The energy-coupling factor (ECF) transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. Their central role in the metabolism of bacteria and absence in humans make the ECF transporters a potential antibacterial target, which can be further investigated making use of a selective chemical probe. Here, we report on the virtual screening, design, synthesis, structure-activity relationships (SARs) and coarse-grained molecular dynamics simulations of the first class of inhibitors of the ECF transporters. We investigated the mechanism of action of this chemical class and profiled the best hit compounds regarding their pharmaceutical properties. The optimized hit has a minimum inhibitory concentration (MIC) value of 2 µg/mL against Streptococcus pneumoniae, which opens up the possibility to use this chemical class to investigate the role of the ECF transporters in health and disease.

Targeting the energy-coupling factor (ECF) transporters: identification of new tool compounds

Eleonora Diamanti, ‡a,b Inda Setyawati, ‡c,d Spyridon Bousis, ‡a,b Paulo C. T. Souza, e,f Leticia M. Gómez, b Lotteke J.Y.M. Swier, c Jörg Haupenthal, a Paddy S. Gibson, g Carsten Volz, h Weronika K. Stanek, c Manuel Jäger, b Jennifer Herrmann, h Siewert J. Marrink, e Jan-Willem Veening, g Rolf Müller, g,i Dirk J. Slotboom* c and Anna K. H. Hirsch* a,b,i

Introduction

Energy-coupling factor (ECF) transporters are a recently discovered subclass of the superfamily of adenosine 5'triphosphate (ATP)-binding cassette (ABC) transporters. While ABC transporters are present in all prokaryotic and eukaryotic species, [1][2][3][4] mediating uptake or extrusion of compounds into and from cells, the ECF transporters are absent in humans but present in approximately 50% of prokaryotic species. This class of proteins mediates the uptake of essential micronutrients such as water-soluble vitamins (e.g., folate, 5 riboflavin, 6 cobalamin, 7 biotin, 8 niacin, 9 thiamine, 10 pantothenate 11 ) and metal cations (Ni 2+ and Co 2+ ) 12,13 into bacteria and archaea. They are widely distributed in the Firmicutes phylum of Gram-positive species 14 and contribute to the survival and growth of the bacteria. 15 The ECF transporters are transmembrane proteins consisting of two modules: an integral membrane domain dedicated to the binding of a single substrate (the S-component), and an ECF module formed by an integral membrane protein, the T-component (EcfT) and two intracellular ATPases (EcfA and EcfA', Fig. 1). ECF transporters are classified into two groups, group-I and -II. 16 In group I, the ECF module interacts exclusively with a single "dedicated" S-component, whereas the module in group-II interacts with different ones. In group-II, the same ECF module can associate with distinct S-components, opening up the possibility to block the uptake of several vitamins with a single inhibitor. A recently proposed mechanism of transport postulates that the S-component can dissociate from the ECF module, which allows for exposure of the substrate binding pocket to the extracellular environment. 17,18 After binding, the substrate is buried inside a cavity, and the S-component can rotate (topple over) by ~ 90 degrees in the plane of the membrane. In the toppled state, the S-component binds to the ECF module, which leads to the release of the substrate inside the cytosol. 19 Subsequent pinching of two long alpha helical elements, the coupling helices, of the EcfT subunit is predicted to occur by hydrolysis of ATP, and causes re-orientation and dissociation of the substrate-free S-component, marking the end of a transport cycle. Hence, inhibitors that specifically block the movements taking place in the ECF module, in particular the coupling helices, could impact the uptake of various vitamins (mediated by different S-components) by the bacterium.

Fig. 1 Architecture of ECF group-II transporters. In green ECF module (EcfT, EcfA and EcfA´) and in blue the S-components (e.g., the ones specific for folic acid (FolT), panthotenate (PanT)). The S-components interact with a shared ECF module. Genes coding for Scomponents are scattered around the chromosome, while the ECF-module genes are usually clustered.

Given that this inhibition likely affects growth and survival, it constitutes a new avenue for intervention in bacterial infections. Despite the important functions of ECF transporters, no inhibitor targeting the unique transport mechanism has been reported to date, only substrate-mimicking competitive inhibitors for the S-component ThiT from Lactococcus lactis have been reported. [20][21][22] Here, we describe the discovery of unexplored chemical structures, with compound 20 (Table 1) as the best in the series, as the first inhibitors of ECF-transporter activity. We further show that this compound is able to inhibit the uptake of more than one vitamin and may be used in vitro to explore the function of ECF transporters.

Results and discussion

The absence of known inhibitors, as well as the lack of a robust high-throughput activity assay hampered a medicinalꟷchemistry exploration of ECF transporters. Despite the relatively low resolution (3.0 Å) of the available crystal structure of a folate-specific transporter ECF-FolT from Lactobacillus delbrueckii (PDB ID 5JSZ), 23 and the fact that we are dealing with transmembrane protein with a highly dynamic mechanism of action, we decided to perform a structure-based virtual screening (SBVS) as the only viable hit-identification approach. Here, we report on the SBVS campaign using the X-ray crystal structure of ECF-FolT (PDB ID 5JSZ), 23 which yielded compounds 1 and 2 as inhibitors of ECF-mediated vitamin uptake (Fig. 2). We selected the structure of the transporter from L. delbrueckii, because for this protein a robust, albeit low throughput activity assay is available. The first step in the identification of new hits for the ECF transporters was the selection of druggable pockets, for which we used a fully automated algorithm, DoGSiteScorer, (Fig. S1). 24 A strategic localization near the predicted surface of the membrane, where the coupling helices of the EcfT component are expected to change conformation during transport, and a score of 0.81, guided us to select the so-called P2 pocket as the most promising allosteric binding site (Fig. S2). Compounds binding to this pocket were expected to interfere with the transport cycle and inhibit uptake of all substrates of the ECF transporter in question. We performed the virtual screening on the Express Collection of Princeton BioMolecular Research (1.3 million compounds) using the KNIME Analytics Platform 25 and LeadIT [START_REF]LeadIT. version 2.1.8[END_REF] with the scoring function HYDE implemented in SeeSAR [START_REF] Gmbh | [END_REF] and followed a typical workflow (details are provided in the Supplementary Information), applying a range of filters to focus on drug-like compounds and exclude frequent hitters. [28][29][30] The final selection of the 100 top-ranked compounds included visual inspection, assessment of the docked pose and estimation of the drug-like properties. As a result, we selected twelve distinct molecular scaffolds (1-12, Fig. 2 andS3) for biochemical screening. A transport-activity assay on purified ECF-FolT2 reconstituted in proteoliposomes using radiolabeled folate showed that 1 and 2 are able to reduce translocation of the vitamin across the membrane with IC50 values of 282 M and 1.2 mM, respectively. Hit 2, despite its weak inhibitory activity, has a comparable ligand efficiency to hit 1 and it may be bound in the same region, but it suffers from solubility issues given its zwitterionic nature. Fig. 2 Chemical structures of hit compounds (1 and 2) identified through structure-based virtual screening using the crystal structure of Lactobacillus delbrueckii ECF-FolT2 (PDB ID 5JSZ). 23 As a result, we selected hit 1 for our study because of its chemical tractability and more favorable physicochemical properties. The fragment-sized hit compound 1 with a MW 294.3, cLogP 3.5 and ligand efficacy (LE) of 0.22 was selected as starting point for subsequent hit validation through resynthesis, retesting and evaluation of its in vitro pharmacokinetic properties. Assessment of the inhibition of the ECF transporters, along with in vitro metabolism and cytotoxicity studies supported the potential of this chemotype initially as chemical probe and ultimately for the development of agents that target the ECF transporters.

Biochemical assay to validate HIT 1

This assay was performed by the purification and reconstitution into proteoliposomes of ECF-FolT2 and ECF-PanT, which share the same ECF module, but use different S-components, for folic acid and pantothenate, respectively. At an inhibitor concentration of 5 mM, compound 1 was able to completely inhibit the transport of both folate and pantothenate, indicating that it binds to the shared ECF module. To further validate that the inhibition values of 1 are not due to an artifact like disruption of the lipid bilayer or chelation of Mg 2+ , which is crucial for the hydrolysis of ATP, or direct interference with the hydrolysis of ATP, we used the ABC transporter OpuA from L. lactis, a classical ABC transporter that mediates the uptake of glycine betaine, with membrane domains unrelated to ECF transporters, as negative control. Since the ATP hydrolysis sites are well-conserved among all ABC transporters, OpuA can be used to evaluate the selectivity of the compounds. OpuA was purified and reconstituted in liposomes. The uptake of glycine-betaine into the proteoliposomes was not affected by the presence of 1, providing the first indication that our compound neither inhibits the hydrolysis of ATP by ABC transporters nor does it disrupt the lipid bilayer of the proteoliposomes or interfere with the accessibility of magnesium. In conclusion, we demonstrated that compound 1 is able to inhibit the transport of two different vitamins and selectively inhibit the family of ECF proteins and not ABC transporters in general.

Structure-activity relationship (SAR) study around 1

Encouraged by the potency and drug-likeness of 1, we sought to confirm structural features that are critical for inhibition of the ECF transporter. The low resolution of the available crystal structure might lead to unreliable docking predictions approach; therefore we initiated a focused SAR study first aimed at exploring the role of 2hydroxybenzoic acid with a small set of compounds (13-21) prepared as described in Scheme S1. Removal of one group at the time yielded monosubstituted derivatives 13 and 14. Loss of inhibitory activity was observed in both cases, supporting the key role played by the salicylic acid portion. The reduction of the carboxylic acid to a primary alcohol, its methyl esterification and amidation yielded compounds 15-17. While compound 15 is twofold less potent than 1 (14% inhibition at a concentration of 250 μM), the replacement of the carboxylic acid with an amide (17) had no significant effect (27% inhibition at a concentration of 250 μM). Unfortunately, the solubility of 16 in the assay buffer is substantially decreased. Next, we turned our attention to the role of the hydroxyl group and found that its replacement with a methyl group (18) or methylation (19) reduced the inhibitory activity. By contrast, the finding that 20 is able to inhibit ECF activity with a two-fold higher potency compared to 1 (20, IC50 = 134 μM) supported the notion that a sterically bulky carbamate moiety is well-tolerated. After demonstrating that this chemical class interferes with the uptake of folic acid and pantothenate via inhibiting the ECF transporter in vitro (Fig. 3bꟷc), we investigated whether 20 might inhibit ECF activity in a cellular set up. Therefore, we developed a cell-based transport assay using E. coli MC 1061 heterologously producing ECF_FolT2 and evaluated the ability of 20 to interfere with the uptake of radiolabeled folic acid. As a control, we used E. coli MC 1061 that was not induced for expression. By analogy with the in vitro experiment, also in a cell-based assay, our compound 20 showed an inhibition of 93 ± 1% at a concentration of 250 μM (Table 2). Taken together these findings are in agreement with the proposed allosteric mode of inhibition and show that 20 indeed inhibits the ECF transporter. Indicator strains 20 % Inh. @ 250 μM Escherichia coli MC1061 ECF_FolT2 93 ± 1

