fusion events. P indicates the polar heads; schematically, four distinct layers are seen in absence of pepH2 (A), while only three are present when pepH2 is present (B).

.

DISCUSSION

The aim of the present study was to investigate the specific contribution of NS4B to the membrane alterations associated with viral genome replication in HCV-infected cells. We first produced a recombinant full-length HCV NS4B by cell-free expression, in order to overcome the cyto-toxicity of NS4B upon cellular over-expression. Our protocol constitutes a fast and simple procedure for obtaining milligram quantities of this highly hydrophobic protein [START_REF] Fogeron | Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus[END_REF]. Several laboratories have already reported the purification of recombinant NS4B from different sources (Sf9 insect cells, bacteria), but detailed structural studies were hampered by low levels of expression [START_REF] Einav | A nucleotide binding motif in hepatitis C virus (HCV) NS4B mediates HCV RNA replication[END_REF][START_REF] Einav | Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis[END_REF] or poor purity [START_REF] Thompson | Biochemical characterization of recombinant hepatitis C virus nonstructural protein 4B: evidence for ATP/GTP hydrolysis and adenylate kinase activity[END_REF]. The high yield and purity of our preparation allowed for an in-depth biophysical study of the effects of NS4B on model membranes.

X-ray diffraction and electron microscopy studies demonstrate that the most important changes induced by NS4B concern the order within membranes. DMPC membranes regularly stack, forming lamellar phases at low water content or liposomes at higher water content. In the first case, the spacing between membranes is regular enough to give rise to rather sharp Xray diffraction patterns, while concentric membranes are observed in cryoEM images for the second. When NS4B is present at LPR lower than 500, DMPC phases exhibit broadened Xray diffraction patterns and disordered membrane stacks are observed by cryoEM. The membrane staking default in presence of NS4B appears as mainly due to membrane destabilization.the membrane disorder. While DMPC membranes are contiguous, NS4B induces the interruption of membranes, leading to the formation of membrane fragments and merged membranes. Fragmentation and merging of membranes decreases their homogeneity, potentially explaining the loss of cooperativity of the phase transitions. NS4B, destabilizing and fragmenting liposomes, exhibits detergent-like properties. While this could be an artefact of incomplete detergent removal, at least two factors can exclude this hypothesis. Firstly, BioBead treatment was performed in optimal conditions (concentration, time course, temperature), under which DDM, the detergent used here, is totally absorbed [START_REF] Rigaud | Bio-Beads: an efficient strategy for two-dimensional crystallization of membrane proteins[END_REF]. Secondly, all our samples, liposomes and proteo-liposomes were reconstituted in the same conditions that led to the formation of liposomes when only pure lipid solutions were treated. The effect of NS4B on membranes is thus an intrinsic property of the protein.

The disorder introduced by NS4B on model membranes is similar but not identical to that observed in-situ. This difference is not surprising, and several studies have shown that multiple factors may influence the formation and morphology of MW (see [START_REF] Salloum | Treating hepatitis C infection by targeting the host[END_REF]): lipid composition, recruitment of host-cell factors, the abundance of the different HCV NS proteins, the formation kinetics of the HCV viral replication complex... The primary difference is the lipid composition in our model membranes. MW have been shown to be enriched in PI(4)P [START_REF] Hsu | Viral reorganization of the secretory pathway generates distinct organelles for RNA replication[END_REF], and to be localized in cholesterol-rich lipid rafts [START_REF] Aizaki | Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts[END_REF], and these specific lipids can strongly influence the membrane architecture. Moreover, the local concentration of NS protein can also contribute to various morphologies of MW. Indeed, over-expression of NS proteins induced distinct membrane alterations, revealed by electron microscopy studies, and these membrane rearrangements change during the viral replication process [START_REF] Alvisi | Hepatitis C virus and host cell lipids: An intimate connection[END_REF][START_REF] Romero-Brey | Three-dimensional architecture and biogenesis of membrane structure associated with hepatitis C virus replication[END_REF].

The domains corresponding to the C and N-terminal ends of NS4B have been proposed to be formed by amphipathic helices involved in membrane association: H2, AH1 and AH2 (H1, a small peptide, has not been studied). We confirmed that peptides corresponding to these regions of NS4B perforate liposome membranes. Cryo-EM images of perforated liposomes show pores where the internal aqueous medium is not resolved (Figure SI 6). The pore diameter is thus less than the dimension of the smallest object that can be visually detected by CryoEM, about 3 nm.Our data suggest that pepH2 is sufficient to induce membrane merging.

Although fusion events could not be observed, their occurrence cannot be discarded. In the absence of tension on the membrane undergone by protein [START_REF] Libersou | Distinct structural rearrangements of the VSV glycoprotein drive membrane fusion[END_REF], the fusion process appears nevertheless blocked in hemi-fusion or at the step where an initial fusion pore is formed. The membrane fusion and perforation properties of NS4B reasonably explain its ability to disorder membranes, both in vivo and in vitro. While double membrane vesicles have been observed in MW [START_REF] Romero-Brey | Three-dimensional architecture and biogenesis of membrane structure associated with hepatitis C virus replication[END_REF][START_REF] Paul | Morphological and biochemical characterization of the membranous hepatitis C virus teplication compartment[END_REF], such vesicles appear to be induced by the amphipathic domains of NS4B. All activities of NS4B on membranes could be explained by the presence of amphiphilic domains that insert into biological membranes. Because such domains are present in many proteins, the membrane activity of NS4B can thus be generalized -rearrangements of membranous compartments associated with the replication of positive RNA viruses may be due to the presence of amphiphilic domains. Due to their potential importance in the viral cycle, such amphiphilic domains are excellent targets for developing anti-viral agents.

In summary, we have shown that NS4B disorders biological membranes at different levels.

Firstly, NS4B inhibits regular membrane stacking, impeding in particular their parallel arrangement. Secondly, while the boundaries of native membranes are continuous surfaces, the presence of NS4B introduces interruptions in the membrane and anastomosed network.

All studied peptides corresponding to the N-and C -terminal ends of NS4B perforate membranes. The peptide corresponding to H2 also induces membrane fusion and the formation of double membrane vesicles. Perforation and fusion of membranes induced by the termini of NS4B, and by any exposed amphiphilic domains in general, are sufficient to explain the membrane disordering observed during formation of the virus replication complex.

MATERIAL AND METHODS

Materials

Detergents were purchased from Calbiochem (Brij35) and Interchim (DDM); all other chemicals were from Sigma. Solutions were filtered through 0.22 µm Millipore filters.

For fusion assays and liposome cryo-EM, peptides were dissolved in DMSO (Sigma) at 100 mM or lower. The maximum concentration of DMSO in the presence of lipid (Avanti) sample was 1 %.