Cmpd

Escherichia coli MC1061 no uptake

The lack of uptake in the absence of heterologous expression (Figure 4A) indicates that there is no alternative transporter for folic acid that facilitates the uptake of it at nanomolar concentration. Lastly, we corroborated the allosteric mode of inhibition of 20 in the in vivo set up by using a 2.5-fold excess of the vitamin substrate (Fig. 4B), which did not affect the inhibition significantly. These encouraging results prompted us to measure the effect of compound 20 on the human pathogen Streptococcus pneumoniae. This organism is auxotrophic for pantothenate and biotin and depends on ECF transporters for their uptake. Therefore, we predicted that inhibition of the ECF transporter would reduce its viability. Consistently, compound 20 inhibited growth of S. pneumoniae, but the MIC (minimum inhibitory concentration) was remarkably low compared to its in vitro potency (Table 3) presumably, because the uptake of more than one vitamin was affected. In fact, in the cell-based assay the effect of a diminished uptake of multiple micronutrients may have a synergistical impact on the cell survivability compared to the inhibition of uptake of only one micronutrient we monitor in the in vitro assay. As a negative control, we used a Gram-negative strain that does not express the ECF transporter (E. coli K12) and expectedly, we did not observe an antibacterial effect of compound 20. Indicator strains 20 MIC [µg/mL] Streptococcus pneumoniae DSM-11865 [a] 2 Escherichia coli K12 >100

[a] PRSP: Penicillin-resistant Streptococcus pneumoniae.

Compound 20 was tested for cytotoxic effects on a set of cell lines that includes HEK293 (human embryonic kidney cells), HepG2 (human liver cancer cells), and A549 (adenocarcinomic human alveolar basal epithelial cells). Notably, while 20 did not show any considerable cytotoxic effects in A549 cells at 100 µM (Fig. 5), a moderate decrease in viability was observed in HepG2 and HEK293 cells. This effect was in the same range observed with the positive control doxorubicin at a 20-fold lower concentration. The approximate IC50 value of compound 20 in HepG2 and HEK293 cells was ~100 µM and thus much higher than the antibacterial effects observed in Streptococcus pneumoniae (MIC of 2 µg/mL) leading to a good therapeutic window. Furthermore, we assessed the stability of compound 20 in human plasma and liver microsomes (Table S1) and found an adequate stability in plasma (20, t½ > 2.5 h) and metabolic stability (20, hepatic Clint < 12 µL/min/mg). Thus, our results confirm compound 20 as the first molecular probe that may be used to investigate the role of the ECF transporters both in vitro and in cellulo. Lastly, by using a fluctuation test we examined the ability of S. pneumoniae (strain D39V) to become resistant to compound 20. In contrast to the rifampicin control, where we could readily select resistant mutants (experiment was performed at 2-fold MIC), no mutants resistant to compound 20 (at 2-fold MIC) could be isolated, indicating a very low potential for resistance development against this type of ECF transporter inhibitors (Table S2).

Searching for evidences of the action mechanism

Although, all experimental data point to selective inhibition of the ECF transporters by the hit compounds, it was not possible to experimentally confirm their binding mode. Mutagenesis experiments in P2 pockets were inconclusive as they resulted in low uptake levels of the radiolabelled vitamin, indicating the importance of the selected amino acids in the mechanism of action (Fig. S30). In addition, extensive attempts to co-crystallize the inhibitors with the ECF protein, have been unsuccessful so far. Nonetheless, some of our previous experimental studies provide indications. Given that the compounds inhibit the uptake of both folic acid and pantothenate into the liposomes in the in vitro assay (Fig. 3 AꟷC) and, that two different S-components are involved in the transport of the two substrates, we suggest that the mode of inhibition does not involve high-affinity binding to the substrate binding pockets of the S-component. It should rather involve interaction with the ECF module of the transporter or with one of its interface with the S-component.

To further explore all possible binding pockets of the ECF transporter, we performed coarse-grained (CG) molecular dynamics simulations using the recent unbiased sampling approach 31 based on the new version of the Martini force-field, called Martini 3. 32 The model has so far accurately predicted binding pockets and binding modes for pharmaceutical relevant targets such as nuclear receptors, GPCRs and kinases. Predictions of binding affinities are also possible, in case of sufficient sampling. 31 The bacterial membrane model, ECF transporter and inhibitors were included in the system, which was simulated for a total of 0.3 milliseconds, allowing not only to explore all possible pockets, but also to capture competition with the membrane and enough association and dissociation events to estimate binding free energies (ΔGbind). For transmembrane targets this is a crucial component that need to be considered but is neglected in docking approaches. The results indicated that the inhibitors not only can bind in pocket P2, but also in two additional pockets: at the entrance of P11, which is the main cavity of the S-component pocket, and in P9 which is a narrow, hydrophobic and partially hidden pocket at the interface between EcfT and the S-component (Fig. 6A and Figure S30). Although the binding to P2 is not so favourable, a negative ΔGbind in relation to water for compound 1 (ΔGP2water = ꟷ11.6 ± 1.1 kJ/mol) may explain this compound being found as a hit in the SBVS campaign. The entrance of P11 seems to be a more relevant pocket in relation to water than P2, with a ΔGP11-water of ꟷ13.1 ± 0.4 kJ/mol. However, the competition with the membrane also makes this pocket less relevant, as the pocket is embedded in the lipid environment. Indeed compound 1 can easily escape as the ΔGP11-memb is 4.2 ± 0.4 kJ/mol. In addition, this pocket varies between different S-components as each one of them binds a specific vitamin and is therefore not in agreement with the observed inhibition of multiple substrates. Prediction of binding free energy in relation to water or membrane places P9 as the most probable binding pocket, with compound 1 showing a ΔGP9-water and ΔGP9-memb ꟷ18.8 ± 0.8 and ꟷ1.5 ± 0.8 kJ/mol, respectively. As P2, the binding to P9 is also in line with the strategy of blocking the movements of the coupling helices taking place in the ECF module that are probably involved in the toppling and/or dissociation of the S-component. A representative pose of 1 in P9 (Fig. 6B) shows that certain polar/charged residues (like Ser 173 and Lys 102), that are not so near to this pocket in the crystal structure, can form a hydrogen bond network with the salicylic acid moiety, which suggests an important role for this substituent in the inhibitory action.

The comparison of ΔGP9-water estimates of compound 1 with compounds 14, which shows no inhibition activity, and 20, the best inhibitor obtained in the study (IC50 = 134±26 M) seems to reinforce the hypothesis that P9 is the most probably pocket. For pocket P9, the results nicely correlate with the inhibitory activity, while for the other pockets compound 14 emerges with highest binding affinity in relation to water. The proximity of P9 and its competition with the membrane may also explain the lack of inhibitory activity of 14 as its ΔGP9-memb is unfavorable (+8.2 ± 1.41 kJ/mol), while compounds 1 and 20 show almost zero ΔGbind for P9 in relation to the membrane. As indicated by the water/partitioning, all compounds simulated tend to be located in the membrane. However, the removal of the carboxylate group (in relation to compound 1) makes compound 14 too hydrophobic. The replacement of the hydroxyl group in compound 1 for the NHBoc group in compound 20 seems to promotes additional hydrophobic interactions of the tert-butyl group with Leu 172 (Fig. 6C). At the same time, the hydrogen bond of OH group is mantained by the NHBoc group of compound 20. These combined effects possibly explain the higher affinity of 20. S1). The green isosurfaces correspond to regions with high occupancy (100 times higher than compound 1 density in the membrane). As compound 1 binds only with low affinity to P2, no high occupancy density is observed. The figure also shows bar plots with calculated binding free energies (ΔGbind) of compounds 1, 14 and 20 for pockets P2, P9 and P11 in relation to water and membrane. In the case of compound 14 in pocket P2, the binding events are so reduced that it is only possible to estimate the upper limit (indicated by "≥" in the plots). Estimates of the partitioning free energies of the inhibitors between water and lipids (ΔG lipids/water) are also show in the figure. (B) and (C) Representative poses of compounds 1 and 20 inside pocket P9, respectively. Backmapped from the coarse-grained to the atomistic resolution. Some residues from EcfT (salmon) Sꟷcomponent (gray) are highlighted, including the hydrogen bond network (black dashed lines). Hydrogen atoms have been omitted for clarity.

This complete view including protein flexibility, and competition between environments and (hidden) protein pockets can only be properly estimated in the MD simulations as they consider all of these effects. 33 Additional redocking calculations (starting from relaxed configurations from the simulations) performed now in P2, P9 and P11, did not provide new evidence for the preference for P9 in relation to the other pockets (Figure S32, Table S4). However, future experimental evidence is still necessary to confirm the binding pocket and provide the complete molecular mechanism involved in the inhibition of the ECF transporters promoted by our newly discovered compounds.

Conclusions

In conclusion, we report the identification of the hydroxy benzoic acid and phenyl furan chemical classes as the first inhibitors of ECF transporters. The focused SAR study around the salicylic acid scaffold provided a first insight into the shape requirements for fragment growing. We showed both in vitro and in cellulo that this chemical class has an allosteric mode of inhibition by inhibiting the ECF module and potentially binds to the ECF-T domain. Results from unbiased coarse-grained simulations were in agreement with the design strategy, showing that the ligand probably binds at the interface involving the S-component and ECF module. Although our experimental data strongly indicate that the ECF transporter is the target and that the design hypothesis is correct , future work must include more direct assessment of binding pocket and mechanism of inhibition of the inhibitors. The 2-hydroxybenzoic acid is mandatory for activity whilst introduction of a carbamate function in the 2-position balances potency, metabolic stability and influences the growth of S. pneumoniae as shown by compound 20. The lack of cytotoxicity, the promising plasma and metabolic stability and the lack of resistance development observed under the conditions tested, establishes compound 20 as an excellent chemical probe to foster probe-and drug-discovery efforts in the field of the ECF transporters, which may open a new avenue and promising target in the antibacterial field.