Cell-free protein expression and purification

The encoding region of NS4B (genotype 1a, strain HCV H77C) was cloned between the NcoI and SmaI restriction sites of the pIVEX 2.3d and 2.4d vectors (5PRIME).The genome codes for a polyprotein containing 3011 amino acids (aa). NS4B starts at aa 1712 and is made up of 261 aa. These plasmids were designed to add a C-or N-terminal 6xHis tag respectively.

Constructs were checked by sequencing, and plasmid DNA templates for cell-free production were purified using the Qiagen MidiPrep kit with an OD 260/280 >1.8. The plasmids was aliquoted and stored at -20 °C.

Cell-free expression was performed using the commercial 5PRIME kits RTS100 (small volume batch system BCF) and RTS500 (medium and preparative scale with continuous exchange CECF), according to the manufacturer's instructions, and the mixtures were incubated in the Eppendorf Thermomixer comfort device.

For initial screens, BCF reactions were performed in 50 µL reconstituted RTS100 E.coli HY lysate with 1 µg plasmidic DNA, supplemented with 1 % (w/v) Brij35, and incubated for 4 h at 30 °C and 660 rpm. The mixtures were loaded and separated on 12 % SDS-PAGE gels, then either stained with Coomassie blue or transferred onto nitrocellulose membranes for immunodetection with anti-His tag antibodies conjugated to peroxidase (Roche Applied Science).

For preparative-scale synthesis, CECF reactions were performed using 1 ml reconstituted RTS500 E.coli HY lysate with 15 µg plasmidic DNA, supplemented with 1 % (w/v) Brij35 per ml of reaction mix, and incubated for 20 h at 30 °C and 660 rpm.

At the end of expression, reaction solutions were pooled, diluted 1:10 in buffer A (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.05% DDM), and incubated for 30 min at room temperature. After centrifugation for 30 min at 20,000 x g, the supernatant was loaded onto a Ni-NTA loaded HiTrap HP column (GE Healthcare) equilibrated with buffer A. Affinity chromatography was performed at 1 mL.min -1 flow rate, with washing steps of 10 column volumes of buffer A supplemented with 50 mM imidazol. Proteins were eluted with a linear (0.05-0.5 M) imidazol gradient in buffer A (3 column volumes). NS4B fractions were concentrated on Amicon Ultracel® -30kDa membrane concentrators (Millipore) and further purified on a gel-filtration column (Superdex 200, GE Healthcare), equilibrated with buffer B (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM β-mercaptoethanol, 0.03% DDM), at a flow rate of 0.5 ml.min -1 .

Each of the purification steps was followed by Coomassie-blue-stained 12 % SDS-PAGE gel, according to the Laemmli method, to check sample purity.

Concentration of purified cell-free-expressed NS4B was determined by its absorbance at 280 nm, using extinction coefficient 39 440 M -1 cm -1 .

Proteoliposome reconstitution

To solubilize DMPC lipids, DDM was added in 50 mM Tris-HCl pH 7.5, 150 mM NaCl buffer at a detergent-to-lipid weight ratio of 2.5:1, followed by vigorous vortexing.

Purified NS4B was mixed with detergent-solubilized lipids at the desired final molar ratio; the final lipid concentration was 2 mg/ml. To remove the detergent, appropriate quantity of wet Bio-Beads (SM-2 Bio-Rad) were added every 30 min for 3 hours, with continuous stirring at room temperature to achieve the final Bio-Beads-to-detergent weight ratio of 40:1 (BioRad Instruction sheet). Following Bio-Bead removal, reconstituted proteoliposomes, in the presence or absence of NS4B, were then analyzed by electron microscopy.

Synthesis of NS4B terminal regions

Peptides corresponding to the N-and C-terminal amphipathic helices of NS4B (AH1 & AH2, and H2, respectively) were chemically synthesized as previously described [START_REF] Gouttenoire | An amphipathic alpha-helix at the C terminus of hepatitis C virus nonstructural protein 4B mediates membrane association[END_REF][START_REF] Gouttenoire | Aminoterminal amphipathic alpha-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and viral production[END_REF]. AH1,

Liposome perforation assays

Liposomes containing carbofluorescein (CF) were prepared as previously described [START_REF] Nandi | Interaction of rotavirus particles with liposomes[END_REF].

Briefly, phosphatidylcholine was suspended 10 mM NaCL and 10 Hepes buffer (pH 7.4). A concentrated solution of carbofluorescein was then added and the solution vortexed for at least 1 minute and then sonicated in a Besnson bath sonicator. Liposomes containing fluorescein were separated from free carboflurescein by eluting the mixture through Sepharose column equilibrated with 150mM NaCl and 10 mM Hepes, pH 7.4.

The release of CF upon peptide addition was monitored as an increase in fluorescence at 520 nm, with excitation at 492 nm in a thermostatically controlled Perkin-Elmer LS50B spectrofluorimeter. 100 % CF release measured by addition of Triton X-100 at the end of the reaction. All fluorescence experiments were carried out at least three times.

Liposome fusion assays

Preparation of liposomes: 100 µg each of PE dissolved in organic solvents were mixed and dried in vacuo. To prepare fluorescent liposomes for fusion assays, 2.5 µg BODIPY 500/510 C12-HPC (as fluorescence donor) and 5 µg BODIPY 530/550 C5-HPC (as fluorescence acceptor) were added in the mixture before drying. The lipid film was resuspended in 0.2 ml buffer (150 mM NaCl, 5mM Tris-HCl pH 8), and the mixture was bath-sonicated for 20 minutes. Fluorescent and non-fluorescent liposomes were extruded through an Avanti extruder using 0.2 µm membranes; in general, 40 extruding passages were performed.

Fusion assay: fusion was assayed as previously described [START_REF] Malinin | The rate of lipid transfer during fusion depends on the structure of fluorescent lipid probes: a new chain-labeled lipid transfer probe pair[END_REF][START_REF] Roche | Evidence that rabies virus forms different kinds of fusion machines with different pH thresholds for fusion[END_REF]. Briefly, 10 µl fluorescent liposomes were mixed with 980 µl phosphate-citrate buffer at the required pH (prepared from 100 mM citric acid and 200 mM dibasic sodium phosphate solutions), in the cuvette of a thermostated Perkin-Elmer LS50B spectrofluorimeter. 10 µl non-fluorescent liposome suspension were added, followed by 10µl peptide solution, and the increase in BODIPY 500/510 C12-HPC fluorescence was monitored continuously. Excitation was at 485 nm (slit width 5 nm) and emission was at 518 nm (slit width, 5 nm). The mixture was kept under continuous stirring during the experiment.

X-ray diffraction

Samples were prepared and the patterns recorded with a Guinier camera, as described previsouly [START_REF] Tardieu | Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases[END_REF].

Electron microscopy

Liposomes were prepared as for the perforation experiments at a concentration of 1 mg/ml.

Peptide dissolved in DMSO at a concentration of 100mM was added was diluted 1000-100 times in the liposome sample. 1% DMSO did not change the liposome morphology (not shown).

Most samples were checked by conventional electron microscopy using the negative staining method. 3 µl sample were deposited on an airglow-discharged, carbon-coated grid. The excess was blotted and the grid rinsed with 2 % phosphotungstic acid. Grids were observed in a Philips CM12 microscope operated at 80 kV, and images were recorded on Kodak electron image plates, which were then developed for 5 minutes in D19.

CryoEM was performed as described [START_REF] Lepault | Electron microscopy of frozen biological suspensions[END_REF] using R2/2 quantifoil carbon film (Electron Microscopy Science). 3 µl of the sample were deposited on airglow-discharged quantifoil.

The sample excess was blotted with a filter paper, and the grid plunged into liquid-nitrogencooled ethane. The grid was rapidly transferred and kept under liquid nitrogen. For observation, the grids were mounted in a 636 Gatan holder using its cryo-transfer device. The observations were made in a Technai 200 equipped with a field-emission gun. Images of the protein samples were typically recorded at 35,000 x magnification on SO163 films, which were developed for 12 minutes in D19 (Kodak). Each micrograph was recorded with about 20 electrons/A 2 . Micrographs were scanned with a Nikon Coolscan 8000, at a resolution of 4000 dots per inch. Some images were low-and high-pass-filtered using Imagic [START_REF] Van Heel | A new generation of the IMAGIC image processing system[END_REF]; these filters were set to optimize the observed contrast of membranes, typically 0.03 and 0.5 times the Nquist frequency, respectively. The images of pepH2 were recorded with a direct dtection camera (K2, Gatan) operated in movie mode. The images were aligned and summed as recommended by manufacturer. They were recorded at 29,000 magnification (pixel size 1.72Å) using a total dose less than 30 electrons/Å 2

Figure 1 :

 1 Figure 1: CryoEM. Liposomes reconstituted at different lipid to NS4B protein ratio (LPR) and observed by cryo-EM. A no protein. B LPR=500. C LPR=100. D LPR=10. Pure liposomes show regular stacks of membranes. The more NS4B is present, the more the stacking is perturbed due to the membrane deformation imposed by NS4B and the less are the visualized membranes.

Figure 2 A

 2 Figure 2 A-D: Enlarged views of membrane structures formed at different LPR, no protein (A), 300 (B), 100 (C) and 10 (D). Decreasing LPR correlates with increasing membrane distortion and a reduction of the membrane contrast. For LPR below 50, small particulate objects are detected that are referred as lipid micelles containing NS4B.

Figure 3 :

 3 Figure 3: X-ray diffraction of NS4B and DMPC aqueous mixtures at different temperatures. All samples contain 30 % water. In A, B and C no protein is present. In D the LPR is equal to 100. The temperature is equal to 25°C in A, 20°C in B, 10°C in C and 20°C in D. While the pattern is sensitive to temperature for pure DMPC, it is similar for NS4B-DMPC mixture in the temperature range 10-30°C.

Figure 4 :

 4 Figure 4: Perforation (A) and fusion (B) activities of pepH2. Perforation analysis was performed using pepH2 concentrations, in DMSO, of 1 (blue), 0.1(red), 0.01 (light green), 0.085 (yellow), 0.075 (light blue), 0.005 (orange), 0.003 (green), and 0 (pink) mM (for 0 mM, pure DMSO was added). Fusion experiments were carried out at pepH2 concentrations of 1 (blue, violet), 0.5 (red), 0.2 (greeen), and 0.1 (yellow) mM. In the absence of non-fluorescent lipids (violet), no fluorescence increase is observed.

Figure 5 :

 5 Figure 5: Effects of peptide H2 on liposomes. The scale bar represents 100 nm in A-B, and 33 nm in C-D. In the presence of pepH2 (B-D), liposomes have a strong tendency to aggregate; double concentric liposomes are also more often found. Some interacting membranes present a particular structure: the interacting membrane leaflets exhibit reduced contrast, and density variations appear within the external leaflets (C and D), indicating hemi-fusion-like events.

  AH2 and H2 correspond to amino acids 1-34, 41-65 and 227-254 in the NS4B sequence, in DMSO to a concentration of 1 mM and kept at -20°C until use.

ACKNOWLEGEMENTS

We thank Drs Thierry Meinnel and Félix Rey for their interest in this work that was supported by grants from Agence National contre le Sida et l'Hépatite C (ANRS), Agence National pour la Recherche (ANR). We also thank Dr. Andrew Pascal for reading the manuscript.


Géraldine Bossard
email: bossard@cirad.fr

Valérie Rodrigues

Eleonore Tour

Anne Geiger

Mice immunization with Trypanosoma brucei gambiense translationally controlled tumor protein modulates immunoglobulin and cytokine production, as well as parasitaemia and mice survival after challenge with the parasite

Keywords: Trypanosoma brucei gambiense, Translationally Controlled Tumour Protein (TCTP), host immunization, parasitaemia, cytokines, antibodies

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Human African trypanosomiasis (HAT), the well-known sleeping sickness, is caused by two protozoan species belonging to the genus Trypanosoma, respectively Trypanosoma brucei gambiense (Tbg) causing the chronic form of the disease in West and Central Africa, and Trypanosoma brucei rhodesiense (Tbr) causing the acute form in East Africa. These two trypanosome species are transmitted to humans by two Glossina (tsetse flies) species, Glossina palpalis and Glossina morsitans, respectively. Both forms of the disease have severe social and economic impacts on populations living within or near HAT foci.

Despite the disease has been described more than one hundred years ago, it has not been eradicated yet. Sleeping sickness is a vector-borne disease, thus control strategies may be focused either on the vector or on the patient. Regarding the latter, two complementary strategies are implemented, respectively, a curative one that needs the use of various drugs that often are not easily supported by the patients, or a preventive one which in general consists of the vaccination of populations that are at risk. Unfortunately this technique does not work in the case of sleeping sickness since the bloodstream form of the trypanosome is covered by a coat that consists of a glycoprotein called variant surface glycoprotein (VSG).