Fig. 3

 3 Fig.3 Effects of compounds 1 and 2 on the transport activity of ECF transporters (ECF-FolT2 and PanT) and an unrelated ABC transporter (OpuA). (A) Folate uptake by ECF-FolT2 reconstituted in proteoliposomes filled with 5 mM MgATP (black), 5 mM MgADP (purple) or 5 mM MgATP plus 5 mM of compound 1 (red), with a final DMSO concentration of 10% (v/v) in all experiments. (B) Same as in Panel A, including proteoliposomes filled with 5 mM MgATP plus 5 mM of compound 2 (green). (C) Pantothenate uptake by ECF PanT in proteoliposomes filled with 5 mM of MgATP (black), 5 mM of MgADP (purple), 5 mM MgATP plus 5 mM of compound 1 (red), and a final DMSO concentrations of 5% (v/v). The blue curve represents proteoliposomes filled with 5 mM MgATP and no DMSO. (D) Glycine betaine uptake by OpuA in proteoliposomes filled as described in Panel C. The error bars represent the standard deviation of three independent measurements, except for the data in panel C, where they represent the spread of the data from two independent measurements.

Fig. 4 A

 4 Fig. 4 A) Uptake of radiolabeled folic acid (concentration 50 nM) by Escherichia coli cells in presence and absence of compound 20. The E. coli MC1061 strain does not show endogenous uptake of folic acid (blue bars), in contrast to the E. coli cells that heterologously express the ECF-FolT2 transporter from L. delbrueckii (MC1061 ECF_FolT2, red bars). At a concentration of at 250 μΜ, inhibitor 20 blocks the uptake of radiolabeled folic acid (concentration 50 nM) by 93%. B) Uptake of radiolabeled folic acid (concentration 125 nM) in presence and absence of compound 20. Inhibitor 20 blocks the uptake by 88 ± 2 % at a concentration of 250 μΜ. The values represent the average of two independent experiments, and the error bars indicate the range.

Fig. 5

 5 Fig. 5 Cytotoxicity determination of compound 20.

Fig. 6

 6 Fig.6Unbiased coarse-grained molecular dynamics simulations of inhibitors binding to ECF transporter. (A) Crystal structure of ECF FolT2 (PDB ID 5JSZ), in complex with EcfA and EcfA' shown in blue and red, EcfT in white and S-component in gray. Pocket P2, used in the structurebased virtual screening campaign is highlighted in yellow. Compound 1 densities obtained from the simulations are shown in green, which indicates the existence of two other pockets, P9 and entrance of P11 (pockets names according to Fig.S1). The green isosurfaces correspond to regions with high occupancy (100 times higher than compound 1 density in the membrane). As compound 1 binds only with low affinity to P2, no high occupancy density is observed. The figure also shows bar plots with calculated binding free energies (ΔGbind) of compounds 1, 14 and 20 for pockets P2, P9 and P11 in relation to water and membrane. In the case of compound 14 in pocket P2, the binding events are so reduced that it is only possible to estimate the upper limit (indicated by "≥" in the plots). Estimates of the partitioning free energies of the inhibitors between water and lipids (ΔG lipids/water) are also show in the figure. (B) and (C) Representative poses of compounds 1 and 20 inside pocket P9, respectively. Backmapped from the coarse-grained to the atomistic resolution. Some residues from EcfT (salmon) Sꟷcomponent (gray) are highlighted, including the hydrogen bond network (black dashed lines). Hydrogen atoms have been omitted for clarity.

Table 1 :

 1 Inhibitory potencies (IC50 values and percentages of inhibition) of compound 1 and analogues 13-21 on ECF FolT-2.

Table 2 :

 2 Inhibition of radiolabeled folic acid in Escherichia coli MC1061 by 20 with and without induction of heterologously expressed ECF_FolT2.

Table 3 :

 3 Antibacterial profile of compound 20.

Acknowledgements

A.K.H.H. and D.J.S. gratefully acknowledge funding from the Netherlands Organization for Scientific Research (ChemThem and VIDI grants). A.K.H.H. acknowledges funding from the European Research Council (ERC starting grant 757913) and the European Union's Horizon 2020 research and innovation programme (COFUND-ALERT, Grant No. 665250 ). D.J.S acknowledges support from the European Research Council (ERC PoC grant 812867). I.S received a scholarship from the Indonesia Endowment Fund for Education (LPDPLembaga Pengelolaan Dana Pendidikan, Departemen Keuangan, Republik Indonesia). Work in the Veening lab is supported by the Swiss National Science Foundation (SNSF) (project grant 31003A_172861), a JPIAMR grant (40AR40_185533) from SNSF, and ERC consolidator grant 771534-PneumoCaTChER. The authors thank Dr. Teresa Röhrig and Tabea-Catharina Schramm for the plasma and metabolic stability measurement, Jeannine Jung for the cytotoxicity evaluation, Dr. Daniel Krug and Patrick Haack for the analytical measurement, and H. Sikkema for glycine betaine uptake assays. The authors would like to thank also Viktoria Schmitt and Arnau Domenech for antibacterial testing. P.C.T.S. and S.J.M. acknowledge the National Computing Facilities Foundation (NCF) of The Netherlands Organization for Scientific Research (NWO) for providing computing time.


Emeline Richard
email: emeline.richard@cermav.cnrs.fr

Aurélien Traversier

Thomas Julien

Manuel Rosa-Calatrava

Jean-Luc Putaux

Isabelle Jeacomine

Eric Samain

Biotechnological production of sialylated solid lipid microparticles as inhibitors of influenza A virus infection

Influenza viruses bind to their target through a multivalent interaction of their hemagglutinins (HAs) with sialosides at the host cell surface. To fight the virus, one therapeutic approach consists in developing sialylated multivalent structures that can saturate the virus HAs and prevent the binding to host cells. We describe herein the biotechnological production of sialylated solid lipid microparticles (SSLMs) in three steps: (i) a microbiological step leading to the large-scale production of sialylated maltodextrins by metabolic engineering of an Escherichia coli strain, (ii) a new in vitro glycosylation process using the amylomaltase MalQ, based on the transglycosylation of the terminal sialoside ligand of the sialylated maltodextrin onto a long-chain alkyl glucoside, and (iii) the formulation of the final SSLMs presenting a multivalent sialic acid. We also describe the morphology and structure of the SSLMs and demonstrate their very promising properties as influenza virus inhibitors using hemagglutination inhibition and microneutralization assays on the human A/H1N1 pdm09 virus.

).

Introduction

Influenza A viruses (IAVs) are highly contagious pathogens that cause annual epidemics and pandemics in the human population [START_REF] Salomon | The influenza virus enigma[END_REF]. The infection cycle of IAV is initiated when the viral hemagglutinin (HA) glycoprotein interacts with complex sialoside structures present on the glycoproteins and glycolipids on the surface of the host cells [START_REF] Karakus | Breaking the convention: Sialoglycan variants, coreceptors, and alternative receptors for Influenza A virus entry[END_REF][START_REF] Vries | Influenza A virus hemagglutininneuraminidase-receptor balance: Preserving virus motility[END_REF]. This attachment process is based on multivalent bonds to gain stability and prevent the dissociation of viruses [START_REF] Zhang | Emerging and state of the art hemagglutinin-targeted influenza virus inhibitors[END_REF].

Neuraminidase inhibitors (NAIs) are antivirals recommended worldwide against influenza infection (e.g. oseltamivir and zanamivir; [START_REF] Zhang | Emerging and state of the art hemagglutinin-targeted influenza virus inhibitors[END_REF]. They target the viral protein NA, which cleaves the terminal sialic acid from glycan structures for the release of progeny viruses, thereby spreading the virus from the infected host cell to uninfected surrounding cells.

However, NAIs have serious side effects and their efficiency has recently come under debate because their use over the past decades has led to the emergence of circulating strains harboring NA mutations [START_REF] Pan | Insights into susceptibility of antiviral drugs against the E119G mutant of 2009 influenza A (H1N1) neuraminidase by molecular dynamics simulations and free energy calculations[END_REF][START_REF] Abed | A review of clinical Influenza A and B infections with reduced susceptibility to both oseltamivir and zanamivir[END_REF], conferring resistance to these drugs [START_REF] Hussain | Drug resistance in influenza A virus: the epidemiology and management[END_REF]. Importantly, the more recent FDA-approved baloxavir antiviral targeting the viral polymerase [START_REF] Ison | Influenza polymerase inhibitor resistance: Assessment of the current state of the art -A report of the isirv Antiviral group[END_REF] although not yet widely used, will probably suffer the same limitations, because baloxavir-resistant strains harboring specific mutations in the polymerase are already circulating [START_REF] Hayden | Baloxavir Marboxil for Uncomplicated Influenza in Adults and Adolescents[END_REF][START_REF] Omoto | Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil[END_REF].

One of the alternative methods of blocking the influenza virus infection is to inhibit virus adhesion by saturating the host HA binding sites with synthetic sialoside ligands. However, the binding energy between a single HA-sialoside ligand pair is too weak to allow the use of a monovalent sialoside as a successful drug candidate. Because of the cluster glycoside effect [START_REF] Lundquist | The cluster glycoside effect[END_REF], much better binding is observed between HA and multivalent structures that display many copies of specific sialoside ligands. A variety of synthetic multivalent sialoside inhibitors of influenza virus adhesion has already been developed, that includes liposomes [START_REF] Kingery-Wood | The agglutination of erythrocytes by Influenza virus is strongly inhibited by liposomes incorporating an analog of sialyl gangliosides[END_REF], dendrimers [START_REF] Reuter | Inhibition of viral adhesion and infection by sialic-acidconjugated dendritic polymers[END_REF], synthetic polymers [START_REF] Sigal | Polyacrylamides bearing pendant α-sialoside groups strongly inhibit agglutination of erythrocytes by influenza virus: The strong inhibition reflects enhanced binding through cooperative polyvalent interactions[END_REF][START_REF] Gambaryan | Polymer-bound 6 sialyl-N-acetyllactosamine protects mice infected by influenza virus[END_REF], polysaccharides [START_REF] Li | Carbohydrate-functionalized chitosan fiber for influenza virus capture[END_REF], and nanoparticles [START_REF] Papp | Inhibition of Influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles[END_REF].

However, the large-scale synthesis of these multivalent sialoside structures represents a double technological challenge, which may explain why this approach has not yet resulted in the development and FDA approval of efficient sialoside-based antiviral drugs against influenza infection.