Despite the trypanosome genome includes over 1,000 different VSG genes, due to a strict allelic exclusion, only one gene is expressed at any time so that the coat is composed of a single type of VSG. However, an antigenic variation, associated with successive waves of parasitaemia, occurs in vivo (in the infected human), which consists in the replacement of a given VSG type, for example VSG1 by a VSG2, resulting from the silencing of the VSG1 gene and the concomitant activation of the VSG2 gene [START_REF] Dubois | Trypanosomes expressing a mosaic variant surface glycoprotein coat escape early detection by the immune system[END_REF][START_REF] Geiger | Escaping Deleterious Immune Response in Their Hosts: Lessons from Trypanosomatids[END_REF]. Since VSG2 is not recognized by the anti-VSG1 antibodies the patient may have produced, the trypanosome escapes the host defense and continues to multiply up to the next wave of parasitaemia which induces the replacement of VSG2 by a VSG3. Thus another approach should be considered.

The idea is to immunize the patient not against the trypanosome itself, but against a major component of its pathogenicity. With this respect, a comparative inventory of the excreted / secreted proteins (ESPs) produced in vitro respectively by the procyclic form (hosted by the vector) [START_REF] Atyame Nten | Excreted/secreted proteins from trypanosome procyclic strains[END_REF] and the bloodstream form (pathogen for humans) [START_REF] Geiger | Exocytosis and protein secretion in Trypanosoma[END_REF] of the parasite was carried out. More than 400 ESPs have been identified in the secretome of the bloodstream form of the trypanosome. Some of them have also been identified in secretion vesicles isolated from the serum of trypanosome-infected mice, demonstrating that these proteins are excreted not only in vitro but also in vivo. Furthermore, the total secretome has been shown to be involved in an immunosuppression mechanism impairing dendritic cells maturation, altering their function, leading to a dramatic reduction of the secretion of TNFα which plays a key role in regulating parasitemia along Trypanosoma brucei infection [START_REF] Magez | Tumor necrosis factor alpha is a key mediator in the regulation of experimental Trypanosoma brucei infections[END_REF], interleukin 10 and 6 production as well as that of cytokines Th1 / Th2 [START_REF] Garzón | The Trypanosoma brucei gambiense secretome impairs lipopolysaccharideinduced maturation, cytokine production, and allostimulatory capacity of dendritic cells[END_REF]). It should be noted that Trypanosoma infection induces activation of polyclonal cells of B cells, clonal exhaustion of B cells, depletion of Marginal Zone B and Folicular B cells, and finally destruction of the memory compartment of B cells [START_REF] Bockstal | T. brucei infection reduces B lymphopoiesis in bone marrow and truncates compensatory splenic lymphopoiesis through transitional B-cell apoptosis[END_REF]. Among the ESPs we identified number of proteases that may contribute to the parasite's virulence [START_REF] Atyame Nten | Excreted/secreted proteins from trypanosome procyclic strains[END_REF].

However our attention was drawn to the presence in the trypanosome secretome of a particular protein, the Translationally Controlled Tumor Protein (TCTP). TCTP is a highly conserved protein distributed ubiquitously in eukaryotes, whether plants or animals. It was reported to be involved in cellular processes such as stress responses [START_REF] Brioudes | Translationally controlled tumor protein is a conserved mitotic growth integrator in animals and plants[END_REF], cell proliferation [START_REF] Stierum | Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2[END_REF][START_REF] Arcuri | Translationally controlled tumor protein (TCTP) in the human prostate and prostate cancer cells: expression, distribution, and calcium binding activity[END_REF], embryo development [START_REF] Roque | Tumor protein Tctp regulates axon development in the embryonic visual system[END_REF], apoptosis [START_REF] Susini | TCTP protects from apoptotic cell death by antagonizing bax function[END_REF] and physiological disorders [START_REF] Kadioglu | Peptide aptamer identified by molecular docking targeting translationally controlled tumor protein in leukemia cells[END_REF][START_REF] Seo | Interaction of antihistaminic drugs with human translationally controlled tumor protein (TCTP) as novel approach for differentiation therapy[END_REF].

TCTP was not only identified in the secretome of the bloodstream form of the trypanosome, but also in the secretome of its procyclic form which is hosted by its Glossina vector [START_REF] Atyame Nten | Excreted/secreted proteins from trypanosome procyclic strains[END_REF]. Moreover the corresponding gene (TbgTCTP) was shown to be overexpressed in Tbg-infected Glossina palpalis flies as compared to non-infected flies [START_REF] Hamidou Soumana | RNA-seq de novo assembly reveals differential gene expression in Glossina palpalis gambiensis infected with Trypanosoma brucei gambiense vs non-infected and self-cured flies[END_REF] which could indicate TCTP to be involved in fly infection process, inasmuch the protein was also identified in total gut extracts from Tbg-infected flies [START_REF] Geiger | Differential expression of midgut proteins in Trypanosoma brucei gambiensestimulated vs. non-stimulated Glossina palpalis gambiensis flies[END_REF]. Recently we have produced the recombinant rTbgTCTP protein, which was shown to be able to modulate the growth of bacteria isolated from the gut of tsetse flies [START_REF] Bossard | Characterization of recombinant Trypanosoma brucei gambiense Translationally Controlled Tumor Protein (rTbgTCTP) and its interaction with Glossina midgut bacteria[END_REF].

Finally TCTP seems to be involved in various steps governing the interactions between the parasite and either its insect vector or its human host.

To attempt getting a better insight into the role of TbgTCTP as a possible effector of the parasite pathogenic process, we have investigated the effect of the immunization of mice with the rTbgTCTP on the modulation of immunoglobulins and cytokines/chemokines production in parallel with the evolution of parasitaemia and mice survival.

Material and methods

Recombinant rTbgTCTP protein production

The whole production process was described in [START_REF] Bossard | Characterization of recombinant Trypanosoma brucei gambiense Translationally Controlled Tumor Protein (rTbgTCTP) and its interaction with Glossina midgut bacteria[END_REF]. Briefly, the Tbg tctp gene was synthesized and cloned into the baculovirus vector pAcGHLT-A; the vector was then transfected into Spodoptera frugicola (strain 9) cells in which the gene was expressed.

The recombinant rTbgTCTP was recovered and purified using the AKTAprime plus system.

Mice immunization schedule

Mice were kept under strict ethical conditions according to the international guidelines for the care and use of laboratory animals. The experiments designed for this study were approved by the regional Ethic Committee for Animal Experimentation CEEA-LR 36 under project number 02187.01 and authorized by French Ministry for Higher Education and Research. The immunogenicity and antigenicity of rTbgTCTP were evaluated on 6-weeks-old female mice (OF-1 mice) purchased from Charles River Laboratory, France. Three doses of rTbgTCTP, 20µg, 40µg and 80µg, in 70µl of Complete or incomplete Freund's adjuvant were tested. For each dose, the immunization was performed with three successive sub-cutaneous injections separated by a 2-weeks interval. Controls were processed similarly with adjuvant alone or PBS.