The first challenge is the synthesis of the sialosides. The avian IAVs virus preferentially binds to sialic acid α(2,3)-linked to galactose, but human-adapted influenza preferentially binds to sialic acid α(2,6)-linked to galactose. The stereoselective synthesis of these sialosides is a notoriously difficult problem in carbohydrate chemistry, resulting in very low yields and high synthesis costs. An efficient alternative is the microbiological approach that has been developed in CERMAV for the production of 3'-sialyllactose [START_REF] Fierfort | Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides[END_REF] and 6'sialyllactose [START_REF] Drouillard | Efficient synthesis of 6'-sialyllactose, 6,6;-disialyllactose, and 6'-KDO-lactose by metabolically engineered E. coli expressing a multifunctional sialyltransferase from the Photobacterium sp. JT-ISH-224[END_REF]). This technology is based on the expression of specific recombinant sialyltransferase genes in Escherichia coli strains that have been genetically engineered to overproduce the cytidine monophosphate-N-acetylneuraminic acid (CMP-NeuAc) sugar nucleotide used in the sialylation reaction. These engineered recombinant strains are cultivated at high cell density in the presence of lactose which is internalized and sialylated in the cell to produce sialyllactose at a very high yield.

The second challenge is the multivalent display of the sialoside on a macromolecular multivalent architecture. Many chemical methods have been developed to conjugate carbohydrate structures to various multivalent structures. Among the most effective ones, clickchemistry reactions -which are highly selective and proceed under smooth conditions -have emerged as very powerful techniques for the efficient coupling of functionalized oligosaccharides to complementary activated scaffolds [START_REF] He | Carbohydrate CuAAC click chemistry for therapy and diagnosis[END_REF]. Even though this sophisticated chemistry is easy to perform with high yields at the laboratory scale, the largescale production and availability of the functionalized oligosaccharides (azide or propargyl sialoside) still hamper the practical development of new drugs based on these technologies.

In the present article, we describe an alternative global biotechnological approach for the large-scale production of sialylated solid lipid microparticles (SSLMs) that shows efficient in vitro antiviral action against the A/H1N1 pdm09 influenza virus. As described in Figure 1, this approach combines a microbial step for the efficient production of sialylated maltodextrin chains with a new glycosylation method allowing the enzymatic transfer of the terminal sialoside ligand of the sialylated maltodextrin onto a long-chain alkyl glucoside. The resulting amphiphilic structures can then be used to construct SSLMs whose surface is covered with sialoside ligands.

Results and discussion

Microbial production of sialylated maltodextrins

The strategy for the microbiological production of sialylated maltodextrin is similar to that of our previously described process of sialyllactose synthesis [START_REF] Fierfort | Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides[END_REF], except that maltotetraose (M4) was used as the acceptor here instead of lactose. The production strain MQZ-2 is a derivative of the E. coli K12 strain DH1 containing null mutations in the following genes: lacZ, nanKETA, lacA, malPQ, and malZ. The MQZ-2 strain also expresses the heterologous neuABC genes from Campylobacter jejuni, lgtE from Neisseria meningitis, and the gene encoding 6ST from Photobacterium sp. JT-ISH-224. The MQZ-2 strain was cultivated at high cell density in the presence of an enzymatic starch hydrolysate mainly composed of M4.

M4 was actively internalized by the maltose/maltodextrin transport system and accumulated in the cytoplasm due to the inactivation of the malPQ and malZ genes involved in intracellular catabolism of maltodextrins [START_REF] Boos | Maltose/Maltodextrin System of Escherichia coli: Transport, metabolism, and regulation[END_REF]. A galactose residue was then added on the terminal glucose of the M4 internalized by β(1,4)-galactosyltransferase (encoded by lgtE), creating a terminal lactosyl motif which the α(2,6)-sialyltransferase subsequently used as an acceptor to produce an oligosaccharide structure (M4-S) composed of a M4 chain at the reducing end and a sialyllactoside motif at the non-reducing end. At the end of the culture, thinlayer chromatography (TLC) analysis (butanol:formic acid:water; 4:8:1) showed that M4 had been totally converted into a longer oligosaccharide, which migrated as the expected structure

Neu5Acα2-6Galβ1-4Glcα1-4Glcα1-4Glcα1-4Glc (M4-S). The mass spectrometry analysis of the crude extract of MQZ-2 (Figure 2) confirmed the presence of this structure, showing a main peak at m/z 1168 corresponding to the quasi-molecular ions [M-H]-derived from M4-S. The mass spectrum also showed the presence of a minor peak at m/z 956 and at m/z 795 resulting from the glycosylation of small amounts of maltotriose and maltose initially present in the M4 preparation: M3-S and M2-S, respectively.

Enzymatic transfer of sialoside ligands on long-chain alkyl glucosides Amylomaltase MalQ (4-α-glucanotransferase; EC 2.4.1.25) is a transglycosidase that catalyzes the glucan transfer from one α(1,4)-glucan to another α(1,4)-glucan or to glucose. The smallest donor substrate recognized by amylomaltase is maltotriose [START_REF] Palmer | The action pattern of amylomaltase from Escherichia coli[END_REF]). Acting on maltotriose, it releases glucose from the reducing end, forms a maltosyl-enzyme complex, and transfers the maltosyl residue onto the nonreducing end of an acceptor. Using longer maltodextrin as donor, the amylomaltase MalQ is also able to release maltose or longer maltodextrin [START_REF] Weiss | Structural basis for the interconversion of maltodextrins by MalQ, the amylomaltase of Escherichia coli[END_REF]. The natural acceptors of MalQ are glucose, maltose or any larger maltodextrin. Interestingly, MalQ can also recognize a large variety of αor β-glucosides as acceptors [START_REF] Kitahata | Production of rubusoside derivatives by transgalactosylation of various β-galactosidases[END_REF]). The terminal α(1,4)-glucan chain of the M4-S sialoside should be long enough to be recognized as a substrate donor by the amylomaltase MalQ, which may thus be able to release one glucose or maltose from the reducing end of the sialoside and transfer the remaining sialoside chain onto a glucoside acceptor. Using a long-chain alkyl glucoside as the acceptor, this process could provide easy access to the synthesis of sialylated glycosides. To test this possibility, a crude M4-S extract from strain MQZ-2 was incubated with MalQ in the presence of n-dodecyl-β-D-maltoside (C12-M2) as the acceptor. To displace the equilibrium to favor the formation of the desired transglycosylated products, the reaction was conducted in the presence of a glucose oxidase-catalase mixture (Megazyme E-GOXCA) to consume glucose as soon as it was formed. The reaction was conducted with an excess of M4-S, and the TLC analysis showed that C12-M2 disappeared, being correlated with the consumption of M4-S and the formation of a series of glycosides that migrated slower than C12-M2. The reaction was stopped after the total consumption of C12-M2, and the transglycosylation products were purified using reverse phase chromatography. Mass spectrometry revealed a series of amphiphilic sialosides ranging from C12-M4-S to C12-M7-S (Figure 3A). The major peak at m/z 1448 corresponds to the C12-M5-S structure and NMR spectroscopy indicated that the average degree of polymerization (DP) of the maltodextrin chain was 5.0 monomer units (Figure S1). The glycoside structure was unambiguously confirmed by NMR spectroscopy. In the 1 H NMR spectrum, the presence of Neu5Ac was indicated by its axial and equatorial H3 signals at d = 1.80 and 2.77 ppm, respectively. Moreover, the C6 signal of the Gal unit is shifted downfield from 60.94 ppm in the free form to 63.71 ppm, which thus confirms the establishment of a glycosidic linkage at this position. The presence of the alkyl chain is proven by the characteristic signals between 0.93 and 1.68 ppm in the 1 H NMR spectrum, and between 13.51 and 31.25 ppm in the 13 C NMR spectrum (Table S1).

The formation of C12-M5-S as the major product, and C12-M4-S is consistent with the expected transglycosylation reaction of M4-S with C12-M2 as the acceptor and the liberation of one glucose or maltose respectively (Figure 4 (1,2)). The formation of C12-M6-S and C12-M7-S is more complex and can be explained by a series of transglycosylation reactions and the fact that the liberated M2 can then be used as an acceptor in another disproportionation reaction with M4-S resulting in the formation of M5-S and glucose (Figure 4 (3)). Finally, a third transglycosylation reaction would explain the formation of C12-M6-S from M5-S and C12-M2 To produce sialylated glycosides with a longer lipid chain, a transglycosylation reaction was conducted with cetearyl-glucoside (Tego Care CG90 from Evonik) as the acceptor.

Cetearyl-glucoside is a mixture of hexadecyl and octadecyl glucoside, which is commercially used as an emulsifier for cosmetic applications. Because CG90 is poorly soluble in water, it was added in a large excess in the transglycosylation reaction. Sialylated glycosides were separated from unreacted alkyl glucoside using reverse phase chromatography, and mass spectrometry revealed a series of amphiphilic sialosides ranging from C16-M3-S to C16-M7-S (Figure 3B). No peak originating from C18 glycosides was detected, indicating that only the hexadecyl glucoside fraction of the CG90 had successfully been used as the acceptor and that octadecyl glucose was probably not soluble enough in water to serve as the acceptor. Similar to the C12 series, the main product of the C16 series was centered on C16-M5-S. NMR spectroscopy unambiguously confirmed the glycoside structure (Table S2). Preparation and characterization of sialylated solid lipid microparticles (SSLMs)

SSLMs were prepared using the hot homogenization technique [START_REF] Naseri | Solid lipid nanoparticles and nanostructured lipid carriers: Structure preparation and application[END_REF] with hexadecan-1-ol (cetyl alcohol), hexadecyl hexadecanoate (cetyl palmitate), octadecan-1-ol (stearyl alcohol), and propane-1,2,3-triyl trioctadecanoate (tristearin) as the core lipid fraction.

All these lipids were solid at physiological temperature with melting temperatures of 49.3, 50, 59.8, and 55 °C, respectively. An oil-in-water microemulsion was prepared at high temperature with ultrasonication of the melted core lipids in the presence of sialylated glycosides.

Immediately after ultrasonication, the microemulsion was quenched to 4 °C, resulting in the crystallization of the core lipid and the formation of a stable aqueous suspension of SSLMs. To estimate the amount of glycosides incorporated in the SSLMs, the microparticles were dialyzed and the concentrated filtrate was analyzed by TLC. The absence of glycosides in this fraction proved their total incorporation into the SSLMs when they were used at 1 mM or below.

When cetyl alcohol was used as the core lipid, the particle size analysis showed that SSLMs prepared with C12-M5-S (CP1) and C16-M5-S (CP2) at 1 mM had a similar average size of around 350 nm with a narrow polydispersity index (PI) below 0.13. After 90 days, the average size of CP1 increased from to 360 to 433, whereas the average size of CP2 increased from 332 to 387. The increase in PI was higher for CP1 (PI = 0.37) than for CP2 (PI = 0.19), indicating that SSLMs prepared with C16-M5-S were more stable than those prepared with C12-M5-S.