The experimental design included 30 mice randomly distributed in 5 groups of 6 mice. The mice were then identified by numbering. The groups were the followings:

-PBS Control group (mice numbers 1 to 6): each mouse became three successive injections of 70µl PBS; -Freund's control group (mice numbers 7 to 12): each mouse received a first injection of a mixture of 35µl of complete Freund's adjuvant and 35µl of PBS. Second and third injections, were of 70µl incomplete Freund's adjuvant; -in the three groups called "20TCTP" (mice numbers 13 to 18), "40TCTP" (mice numbers 19 to 24) and "80TCTP" (mice numbers 25 to 30), the mice received three successive injections of 20, 40 or 80µg rTbgTCTP protein, respectively, emulsified in 70µl of either complete Freund's adjuvant (first injection) or incomplete Freund's adjuvant (second and third injections).

Mice infectious challenge with trypanosomes

Eighteen days after the last injection, each mouse was infected intraperitoneally with 3000 trypanosomes belonging to the T. b. gambiense species (ABBA strain) in a total volume of 100µl blood from one previously infected mouse. The ABBA strain has been isolated in Côte d'Ivoire, in 1983, from a patient suffering HAT. After infection, each mouse was monitored every day in order to evaluate parasitaemia (microscopic observation) and survival. Once a week, hematocrit was measured (200µl) and the serum was collected and stored at -80°C until cytokine and antibody analysis.

Cytokine quantification

A commercial BD CBA mouse Th1/Th2 cytokines kit (Becton Dickinson, USA) was used to quantify IL-10, IL-6, IL-4, IL-2, TNF-α and IFN-γ in the sera using the bead array technology as described by the manufacturer. Briefly, a mixture of seven capture beads (with distinct APC fluorescent intensities) coated with capture antibodies specific for each cytokine was used. Fifty µl of the bead mixture to which 50 µl of an unknown serum sample (or of a recombinant standard), and with 50 µl of phycoerythrin (PE) detection reagent were incubated to form sandwich complexes. The concentration of each particular cytokine was measured by the intensity of PE fluorescence of each sandwich complex. After washing, samples were processed on the BD Canto II dual laser (488 and 633 nm) flow cytometer and data were analyzed by FCAP Array software. Serial diluted standards were first analyzed to generate calibration curves and the sample cytokine concentrations were calculated with reference to these curves.

ELISA: immunoglobulin detection

Each well of 96 Maxisorp plates (Thermofisher) was coated overnight at 4°C with 0.1µg rTbgTCTP diluted in 100µl. The uncoated antigen was then removed as follows: the plate was saturated with 150µl of PBS containing 5% nonfat milk and 0.1% Tween 20 (blocking buffer solution), and incubated during 45 min at 37°C. Blocking buffer was removed and replaced by mouse serum sample each diluted at 1:100 in blocking buffer solution (100µl per well), and incubated 45 min at 37°C. Plates were then washed 4 times with PBS containing 0.1% Tween 20 (washing buffer solution). Horseradish peroxidase-conjugated sheep anti-mouse IgG, IgG1, IgG2a, IgG2b secondary antibody (Jackson) was added to each well (100µl) at 1:2000 dilution in blocking buffer solution, and incubated 45 min at 37°C. Each immunoglobulin was then tested independently. The conjugate was discarded, and the plates were washed 4 times with the wash buffer solution. Kblue substrate (Neogen Corp.) was then added (100µl per well) and plates were incubated 30 min in the dark at room temperature.

Absorbance at 620nm was measured using a spectrophotometer (Enspire, Perkin Elmer Inc.).

Statistical analysis

Statistical analyses were carried out using R studio software. Data from parasitaemia and mouse serum (ELISA and BD TM CBA mouse Th1/Th2 Cytokine Kit) were compared between groups using non-parametric tests (Kruskall-Wallis and Wilcoxon test). The R package Survival was used to analysis survival with the Kaplan-Meier estimate [START_REF] Harrington | A class of rank test procedures for censored survival data[END_REF].

Results

Production, purification of rTbgTCTP

rTbgTCTP protein purification was processed as reported in [START_REF] Bossard | Characterization of recombinant Trypanosoma brucei gambiense Translationally Controlled Tumor Protein (rTbgTCTP) and its interaction with Glossina midgut bacteria[END_REF], and allowed to recover approximately 174 mg of recombinant protein (95% purity rate) per liter of insect cells culture medium. Based on the rTbgTCTP specific sequence, the B linear epitopes prediction software [Bepipred 1.0 Server [START_REF] Larsen | Improved method for predicting linear B-cell epitopes[END_REF]] predicted epitopic structure covering 46 % of the total protein sequence, thus showing theoretically a large antigenic potential.

Kinetics of T. b. gambiense infection

We have investigated the possibility that mice immunization with rTbgTCTP could have an impact on the parasite multiplication rate (parasitaemia evolution) and, consequently on mice survival.

Parasitaemia in mice

The level of parasitaemia is classically used as a diagnostic parameter to evaluate the evolution of the disease itself in humans (and in animals) [START_REF] Stanghellini | Technics of detection and diagnosis of African trypanosomiasis in humans[END_REF].

Figure 1 shows the mean fluctuation of the parasitaemia in mice of the six different groups after having been challenged with trypanosomes at day(0). The evolution of the control groups (PBS and Freund) was characteristic for Tbg infections [START_REF] Giroud | Murine Models for Trypanosoma brucei gambiense disease progression from silent to chronic infections and early brain tropism[END_REF][START_REF] Parra-Gimenez | Parasitological, hematological, and immunological response of experimentally Infected Sheep with Venezuelan Isolates of Trypanosoma evansi, Trypanosoma equiperdum, and Trypanosoma vivax[END_REF]. Parasitaemia increased from day(5) to reach a first peak at day(7) to day (8). Thereafter it decreased to a base-line and increased again to reach a second parasitaemia peak between day(14) to day(15). A third peak occurred roughly at day(+19) / day(21). Regarding the groups of immunized mice, differences were noticed with reference to the control groups. While the first parasitaemia peak for the group 20TCTP (5.10 8 parasite/ml) was similar to that of the controls (4 to 5.10 8 parasite/ml), those of groups 40TCTP (2,5.10 8 parasite/ml) and 80TCTP (2.10 8 parasite/ml) were 1.5 to 2 times lower.

Nevertheless the main significant difference consisted in the absence of the second parasitaemia peak in the mice of the three immunized groups as compared to the control groups. However, from day(17/18) post infection, parasitaemia increased in mice from all the 5 groups whether control or TCTP immunized. In addition, for the TCTP20 and TCTP80 groups, the 2 nd parasitaemia peak appeared and increased at the time the 3 rd peak of the control mice was increasing.