As shown in Table 1, the concentration of C16-M5-S could decrease to 0.5 mM (CP3), 0.25 mM (CP4), and 0.125 mM (CP5) without affecting the stability of the SSLMs or significantly modifying their average size and PI.

Three other lipids were tested as lipid cores of SSLMs constructed with C16-M5-S as the emulsifier. SSLMs prepared with cetylpalmitate (PP1) and stearyl alcohol (SP1) were both stable, with PIs of 0.19 and 0.24, respectively. In contrast, the use of tristearin (TP1) resulted in the formation of unstable SSLMs with a high PI of 2.66. CP1 was prepared in the presence of 1 mM C12-M5-S. The SSLMs are flat disks with a roundish shape, although some of them are slightly polygonal (Figure 5A). Their diameter ranges between 10 nm and 1 µm. The smallest particles were sometimes lying on their edge, making it possible to measure their thickness (50-300 nm). The shape of the CP5 particles, prepared with 0.125 M C16-M5-S, was more clearly defined. They were mostly flat truncated parallelepipeds with lengths ranging from 300 nm to 1.5 µm, widths from 200 to 700 nm and thicknesses from 50 to 120 nm (Figure 5B). The lateral faces seemed to be perpendicular to the supporting carbon film and the angles between the longer faces were about 68 and 112°. The SP1 sample, prepared from stearyl alcohol and 1 mM C16-M5-S, seemed to contain several populations (Figure 5C). One population contained nearly perfect and truncated parallelepipeds that were smaller than those in CP5 (length: 50-700 nm; width: 150-400 nm, thickness: 40-80 nm). The angles between the faces were the same as those in CP5. As seen in

Figure S5

, when the particle is oriented edge-on, it was sometimes possible to observe a regular lamellar organization, with the lamellae lying parallel to the large surface. Another type of particle was visible, although it seemed thinner and with less defined roundish shapes. PP1 also contained different types of particles. The largest ones were polydisperse bulky spheroids with diameters ranging from 100 to 700 nm (Figure 5D). A second fraction of smaller particles was observed. They were flat, slightly polygonal, their diameters ranging from 20 to 110 nm and thicknesses from 20 to 40 nm.

The Z-average mean value calculated using a dynamic light scattering (DLS) analysis was based on a sphere with a diffusion coefficient equal to that of the scattering particle. Considering that the particles were flat in most systems, the equivalent spheres had a smaller diameter.

Nevertheless, the relative average sizes measured using DLS were in fairly good agreement with those observed from the TEM images. 1).

Structure of bulk compounds and SSLMs

The crystal structure of the samples was determined using small-and wide-angle X-ray scattering (SAXS and WAXS) analyses (Figure 6) by plotting the scattered intensity as a function of the scattering vector q = 2p/d, where d is the repeat distance between the corresponding lattice planes. The SAXS profile of bulk cetyl alcohol showed a series of equidistant peaks, the first one located at q1 = 1.69 nm -1 , and the second and third ones at q2 = 2q1 and q3 = 3q1 (Figure 6A). This profile suggests a regular lamellar organization with a repeat distance of dlam = 2p/q1 = 3.71 nm. The chains in the stearyl alcohol crystal were organized in a similar fashion, but considering the longer fatty moiety, the stronger peak in the SAXS profile was located at q1 = 1.52 nm -1 (Figure 6A), corresponding to a lamellar repeat of dlam = 4.13 nm. For cetyl palmitate, the stronger peak was found at q1 = 1.47 nm -1 , for a lamellar repeat dlam = 4.25 nm. Within the 14-19 nm -1 q-region, the WAXS profiles of bulk cetyl alcohol and stearyl alcohol were identical and exhibited three main peaks at 14.77, 15.57, and 17.62 nm -1 , corresponding to lattice distances of 0.43, 0.40, and 0.36 nm, respectively. The profile of cetyl palmitate contained two main peaks at 15.26 and 16.93 nm -1 , corresponding to lattice distances of 0.41 and 0.37 nm, respectively (Figure 6B). These results show that both bulk cetyl alcohol and stearyl alcohol are crystallized in the monoclinic g allomorph, in agreement with previous studies. They have both a bilayer organization in which the cetyl alcohol and stearyl alcohol molecules are tilted by about 58° with respect to the lamellar plane [START_REF] Abrahamsson | The crystal structure of the monoclinic form of n-hexadecanol[END_REF][START_REF] Métivaud | Hexadecane (C16H34) + 1-Hexadecanol (C 16H33OH) binary system: Crystal structures of the components and experimental phase diagram. application to thermal protection of liquids[END_REF][START_REF] Berwanger | Phase transitions and molecular dynamics of n-hexadecanol confined in silicon nanochannels[END_REF]. Cetyl palmitate appears to be crystallized in the b-form that also corresponds to a monoclinic unit cell [START_REF] Métivaud | Hexadecane (C16H34) + 1-Hexadecanol (C 16H33OH) binary system: Crystal structures of the components and experimental phase diagram. application to thermal protection of liquids[END_REF], sometimes considered as orthorhombic, because the angle of the molecules to the lamellar plane is nearly 90° [START_REF] Lukowski | Crystallographic investigation of cetylpalmitate solid lipid nanoparticles[END_REF].

Figure 6. q´I(q) SAXS (A) and WAXS (B) profiles of bulk compounds and SSLMs. The reference of each sample is given in Table 1.

The crystal structures of the SSLMs prepared from cetyl alcohol or stearyl alcohol were similar, but they differed from those of the parent bulk compounds. The WAXS profiles of CP1, CP2 (not shown), CP5, and SP1 exhibited two main peaks at q = 15.32 and 17.30 nm -1 and a weaker one at 15.82 nm -1 (Figure 6B), corresponding to lattice spacings of 0.41, 0.36, and 0.40 nm, respectively. The structure of the PP1 particles was similar to that of the parent bulk cetyl palmitate (Figure 6B). The SAXS profiles of SSLMs were also typical of bilayer organizations with lamellar repeats of dlam = 4.52 nm (q1 = 1.39 nm -1 ) for CP1, CP2, and CP5, larger than that of the parent bulk cetyl alcohol (Figure 6A). The lamellar repeat of PP1 particles (q1 = 1.47 nm -1 , dlam = 4.27 nm) was nearly identical to that of bulk cetyl palmitate. The peaks detected in the profile of SP1 can be divided into two subsets, each associated with a lamellar structure: a major one with dlam = 5.08 nm (q1 = 1.23 nm -1 ) and a minor one with dlam = 4.13 nm (q1 = 1.52 nm -1 ). The peak positions of the minor phase corresponded to those of the g-phase of bulk stearyl alcohol (Figure 6A). The crystal structures of the SSLMs prepared from cetyl alcohol and stearyl alcohol thus differed from those of the parent bulk compounds. The data from CP1, CP2, CP5, and the major form of SP1 are consistent with an orthorhombic b allomorph, in agreement with the TEM images in which the vertical surfaces of the particles seemed to be perpendicular to the supporting carbon film (Figure 5A-C and Supporting

Material Figure S5). From the X-ray scattering data only, it is not possible to determine if the two phases detected in the SP1 sample coexist in each particle or correspond to two different fractions, although the latter possibility seems to be supported by the variety of particle shapes observed in TEM images (Figure 5C).

Additional control experiments were carried out in order to shed some light on the origin of the structural differences between the bulk n-fatty alcohols and the corresponding SSLMs.

First, two samples of bulk cetyl alcohol were heated to 100 °C. One was allowed to cool down slowly to room temperature, while the other was quench-frozen in ice-cooled water. In both cases, the WAXS profiles were identical to that of bulk cetyl alcohol (Figure S6Ba-c).

However, the SAXS profile of the quench-frozen specimen contained two subsets of equidistant peaks (Figure S6Ac): a major one corresponding to that of bulk cetyl alcohol (Figure S6Aa)

and a minor one to that of CP5 SSLMs (Figure S6Ae). In another experiment, particles were prepared from cetyl alcohol following the same protocol as for SSLMs, but without adding the glycoside. Cetyl alcohol was thus heated, sonicated and quench-frozen (sample referred to as CP in Table 1). On the one hand, the WAXS profile was identical to that of CP5, prepared in the presence of glycoside (Figure S6Bd). On the other hand, the SAXS profile contained two subsets of equidistant peaks, but this time, the major one was the same as that of CP5, while the minor one corresponded to that of bulk cetyl alcohol (Figure S6Ad). Similar observations were noted for particles prepared from stearyl alcohol without adding the glycoside (sample referred to as SP in Table 1). The SAXS and WAXS profiles were the same as those of the SP1 SSLMs (Figures S7Bb,c and S7Ab,c, respectively), including the presence of a minor fraction exhibiting the peak positions of bulk stearyl alcohol (Figure S7Aa). These results show that the b-form of SSLMs can be obtained without glycosides present in the formulation. It is a minor fraction when the bulk compound is melted and quench-frozen, but the major form when the melted compound is dispersed in water into microdroplets under strong sonication before being quench-frozen. The high surface-to-volume ratio of the droplets may induce a confinement effect, promoting the nucleation and growth of the metastable b phase upon fastfreezing. Homogenization/fragmentation of the melted cetyl alcohol and stearyl alcohol by sonication and quench-freezing are thus both important aspects of the protocols, influencing not only the size of the SSLMs, but also their final crystal structure. Long-chain n-fatty alcohols with an even number of carbons have been reported to crystallize in the g-form, but those with an odd number crystallize in the b-form [START_REF] Precht | Kristallstrukturuntersuchungen an Fettalkoholen und Fettsäuren mit Elektronen-und Röntgenbeugung II. Fette, Seifen[END_REF][START_REF] Ventolá | Polymorphism of N-alkanols: 1-heptadecanol, 1octadecanol, 1-nonadecanol, and 1-eicosanol[END_REF]. The fact that CPn and SP1 SSLMs crystallized into the b-form, but their parent fatty alcohols corresponded to the g allomorph is consistent with the previous observation that a metastable b-phase can be obtained by quenching stearyl alcohol from the melt and that this phase has the same structure as the b-phase of the odd fatty alcohols [START_REF] Ventolá | Polymorphism of N-alkanols: 1-heptadecanol, 1octadecanol, 1-nonadecanol, and 1-eicosanol[END_REF].