Mice survival

Figure 2 shows mice survival in the different groups. During the first 6 days following mice infection with T. b. gambiense, 100% of the individuals survived. At day 7, we observed a loss of 50% of mice from the PBS-control group, while 80% survived in the four other groups whether Freund-control or the three groups of TCTP-immunized mice. From day(15) survival rate decreased progressively and regularly except in the 80TCTP group where a 80% mice survival lasted up to day(20), then decreased similarly to the other groups.

Hematocrit and weight of the mice

Mean hematocrit value evolution was similar in all the 5 groups (data not shown). It was stable at 55 % along the 48 days of immunization. Then, it decreased to 50% during the 4 first days post infection and finally to 40 and to 30%, depending on the considered group, between day(4) and day( 8) and remained at these low levels until day(18) post infection.

No particular effect was noticed regarding the weight of mice, which did not vary along the experiment, regardless of the group (data not shown).

Mice antibody and interleukine responses to immunization with rTbgTCTP

Note regarding the Figures: the day

Antibody response

IgG, IgG1, IgG2a and IgG2b antibodies production was measured in order to evaluate the rTbgTCTP antigenic potential. The assays were performed on sera taken from the mice at day(-48), just before the first injection of the PBS, Freund's adjuvant or of the three doses of rTbgTCTP; the next assays were performed at days(-33) and (-18), then at day(0), just before performing parasite inoculation, and at days(+4), (+8) and (+12) post infection. Results, including individual data, are shown in Figures 3456.

Regarding IgG (Figure 3) the seroconversion against rTbgTCTP did not occur before day(+4) in each control group (PBS and Freund) whereas it started before infection, at day(-33) in the 3 TCTP groups, although the results were only statistically slightly significant (p = 0.02), at this day, in 80TCTP. In the PBS, 20TCTP and 40TCTP groups, seroconversion occurred significantly (respectively, p = 0.0001; p = 0.002 and p = 0.02) at day(+4). Finally, IgG production increased from day(0) to day(+8) in all the 5 groups with exception for the 80TCTP group where it was highest from day(0) to day(+8) and then decreased. It is important to note the dispersal of individual data and individual reactions of some mice compared to other mice from the same group. This is, for example, the case of mouse n° 22 from the 40TCTP group; this same mouse was also singled out, although less clearly, for the production of IgG1, IgG2a and IgG2b. Finally, the only statistically significant difference was recorded when comparing the "80TCTP" group with control group: IgG production began earlier and the levels were much higher.

Regarding the IgG1 subclass (Figure 4), we could not detect any production in both control groups (PBS and Freund) throughout the experiment. In contrast seroconversion occurred either at day(-18) (40TCTP / p = 0.0001; and 80TCTP / p = 0.0008) or at day(0) (20TCTP), this latter being however not significant (p > 0.05) because of the high data dispersal.

Maximum amount of IgG1 was recorded at day(0) (80TCTP) or at day(+4) (40TCTP) and then decreased until day(+12).

IgG2a (Figure 5), only 40TCTP (p = 0.02) and 80TCTP (p = 0.03) produced this immunoglobulin at day(-18), i. e. before infectious challenge with the parasite. From this time up to day(+8), IgG2a production increased slowly and finally decreased drastically at day(+12).

Finally, regarding IgG2b (Figure 6), its level was low throughout the experiment, whatever the group. The weak variations observed in 80TCTP were not statistically significant. One may notice that group 20TCTP included a mouse (n° 13) whose behavior seemed distinctly different from the 5 other members of the group which was not reliable with any clinical signs

Cytokines responses

IL-2, IL-4, IL-6, IL-10, TNFα and IFNγ levels were measured in sera recovered from mice respectively at day(-48), just before immunization, at day(0), just before mice challenge with trypanosomes, and at day(+4), day(+8) and day(+12) post infection. Results are shown in IL-2, IL-4, IL-6 and IL-10 data were most often highly dispersed. Amounts of IL-2 (Figure 8) and IL-4 (Figure 9), respectively, were similar over time and experimental design. Whatever the groups, IL-6 (Figure 10) and IL-10 (Figure 7) levels significantly decreased after infection. IFNγ (Figure 11) and TNFα (Figure 12) production, their levels increased at day (0) compared to day (-48). Finally, the rates of IFNγ (Figure 11) and TNFα (Figure 12) decreased significantly after infection. It is interesting to note that for the 80 TCTP group, all the cytokine productions were very low or totally absent all over the whole time of the experiment.

Discussion

The most widely used method, as it is usually the most effective for preventing an infectious disease, is vaccination, the extension of which to the largest number of individuals in a population protects the entire population itself. This approach unfortunately has exceptions, for example, when epitopes of the pathogen are only weakly immunogenic, or when frequent mutations occur that modify the structure of epitopes or when, through an appropriate mechanism, they are regularly renewed. This latter case occurs in the infectious form of trypanosome; that is why it escapes the immune defenses of the host, human or animal. This is also why the principle of vaccination is currently inapplicable because it is not known which of the hundreds of possible variants of VSG will cover the parasite at the time of infection, nor in which sequence (preprogrammed or random) the various genes encoding the variants will be successively activated. The vaccination approach has however not definitively to be discarded, it may be focused on invariant proteins that are essential either for the survival of the parasite or for the expression of its pathogenicity. In this context, the choice of TCTP was based both on data from the literature and on those resulting from our previous researches.

From a general point of view results recorded under some experimental conditions were too dispersed to be statistically validated, mice immunization with rTbgTCTP was working, although limited in intensity and time. In fact, immunization resulted, in a dose-dependent manner, in the production of specific immunoglobulins, in the stimulation of the production and/or in the inhibition of certain cytokines, and finally in modulating parasitaemia kinetics and mice survival.

Regarding this latter aspects, four criteria were measured, the weight of the mice, their hematocrit, their survival throughout the experiment, and the evolution of their parasitaemia after having been trypanosome-infected. The criteria included hematocrit, since anemia was shown to be associated with T. congolense and T. vivax infections of cattle [START_REF] Mbewe | Prevalence of pathogenic trypanosomes in anaemic cattle from trypanosomosis challenged areas of Itezhitezhi district in central Zambia[END_REF].

In this study, neither the evolution of the weight in the immunized / infected mice nor that of the hematocrit did differ significantly from that of the control mice. In contrast, a dosedependant effect of immunization clearly appeared on mouse mortality since immunization with the highest dose of rTbgTCTP (80 μg per mouse) allowed the survival of 80% of individuals at day(+20) post-inoculation, while less than 40% of individuals survived in the other groups of mice at the same time. However, the effect of rTbgTCTP only delayed mice death by 7 days.