Evaluation of SSLMs as inhibitors of influenza A virus

The potential antiviral action of different SSLMs on the human A/H1N1 pdm09 virus was evaluated with standard hemagglutination inhibition (HI) and microneutralization (MN) assays [START_REF] Truelove | A comparison of hemagglutination inhibition and neutralization assays for characterizing immunity to seasonal influenza A[END_REF]. The HI assay utilizes the natural process of HA-induced hemagglutination of the red blood cells; in contrast, the MN assay measures cytopathic effects of the infectious virus through plaque formation [START_REF] Hayden | Influenza virus[END_REF]. These processes are blocked when sufficient amounts of compounds (antibody, decoy) with affinity to the virus particle -especially hemagglutinin -are present. Serum from mice immunized with commercial TIV Vaxigrip and PBS were used as positive and negative controls, respectively for HI and MN assays.

The results presented in Table 2 show that the most promising compound is CP2, which contains 1 mM of C16-M5-S as the sialylated glycoside. Interestingly, this compound harbored a significant capacity to inhibit both virus HA-inducing hemagglutination of red blood cells (HI titer: 512) and virus infection of Madin-Darby canine kidney (MDCK) cells (MN titer, 640). In contrast, CP1, which contains 1 mM of C12-M5-S as the sialylated glycoside, harbored lower activity, suggesting the importance of the size of the lipid chain. CP3, CP4, and CP5 compounds, which contain lower amounts of C16-M5-S, were less active (HI titers: < 256 or 128, MN titers: 40 or 160) indicating that a minimal surface density of sialylated ligand is required for efficient virus binding. The SP1compound, which contains a different lipid core (stearyl alcohol instead of cetyl alcohol in CP1), showed a high capacity to inhibit virus HAinducing hemagglutination (HI titer: 1024), but lower inhibition was observed in the MN assay (MN titer: 80). Although these results are not completely consistent, the SP1 compound is also a promising candidate that could be further evaluated with CP2 and other compounds in in vitro and optionally in in vivo potency assays.

Of note, the PP1 compound showed a high cytotoxicity for red blood cells; therefore, a minimal dilution of 8192 was necessary. At this very low concentration, we did not observe any capacity to inhibit virus HA-induced hemagglutination of red blood cells and its cytotoxicity was too high to test the compound with a MN assay. This toxicity may be related to a difference in the crystalline structure of the particles between active CP2 and SP1, which contain simple aliphatic alcohol as the lipid core and the cytotoxic PP1 in which the lipid core is an ester containing two lipid chains. In this respect, compound TP1, which contains tristearin, was too unstable to be tested further.

Conclusion

Antivirals play a major role in the treatment and control of influenza epidemics and pandemics.

Neuraminidase and polymerase inhibitors have demonstrated clinical benefits, but the emergence of viral resistance may cripple the current arsenal, particularly in a context in which the vaccine options are not optimal either. In this study, we explored an alternative way of blocking the influenza virus infection based on SSLMs. Classically, solid lipid microparticles (SLMs) are used as drug carrier for prolonging the drug release, preventing its degradation and improving its bioavailability. SLMs also present the advantages of being biocompatible and productible at large industrial scale [START_REF] Gugu | Solid lipid microparticles: An approach for improving oral bioavailability of aspirin[END_REF][START_REF] Scalia | Solid lipid microparticles as an approach to drug delivery[END_REF]. Few articles describe the use of decorated solid lipid nanoparticles (SLNs) for drug delivery on a selective target [START_REF] Garanti | RGD-decorated solid lipid nanoparticles enhance tumor targeting, penetration and anticancer effect of asiatic acid[END_REF][START_REF] Sandri | Chitosan-associated SLN: In vitro and ex vivo characterization of cyclosporine A loaded ophthalmic systems[END_REF][START_REF] Hu | Sialic acid-modified solid lipid nanoparticles as vascular endothelium-targeting carriers for ischemia-reperfusioninduced acute renal injury Sialic acid-modified solid lipid nanoparticles as vascular endothelium-targeting carriers for ischemia-reperfusion-induced acute renal injury[END_REF]. To the best of our knowledge, this work describes the first synthesis of decorated SLMs for multivalent display and not for drug vectorization. Indeed, these SSLMs can act as a decoy mimicking the natural epithelium surface. Eventually, formulated in a nasal spray, SSLMs could be a promising new antiviral drug, able to inhibit virus adhesion by saturating the host HA binding sites.

These SSLMs made of sialosides with a C16 alkyl chain are produced using a unique biotechnological approach that provides for their large-scale production at a reasonable cost.

The first microbial production step of sialylated maltodextrins chains is easily scalable to very large quantities, as is the production of human milk oligosaccharides. Interestingly, the enzymatic synthesis of alkyl-sialosides can be carried out in the crude cellular extract without requiring a prior purification of either the sialylated maltodextrins or amylomaltase. Likewise, due to their highly amphiphilic nature, the resulting alkyl-sialosides can be directly purified in one step from the crude reaction mixture using reverse phase chromatography. The final step of SSLM preparation is a self-assembling process which is straightforward to implement.

Other advantages of the process include the very low cost of the raw materials (maltodextrins, alkylglucosides, and lipids) and the composition of the final SSLMs formed by the assembly of natural molecules (glucose, galactose, sialic acid, and lipid) attached by common biochemical glycosidic linkages. Given this natural composition, the SSLMs are likely nontoxic and fully biodegradable.

Our results indicate that SSLMs are promising antiviral candidates because they have shown significant inhibition action against A/H1N1 pdm09 influenza virus in HI and in MN in vitro assays. These results pave the way for further evaluation of SSLMs against other influenza A subtypes and influenza B strains to characterize their putative broad-spectrum antiviral activity against influenza viruses. In addition, other physiological in vitro models, e.g. human reconstructed airway epithelium for example, and in vivo models of infection, e.g. mouse or ferret, must be used to confirm the potential of SSLMs as inhibitors of influenza viruses. This overall approach can also be adapted to the synthesis of other glycosylated solid lipid microparticles containing different sialosides such as a(2,3)-linked sialosides, which are recognized by avian influenza viruses.

Materials and methods

Plasmids and strains

The host strain MQZ was constructed by disrupting the malPQ and malZ genes in the strain ZLKA [START_REF] Fierfort | Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides[END_REF] which was a lacZ, lacA and nanKETA null mutant derived from the E. coli strain DH1 (DSM 4235).

To knockout the malP and malQ genes, a 3.110 kb segment located between nucleotides 371 of malP and 1097 of malQ was deleted and replaced by the 5'TCTAG sequence as follows:

two DNA segments flanking the deleted sequence were amplified by PCR. The upstream 1.080 kb segment was amplified with primers 5'-GTCGACtcgactcaatggcaactgtc and 5'-TCTAGAgtggggtaataccgttggtg and the downstream 0.958 kb segment was amplified with primers 5'-TCTAGaatgccgattggcttgtatc and 5'-GGATCCtctgtccaaatccttcagca. The two amplified fragments were ligated at their terminal XbaI restriction site and cloned together into the BamHI SalI sites of the suicide vector pKO3. The deletion was then carried out according to the pKO3 gene replacement protocol in [START_REF] Link | Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: Application to open reading frame characterization[END_REF].

To knockout the malZ gene, a 0.491 kb segment located between nucleotides 594 and 1084 of malZ was deleted as follows: two DNA segments flanking the deleted sequence were amplified by PCR. The upstream 0.847 kb segment was amplified with primers 5'-GGATCCGCGTATCTCGCTGTATGTCGGTTTC and 5'-GGATCCGCGTATCTCGCTGTATGTCGGTTTC and the downstream 0.843 kb segment was amplified with primers 5'-AAGCTTGATCACCGAAGCGGCGAAAGAAAC and 5'-GTCGACGATTTAGACGCTCATTATGACGCCCTC. The two amplified fragments were ligated at their terminal HindIII restriction site and cloned together into the BamHI SalI sites of the suicide vector pKO3. The deletion was then carried out according to the pKO3 gene replacement protocol in [START_REF] Link | Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: Application to open reading frame characterization[END_REF].

The sialoside production strain MQZ-2 was constructed by transforming the host strain MQZ with the three plasmids, pBS-SS, pSU-6ST and pBBR3-lgtEm. Plasmid pBS-SS was a pBluescript derivative containing the neuABC genes from C. jejuni ATCC 43438 encoding CMP-Neu5Ac synthetase, sialic acid synthase and GlcNAc-6-phosphate 2 epimerase, respectively [START_REF] Drouillard | Efficient synthesis of 6'-sialyllactose, 6,6;-disialyllactose, and 6'-KDO-lactose by metabolically engineered E. coli expressing a multifunctional sialyltransferase from the Photobacterium sp. JT-ISH-224[END_REF]. Plasmid pSU-6ST was a pSU2718 derivative carrying the Photobacterium sp. JT-ISH-224 gene for α(2,6)-sialyltransferase [START_REF] Richard | Chemobacterial synthesis of a sialyl-Tn cyclopeptide vaccine candidate[END_REF]. Plasmid pBBR3-lgtEm containing the N. meningitis lgtE gene for b1,4 galactosyltransferase was constructed as follows: a 0.942 kb DNA fragment containing the sequence of lgtE (GenBank:

AAB48387.1) was amplified by PCR using the genomic DNA of N. meningitis 126E as a

template.

An XbaI site was added to the left primer (5'-TCTAGATACCGGGGCTATTGAAACC) and a SacI site was added to the right primer (GAGCTCGCGGGAATGACAGTGTATC). The amplified fragment was first cloned into pCR4Blunt-TOPO vector (Invitrogen) and then subcloned into the XbaI and SacI sites of plasmid pBBR1-MCS3 plasmid [START_REF] Kovach | Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes[END_REF] to form pBBR3-lgtEm.

To clone the amylomaltase malQ gene, a 2.090 kb DNA fragment containing the malQ sequence was amplified by PCR using the genomic DNA of E. coli K12 as a template and the following primers: 5'-CCATGGAAAGCAAACGTCTGGATAATG and 5'-ACTCTACTTCTTCTTCGCTGCAG. The amplified fragment was first cloned into pCR4Blunt-TOPO vector (Invitrogen) and then sub-cloned into the NcoI and EcoRI sites of expression vector pProEX-HTb (Invitrogen) to form pPro-malQ. For the production of recombinant MalQ, the strain MS was transformed with pPro-malQ plasmid and cultivated in a 3-L bioreactor containing 1.5 L of Terrific broth at 30 °C. MalQ production was induced by adding IPTG (150 mg.L -1 ) when the optical density of the culture reached 5.0. Cells were harvested by centrifugation 4 h after induction and the cell pellet was resuspended in 150 mL of potassium phosphate buffer (10 mM, pH 7.0). Cells were disrupted in a high-pressure cell homogenizer (Constant System Ltd) and the crude extract containing 25 units.mL -1 of MalQ was aliquoted and frozen at -80 °C until its use as a direct MalQ source in enzymatic synthetic reactions. MalQ activity was determined by measuring the initial rate of glucose formation from maltotriose using a glucose enzymatic assay kit from Megazyme. One MalQ unit was defined as the amount of enzyme required to produce 1 µmol.min -1 of glucose from maltotriose at 25 °C.