The evolution of mice survival can be explained by that of their parasitaemia depending on the different groups. In the two control groups, parasitaemia evolved along the classical pattern of Tbg-infected mice: a first peak 7 to 8 days after infection, a second around day(+14) to day(+15) and finally a third one at day(+22) after which all the surviving mice died. In the immunized mice, the first peak occurred also 7 to 8 days post-infection, but it was approximately 50% lower in the mice that were immunized with either 40 or 80 µg of rTbgTCTP, than in the control groups. However, the most remarkable was the increased time that separated the second peak from the first: 15 days (group 80TCTP) versus 7-8 days in the control groups. This result clearly demonstrated the mice immunization with rTbgTCTP to provide an inhibitory, although transient, effect against in vivo Tbg blood stream forms multiplication.

Regarding immunoglobulin productions (IgG, IgG1, IgG2a and IgG2b), a first comment concerns the reality of immunization with rTbgTCTP. Whereas in the control groups the level of immunoglobulins was almost failing, their production was stimulated between days (-33) and (-18) (i. e. after the 2nd injection of rTbgTCTP), and their production increased very significantly between the 3rd injection and the day(0) which corresponded to the mice inoculation with trypanosomes. The dose-dependent effect also appeared very clearly. The ELISA tests being very specific, the IgG tested were anti-rTbgTCTP immunoglobulins, demonstrating: a) that TCTP is really immunogenic, and b) that the immunization procedure was working.

Post-infection stimulation of immunoglobulin production in the mice control group logically meant that the trypanosomes secreted TCTP in vivo, in the infected mouse, in amounts sufficient to stimulate the mice immune response. Theoretically, this interpretation was in agreement with previous results that have shown, among others: a) that TCTP is part of the trypanosome secretome proteins (procyclic and bloodstream forms), b) that it is excreted in vivo and released into the bloodstream of the mouse, c) that the corresponding TCTP gene is also overexpressed in flies infected by the parasite. This effect may also be significant in the 20 and 40TCTP mice immunized with the lowest doses; it was lesser significant in the 80TCTP mice immunized with the highest dose.

The question was why does the level of free IgG decrease from 4 or 8 days post-infection in immunized mice? Immunization had only a partial effect on the evolution of parasitaemia; it lowered the importance of the first peak but did not suppress it; it also significantly decreased the concentration / density of surviving trypanosomes during the period following the parasitaemia peak, but did not completely eliminate them. It may be thought that the first peak of parasitaemia weakens the mouse reaction capacities, reducing, maybe suppressing, the production of total IgG, thus decreasing the free IgGs in the blood, while the molecules still present were mobilized via agglutination with the TCTPs that continued to be produced and excreted by the parasite. Knowing that B lymphocytes play an important role in parasite clearance [START_REF] Magez | The role of B-cells and IgM antibodies in parasitemia, anemia, and VSG switching in Trypanosoma brucei-infected mice[END_REF], the lack of free IgG availability could probably be due to clonal depletion of B cells or even destruction of the memory compartment of B cells as described [START_REF] Bockstal | T. brucei infection reduces B lymphopoiesis in bone marrow and truncates compensatory splenic lymphopoiesis through transitional B-cell apoptosis[END_REF] and thus allowed a new and fatal increase in the trypanosome population, even with the highest dose of TCTPs.

Besides global IgG analyses, we tested also the subclasses IgG1, IgG2a and IgG2b. In the control groups and compared to the immunized groups, IgG1 were not produced at all even after mice infection; this could mean that the in vivo TCTP secretion by the infecting trypanosomes was too low to induce an IgG1 response from the mice. In mice that had been immunized, a dose-dependent effect was shown regarding IgG1 production.

In mice, there are two IgG2 sub-classes, IgG2a and IgG2b. IgG2b were very weakly produced, while IgG2a production evolved quite similarly to IgG1, except that a weak production can be observed in control mice after they have been infected by trypanosomes.

To define the orientation of the immune response using the IgG1/IgG2 ratio, we used only the IgG2a sub-class response, as the production of IgG2b was extremely weak. Before infection, the orientation was towards Th2 type through the production of IgG1. After infection, orientation of the immune response switched to a Th1 type response, but the cytokine productions in our study did not seemed to correlate. Indeed, concerning the Th1, Th2 and inflammatory cytokines tested (INFγ, TNFα, IL-2, IL-4, IL-6 and IL-10), and except for the 80 TCTP group which is discussed further, pre-immunization with TCTP did not induce any specific modulations of cytokine levels when comparing with Freund control group, whereas infection induced a modulation. Once more, the modulation is approximately the same between groups. All groups showed similar variations in the levels of the cytokines before infection with the parasite, whether they received the molecule or not. The decrease of levels of IL-6, IL-10, TNFα and IFNγ after infection was observed in all groups, and we assumed that was due by the presence of the viable parasite in animals. Some previous study showed that, in T. b. rhodesiense-infected mice, IFNγ production correlates with a decrease in parasite load and is implicated in resistance to infection [START_REF] Hertz | Resistance to the African trypanosomes is IFN-gamma dependent[END_REF]. It has also been shown that, during T. congolense infection, massive production of IFNγ appeared to have a deleterious effect on the liver [START_REF] Shi | Experimental African trypanosomiasis: IFN-gamma mediates early mortality[END_REF]. In our experiment, the rapid decrease in IFNγ production after infection was observed for all groups, without any significant difference in pre-immunization with TCTP or not, and therefore cannot explain the relative lengthening of the survival for the TCTP groups.

The TH1/TH2 immune response and its balance may differ depending on the considered parasite. In helminths, for example, the TH2 response is favored [START_REF] Cortés | Th2 and Th1 Responses: Clear and Hidden Sides of Immunity Against Intestinal Helminths[END_REF]. In contrast, in Leishmaniae, the Th1 response will be preponderant; nevertheless it has also been shown that in Leishmania major the Th1 response can switch to a Th2 response [START_REF] Louis | Antileishmania effector functions of CD4+ Th1 cells and early events instructing Th2 cell development and susceptibility to Leishmania major in BALB/c mice[END_REF]. The immune response against parasite is a TH2 response implies IgG1 production together with IL-4 and/or IL-10. In our study, IgG and IgG1 production were only observed in animals which received rTbgTCTP protein with a maximum at day(0) and day(+4) although we didn't find significant levels in IL-4 and IL-10 productions in these animals.