Preparation of M4

The strain of Pseudomonas stutzeri NRRL B-3389 was obtained from the DSMZ collection (DSM 13627) and cultured on starch for the production of maltotetraose-producing amylase as described in [START_REF] Robyt | Isolation, purification, and characterization of a maltotetraose-producing Amylase from Pseudomonas stutzeri[END_REF]. After cell removal by centrifugation, 100 mL of the culture supernatant was filtered-sterilized through a 0.22 µm filter and added to 1 L of a 100 g.L -1 autoclaved gelatinized starch solution. After 48 h of incubation at 30 °C, TLC analysis (butanol:formic acid:water; 4:8:1) confirmed the M4 formation as the major starch hydrolysis product and indicated the presence of small amount of maltose and maltotriose. The mixture was centrifuged and the M4-containing supernatant was autoclaved and kept at room temperature prior to its use as an acceptor for the synthesis of M4-ligands.

Production of sialylated maltodextrins

Strain MQZ-2 was cultured at high cell density as previously described [START_REF] Priem | A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria[END_REF]) in a 3-L reactor containing 1.5 L of mineral culture medium in presence of ampicillin (100 mg.L -1 ), tetracycline (20 mg.L -1 ) and/or chloramphenicol (50 mg.L -1 ). The culture consisted of three phases: an exponential growth phase, starting with the inoculation of the bioreactor and lasting until exhaustion of the carbon substrate (glucose 17.5 g.L -1 ), a 5 h fed-batch phase with a high glycerol feeding rate of 5 g.L -1 .h -1 , and a 25-h fed-batch phase with a lower glycerol feeding rate of 3 g.L -1 .h -1 . Maltotetraose (10 g, 6.7 g.L -1 ) and the inducer (IPTG, 50 mg.L -1 ) were added at the end of the exponential phase. At the end of the culture, bacterial cells were recovered by centrifugation ( 7000´ g, 20 min) and the pellet was resuspended in the same volume of distilled water (2 L). The cells were permeabilized by heating to 100 °C for 45 min and after cooling, the pH was lowered to 4.0 with HCl. After another centrifugation step (7000 ´ g for 20 min), the supernatant containing the sialoside was recovered and the pH was adjusted to 7.0 with NaOH. The M4-S concentration of the crude intracellular fraction obtained was determined using a glucose assay. After acid hydrolysis (4 h in 1 N HCl), the glucose rate was determined using the glucose enzymatic kit from Megazyme at 3 g.L -1 , therefore, the concentration of the sialoside was 5 g.L -1 .

Production of sialylated glycosides

Dodecyl β-D-maltoside (C12-M2) and cetearyl glucoside were obtained from Carbosynth (DD06199) and from Evonik (Tego Care CG90), respectively. The glucose oxidase/catalase mixture was purchased from Megazyme (E-GOXCA).

For the production of C12 glycosides, C12-M2 (1 g, 1.96 mmol) was solubilized in 600 mL of intracellular fraction from the MQZ-2 culture containing M4-S (3 g, 2.68 mmol). MalQ (150 units), glucose oxidase (1200 units), and catalase (30,000 units) were added and the mixture was incubated at room temperature. The reaction was followed by TLC analysis (butanol:acetic acid:water; 2:1:1) and stopped after 24 h (total consumption of dodecyl-maltose) by heating to 100 °C for 20 min to inactivate the enzymes. After 20 min of centrifugation at 9000 rpm, the supernatant was loaded on a C18-column (10 g Reverlis) at 5 mL.min -1 . The column was washed with 100 mL of deionized water and the desired product was eluted with 500 mL of a methanol gradient from 0 to 100% at 5 mL.min -1 . The elution was followed by TLC analysis (butanol:acetic acid:water; 2:1:1). After freeze-drying, 870 mg of purified sialylated glycosides was obtained (yield: 30%).

For the production of C16-glycosides, cetearyl-glucoside (CG90) (2 g) was solubilized in large excess, in 400 mL of the intracellular fraction from the MQZ-2 culture containing M4-S

(2 g, 1.79 mmol). MalQ (200 units), glucose oxidase (1200 units), and catalase (30000 units)

were added and the mixture was incubated at 30 °C for better glycoside solubilization. The reaction was followed by TLC and stopped after 48 h (total consumption of M4-S) by heating at 100 °C for 20 min to inactivate the enzymes. Purification followed the same steps as for the C12 glycoside, and 510 mg of purified sialylated glycosides were obtained (yield: 20%).

DEPTQ and proton NMR spectra were recorded with a Bruker Avance 400 spectrometer operating at a frequency of 100.618 MHz for 13 C and 400.

Preparation of sialylated solid lipid microparticles

The solid lipids (135 mg in 10 mL) were melted at 100 °C in the presence of either C12-M5-S or C16-M5-S. Unless otherwise indicated in the text, the glycoside concentration was 1 mM.

The mixture was dispersed by vortexing and then sonicated (Labsonic, Stedim) at 60 W for 5 min at 90 °C. The oil-in-water emulsion was then immediately poured in a pre-chilled 125 mL sterile Erlenmeyer flask pre-chilled in an ice bath. To estimate the amount of incorporated glycosides, the SSLMs were dialyzed against water (MWCO: 12-14 kDa, ZelluTrans, 24 h).

The concentrated filtrate and retentate were analyzed by TLC (butanol:acetic acid:water; 2:1:1).

Dynamic light scattering

SSLMs were analyzed with regard to particle size and size distribution using DLS. The mean particle size and the polydispersity index (PI) were obtained using a Zetasizer Nano ZS (Malvern Instruments, Malvern, United Kingdom). Samples were diluted 100 times with water before analysis to yield a suitable scattering intensity. The Z-average mean size and PIs were obtained by calculating the average of three measurements in 10 mm diameter disposable plastic cells.

Transmission electron microscopy

Drops of dilute aqueous SSLM suspensions were deposited on glow-discharged carbon-coated copper grids and negatively stained with 2 wt% uranyl acetate. The preparations were observed in a JEOL JEM-2100 Plus microscope operating at 200 kV and images were recorded with a Gatan Rio 16 digital camera.

WAXS and SAXS

Powders of lyophilized SSLMs were poured into 1-mm outer diameter glass capillaries that were flame-sealed and X-rayed with Ni-filtered CuKa radiation (l = 0.1542 nm) under vacuum in a Warhus chamber. Two-dimensional scattering patterns were recorded on Fujifilm imaging plates, and read off-line with a Fujifilm BAS 1800-II bioimaging analyzer. The capillary-todetector distance was set to about 4.5 and 16.5 cm to collect WAXS and SAXS data, respectively. The precise distance was calibrated using calcite and silver behenate powders, respectively. Scattering profiles were calculated by radially averaging the 2D patterns.

Cells and viruses

Influenza virus A/H1N1 pdm09 (A/Lyon/969/09 H1N1, Centre National de Référence des Virus Respiratoires, Lyon, France Sud) was produced in MDCK (ATCC CCL-34) cells using EMEM supplemented with 2 mM L-glutamine (Sigma Aldrich), penicillin (100 U.mL -1 ), streptomycin (100 µg.mL -1 ; Lonza) and 1 µg.mL -1 trypsin at 37 °C and 5% CO2. Viral titers in plaque forming units (PFU.mL -1 ) and tissue culture infectious dose 50% (50.mL -1 TCID) were determined in MDCK cells as previously described [START_REF] Pizzorno | Repurposing of drugs as novel influenza inhibitors from clinical gene expression infection signatures[END_REF].

HI assay

Potential of HI property of SSLMs was evaluated on chicken red blood cells (RBC) using a standard hemagglutination assay, as described previously [START_REF] Mandon | Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection[END_REF]. SSLMs were serially diluted in phosphate buffered saline (PBS) and 50 µL of RBC was added. After 1 h at room temperature, a dilution of each SSLM was determined as the pre-dilution to avoid cytotoxicity on RBCs. Then SSLMs were pre-diluted as determined previously, and 25 µL of each SSLM was serially diluted in PBS. Then, 25 µL of a virus dilution with 4 hemagglutination units (HAu) was added and the plate was incubated for 1 h at room temperature. To each mixture, 50 µL of 0.5% chicken RBCs were added and incubated for 1 h at room temperature.

As previously described in [START_REF] Mandon | Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection[END_REF], serum from mice immunized with commercial TIV Vaxigrip and protecting against an A(H1N1)pdm09 lethal dose infection, and PBS were used as positive and negative controls, respectively. The HI titer was designated as the inverse of the highest dilution at which detectable HI activity was still present.

Microneutralization assay

SSLMs were serially diluted in IM (infection medium as serum-free MEM Eagle with EBSS, Lonza) supplemented with 2 mM L-glutamine (Gibco), penicillin (100 U.mL -1 ) and streptomycin (100 µg.mL -1 ; Gibco) and 1µg.mL -1 trypsin acetylated from bovine pancreas (Sigma). Serial dilutions were mixed with 100 TCID50 of A/ H1N1 pdm09 virus and incubated at 37 °C for 1 h. Then, 50 µL of the SSLM-virus mixture was added to 150 µL of IM on MDCK cells and incubated at 37 °C and 5% CO2. After 96 h of incubation, the presence of cytopathic effects (CPE) was controlled under microscopy observation. As previously described in [START_REF] Mandon | Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection[END_REF], serum from mice immunized with commercial TIV Vaxigrip and protecting against an A(H1N1)pdm09 lethal dose infection, and PBS were used as positive and negative controls, respectively. The MN titer was determined as the inverse of the highest dilution at which no CPE were observed in at least 2 out of 4 wells, as previously described [START_REF] Mandon | Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection[END_REF].

Figure 1 .

 1 Figure 1. Overall process for the biotechnological preparation of SSLMs. Genes encoding the enzymes are shown in italics.

(

  Figure 4 (6)). Similarly, M5-S can react with C12-M2 to produce C12-M4-S with the formation of maltotriose (M3) (Figure 4 (4)) which can itself react with M4-S to form M6-S and glucose (Figure 4 (5)) and explain the formation of C12-M7-S by the transglycosylation of this M6-S and C12-M2 (Figure 4 (7)).

Figure 2 .