Levels of IL-6 and IL-10 were very low (similar than those observed in PBS groups), and no significant modulation of these cytokines was observed, whatever the groups. The IL-6 cytokine was described to have a role in protection against neuropathogenesis of the parasite [START_REF] Lejon | Interleukin (IL)-6, IL-8 and IL-10 in serum and CSF of Trypanosoma brucei gambiense sleeping sickness patients before and after treatment[END_REF]. In addition, it is known that IL-10 plays a role in the immunological balance between protection and pathology of immune response in Human African Trypanosomiasis [START_REF] Shi | Experimental African trypanosomiasis: IFN-gamma mediates early mortality[END_REF][START_REF] Namangala | Relative contribution of interferon-gamma and interleukin-10 to resistance to murine African trypanosomosis[END_REF][START_REF] Bosschaerts | Tip-DC development during parasitic infection is regulated by IL-10 and requires CCL2/CCR2, IFN-gamma and MyD88 signaling[END_REF][START_REF] Bosschaerts | IL-10 limits production of pathogenic TNF by M1 myeloid cells through induction of nuclear NF-kappaB p50 member in Trypanosoma congolense infection-resistant C57BL/6 mice[END_REF][START_REF] Guilliams | IL-10 dampens TNF/inducible nitric oxide synthase-producing dendritic cell-mediated pathogenicity during parasitic infection[END_REF]. The presence or absence of these cytokines in our study may not have an impact in delayed mortality in the TCTP groups.

Compared to the other groups, an extinction of cytokine production is measured in the group injected with 80 µG TCTP. Knowing the strong immunomodulatory properties of this molecule [START_REF] Grébaut | Identification of total and differentially expressed excreted-secreted proteins from Trypanosoma congolense strains exhibiting different virulence and pathogenicity[END_REF][START_REF] Bommer | The translationally controlled tumour protein (TCTP)[END_REF], we hypothesized that the dose injected is very or even too high and that the cells of the immune system are overwhelmed or anergic, and cytokine production was abolished.

Regarding this latter aspects, four criteria were measured, the weight of the mice, their hematocrit, their survival throughout the experiment, and the evolution of their parasitaemia after having been trypanosome-infected. The criteria included hematocrit, since anemia was shown to be associated with T. congolense and T. vivax infections of cattle [START_REF] Mbewe | Prevalence of pathogenic trypanosomes in anaemic cattle from trypanosomosis challenged areas of Itezhitezhi district in central Zambia[END_REF].

In this study, neither the evolution of the weight in the immunized / infected mice nor that of the hematocrit did differ significantly from that of the control mice. In contrast, a dosedependant effect of immunization clearly appeared on mouse mortality since immunization with the highest dose of rTbgTCTP (80 μg per mouse) allowed the survival of 80% of individuals at day(+20) post-inoculation, while less than 40% of individuals survived in the other groups of mice at the same time. However, the effect of rTbgTCTP only delayed mice death by 7 days.

The evolution of mice survival can be explained by that of their parasitaemia depending on the different groups. In the two control groups, parasitaemia evolved along the classical pattern of Tbg-infected mice: a first peak 7 to 8 days after infection, a second around day(+14) to day(+15) and finally a third one at day(+22) after which all the surviving mice died. In the immunized mice, the first peak occurred also 7 to 8 days post-infection, but it was approximately 50% lower in the mice that were immunized with either 40 or 80 µg of rTbgTCTP, than in the control groups. However, the most remarkable was the increased time that separated the second peak from the first: 15 days (group 80TCTP) versus 7-8 days in the control groups. This result clearly demonstrated the mice immunization with rTbgTCTP to provide an inhibitory, although transient, effect against in vivo Tbg blood stream forms multiplication.

To conclude, the results presented here assess the antigenic properties of the rTbgTCTP protein. Injected in mice before infection with trypanosomes, it modulates immunoglobulin production, associated with biological effects such as limitation of parasitaemia level and mice survival. Although the role of TCTP in the parasite's interactions with its host during disease development is still not known, its blocking by immunization-induced immunoglobulins seems to impact directly parasitaemia i.e. the multiplication of the parasite within its host. TCTP may display a higher involvement than calreticulin [another trypanosome secreted protein (tested on T. congolense infected mice) [START_REF] Bossard | Cloning, expression, molecular characterization and preliminary studies on immunomodulating properties of recombinant Trypanosoma congolense calreticulin[END_REF], owing that mouse immunization against TCTP, seems to have higher protective effects, despite only transient, than calreticulin immunization. Therefore research on TCTP deserves pursuing in combination with other trypanosome excreted/secreted proteins in order to improve host protection against parasite infection. Experimental design was as in Figure 3. and 80TCTP) and non-immunized controls (PBS or Freund's adjuvant). Experimental design was as in Figure 7.

  (0) corresponds to the day of mice inoculation with the trypanosomes. The negative and positive values indicate the number of days preceding (period of mice immunization) and following the day(0) of the infection, respectively. Each mouse was identified thus allowing the individual data to be presented in the different Figures (3 to 12) together with the statistics representation.

Figures

  Figures 7 to 12.

  Figures 7 to 12.

Figure 1 .

 1 Figure 1. Kinetics of Trypanosoma brucei gambiense parasitaemia. Three groups of OF-1

Figure 2 .

 2 Figure 2. Survival curves of mice infected with Trypanosoma brucei gambiense (ABBA

Figure 3 .

 3 Figure 3. IgG immunoglobulin production by rTbgTCTP-immunized mice (20TCTP,

Figure 4 .

 4 Figure 4. IgG1 immunoglobulin production by rTbgTCTP-immunized mice (20TCTP,

Figure 5 .

 5 Figure 5. IgG2a immunoglobulin production by rTbgTCTP-immunized mice (20TCTP,

Figure 6 .

 6 Figure 6. IgG2b immunoglobulin production by rTbgTCTP-immunized mice (20TCTP,

Figure 7 .

 7 Figure 7. Cytokine production by rTbgTCTP-immunized mice (20TCTP, 40TCTP and

Figure 8 .

 8 Figure 8. IL-2 cytokine production by rTbgTCTP-immunized mice (20TCTP, 40TCTP and

Figure 9 .

 9 Figure 9. IL-4 cytokine production by rTbgTCTP-immunized mice (20TCTP, 40TCTP and

Figure 10 .

 10 Figure 10. IL-6 cytokine production by rTbgTCTP-immunized mice (20TCTP, 40TCTP and

Figure 11 .

 11 Figure 11. INF γ cytokine production by rTbgTCTP-immunized mice (20TCTP, 40TCTP and

Figure 12 .

 12 Figure 12. TNFα cytokine production by rTbgTCTP-immunized mice (20TCTP, 40TCTP

Funding

This work was supported by CIRAD and IRD (UMR INTERTRYP).

Declarations Ethics approval and consent to participate

The experiments designed for this study were approved by the regional Ethic Committee for Animal Experimentation CEEA-LR 36 under project number 02187.01 and authorized by French Ministry for Higher Education and Research.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

GB and AG conceived and designed the experiments. GB, VR and ET conducted the experiments. GB and VR analyzed the data. GB and AG contributed reagents, materials and analytical tools. GB, VR, ET and AG wrote the manuscript. All authors read and approved the final manuscript.