 2 Figure 2. MALDI-TOF mass spectrum in negative mode of the crude extract of MQZ-2. Peaks at m/z 1118, 956, 794 correspond to the quasi-molecular ions [M-H]-derived from sialylated maltotetraose (M4-S), sialylated maltotriose (M3-S) and sialylated maltose (M2-S), respectively.

Figure 3 .

 3 Figure 3. MALDI-TOF mass spectrum in negative mode of the glycoside fraction purified from the transglycosylation reaction mixture of M4-S in the presence of: A) dodecyl β-D-maltoside (C12-M2) or B) cetearyl-glucoside (C16-M1). Peaks at m/z 1286, 1448, 1610, 1772 correspond to the quasimolecular ions [M-H]-derived from amphiphilic sialosides C12-M4-S, C12-M5-S, C12-M6-S, C12-M7-S. Peak at m/z 1180, 1342, 1504, 1666 and 1828 correspond to the quasi-molecular ions [M-H]derived from amphiphilic sialosides C16-M3-S, C16-M4-S, C16-M5-S, C16-M6-S, C16-M7-S.

Figure 4 .

 4 Figure 4. Schematic representation of possible transglycosylation series catalyzed by MalQ, starting with M4-S as donor and C12-M2 as acceptor.

Figure 5 .

 5 Figure 5. TEM images of negatively stained preparations of particles from CP1 (a), CP5 (b), SP1 (c)and PP1 (d) formulations (Table1).

Figure S3 .

 S3 Figure S3. 1 H-NMR spectrum of C16-M5-S.

Figure S4 .

 S4 Figure S4. 13 C-DEPTQ135-NMR spectrum of C16-M5-S.

Table 1 .

 1 Composition, particle size and structure of bulk materials and sialylated solid lipid microparticles (SSLMs). D-values correspond to the lattice and lamellar spacings deduced from the position of the peaks in the WAXS and SAXS profiles, respectively. "n.d.": not determined.

			Glycoside	Particle size (DLS)	Structure
	Reference	Lipid core	Structure	C (mM)	Average size	Polydispersity	dWAXS (nm)	dSAXS (nm)
					(nm)	index
	Cetyl alcohol	-	-	-	-	-	0.36 / 0.40 / 0.43	3.72
	Stearyl alcohol	-	-	-	-	-	0.36 / 0.40 / 0.43	4.13
	Cetyl palmitate	-	-	-	-	-	0.37 / 0.41	4.25
	C12-M5-S	-	C12-M5-S	-	-	-	amorphous	4.07
	C16-M5-S	-	C16-M5-S	-			amorphous	4.21
	CP *	Cetyl alcohol	-	-	-	-	0.36 / 0.41	4.49
	SP *	Stearyl alcohol	-	-	-	-	0.36 / 0.41	4.13 / 5.00
	CP1	Cetyl alcohol	C12-M5-S	1	360	0.13	0.36 / 0.41	4.52
	CP2	Cetyl alcohol	C16-M5-S	1	332	0.11	0.36 / 0.41	4.52
	CP3	Cetyl alcohol	C16-M5-S	0.5	328	0.16	n.d.	n.d.
	CP4	Cetyl alcohol	C16-M5-S	0.25	290	0.14	n.d.	n.d.
	CP5	Cetyl alcohol	C16-M5-S	0.125	333	0.20	0.36 / 0.41	4.51
	SP1	Stearyl alcohol	C16-M5-S	1	221	0.19	0.36 / 0.41	4.13 / 5.00
	PP1	Cetyl palmitate	C16-M5-S	1	343	0.24	0.37 / 0.41	4.27
	TP1	Tristearin	C12-M5-S	1	422	2.66	n.d.	n.d.

Table 2 .

 2 Evaluation of antiviral properties of SSLMs by standard hemagglutination inhibition (HI) and microneutralization (MN) assays. The hemagglutination inhibition (HI) titer was designated as the inverse of the highest dilution at which detectable HI activity was still present. The microneutralization (MN) titer was designated as the inverse of the highest dilution at which no CPE was observed in at least 2 wells over 4. Serums from mice immunized with commercial TIV Vaxigrip and PBS were used as controls for HI and MN assays.

	Reference	Virus titer
		HI a	MN b
	PBS (Ctrl -)	<2	<20
	C12-M5-S	<8	NT c
	C16-M5-S	<256	<20
	CP1	<256	80
	CP2	512	640
	CP3	<256	160
	CP4	<256	40
	CP5	<128	40
	PP1	<8192	NT
	SP1	1024	80
	TP1	NT	NT
	Serum (Ctrl +)	128	160

a HI: hemagglutination inhibition of red blood cells b Microneutralization: infection inhibition of Madin-Darby canine kidney (MDCK) cells c Not tested

  13 MHz for 1 H. Samples were solubilized in D2O at a temperature of 25 °C. Residual signal of the solvent was used as the

	internal standard: HOD at 4.8 ppm. DEPTQ spectra were recorded using 90 pulses, 20,161 Hz
	spectral width, 65,536 data points, 1.62 s acquisition time, 2 s relaxation delay, and 20,480
	scans. Proton spectra were recorded with a 4000 Hz spectral width, 65,536 data points, 8.19 s
	acquisition times, 10 s relaxation delays, and 16 to 64 scans. The 1 H and 13 C-NMR assignments
	were based on 1 H-1 H homonuclear and 1 H-13 C heteronuclear correlation experiments
	(correlation spectroscopy, COSY; heteronuclear multiple-bond correlation, HMBC;
	heteronuclear single quantum correlation, HSQC). They were performed with a 4000 Hz
	spectral width, 2048 data points, 0.255 s acquisition time, 1 to 1.5 s relaxation delay; 40 to 512
	scans were accumulated.

Table S1 .

 S1 NMR characterization of C12-M5-S.

	C12-M5-S
	NeuAc
			C2	100.33
	H3	1.8 (t, JH,H=12.32 Hz)	C3	40.30
		2.77 (dd, J2,3=J3,4=4.98 Hz)
	H4	3.72	C4	68.38
	H5	3.92	C5	51.85
	H6	3.79	C6	72.58
	H7	3.63	C7	68.47
	H8	3.90	C8	71.24
	H9	3.91 -3.71	C9	62.74
	NHAc : CH3	2.09 (s)	CH3	22.26
			C=O	175
	Gal β
	H1	4.48 (d, J1,2=7.88 Hz)	C1	103.24
	H2	3.60	C2	70.81
	H3	3.70	C3	71.80
	H4	4.01	C4	73.34
	H5	3.86	C5	73.45
	H6	4.03 -3.66	C6	63.62
	Glu α
	H1	5.43 (m)	C1	99.69
	H2	3.6	C2	71.58
	H3	3.93	C3	71.57
	H4	3.69	C4	79.6
	H5	3.63	C5	74.62
	H6	3.89	C6	60.42
	Glu β
	H1	4.52 (d, J1,2=7.94 Hz)	C1	102.08
	H2	3.35 (m)	C2	73.03
	H3	3.82	C3	76.31
	H4	3.70	C4	77.23
	H5	3.72	C5	77.06
	H6	3.88	C6	60.70
	Aliphatic chain
	CH3	0.93 (m)	CH3	13.51
	CH2	1.41 (m)	CH2	25.05
	CH2	1.68 (m)	CH2	28.66
	CH2	1.34	CH2	28.78
	CH2	1.34	CH2	31.25
	CH2	1.34	CH2	22.08

Table S2 .

 S2 NMR characterization of glycosides.

	C16-M5-S
	NeuAc
			C2	100.33
	H3	1.77 (t, JH,H=11.99 Hz)	C3	40.13
		2.76 (m)
	H4	3.71	C4	68.43
	H5	3.89	C5	51.89
	H6	3.74	C6	72.55
	H7	3.61	C7	68.42
	H8	3.93	C8	71.80
	H9	3.93 -3.69	C9	62.69
	NHAc : CH3	2.08 (s)	CH3	22.16
			C=O	174.97
			COOH	173.5
	Gal β
	H1	4.48 (m)	C1	103.17
	H2	3.60	C2	70.86
	H3	3.69	C3	72.57
	H4	3.98	C4	68.30
	H5	3.85	C5	73.70
	H6	4.01 -3.68	C6	63.48
	Glu α
	H1	5.41 -5.45 (m,m)	C1	99.27 -99.6
	H2	3.69 -3.72	C2	71.5 -71.6
	H3	3.88 -4.00	C3	71.51
	H4	3.67 -3.71	C4	77.1 -77.4
	H5	3.60 -3.63	C5	71.22 -71.5
	H6	3.91 -3.85	C6	59.95 -60.2
	Glu α (linked to aliphatic chain)
	H1	4.91 (m)	C1	99.59
	H2	3.62	C2	71.40
	H3	4.01	C3	71.51
	H4	3.67	C4	77.10
	H5	3.90 -3.95	C5	71.70
	H6	3.76	C6	60.20
	Aliphatic chain
	CH3	0.96 (m)	CH3	14.03
	CH2	1.39 (m)	CH2	26.01
	CH2	1.35 (m)	CH2	22.70
	CH2	1.70 (m)	CH2	29.44
	CH2	1.35 (m)	CH2	29.46
	CH2	1.33 (m)	CH2	32.05

Funding and acknowledgments

This work was supported by the French National Research Agency (ANR) through IDEX "Innovation Grant 2017" (ANR-15-IDEX-0002), LabEx ARCANE and CBH-EUR-GS (ANR-17-EURE-0003), Glyco@Alps (ANR-15-IDEX-02), Institut Carnot PolyNat (CARN-025-01) and SATT Linksium "Projet de maturation Glycoflu". The NanoBio-ICMG Platform (UAR 2607, Grenoble) is acknowledged for providing facilities for mass spectrometry (A. Durand, L. Fort, R. Gueret), NMR spectroscopy and electron microscopy.

Abbreviations used in this manuscript CMP-NeuAc, cytidine monophosphate-N-acetylneuraminic acid; CPE, cytopathic effects; Gal, galactose; Glc, glucose; HA, hemagglutinin; DLS, dynamic light scattering; EBSS, Earle's balanced salt solution; EMEM, Eagle's minimum essential medium; HI, hemagglutination inhibition; IAV, influenza virus; IPTG, isopropyl-1-thio-β-D-galactopyranoside; M2, maltose; M4, maltotetraose; M4-S, sialylated maltotetraose; MN, microneutralisation; NA, neuraminidase; NAI, neuraminidase inhibitor; PI, polydispersity index; NeuAc, N-acetylneuraminic acid; NMR, nuclear magnetic resonance; PBS, phosphate buffer saline; RBC, red blood cell; TEM, transmission electron microscopy; TLC, thin-layer chromatography.

Supplementary Data