Maruthi Prasanna

Malgorzata Podsiadla-Bialoskorska

Damian Mielecki

Nicolas Ruffier

Amina Fateh

Annie Lambert

Mathieu Fanuel

Emilie Camberlein

Ewa Szolajska

Cyrille Grandjean
email: cyrille.grandjean@univ-nantes.fr

On the use of adenovirus dodecahedron as a carrier for glycoconjugate vaccines

Keywords: Glycoconjugate vaccine, carbohydrate antigen, virus-like particle, adenovirus dodecahedron, Streptococcus pneumoniae

Virus-Like Particles (VLPs) have been used as immunogenic molecules in numerous recombinant vaccines.

VLPs can also serve as vaccine platform to exogenous antigens, usually peptides incorporated within the protein sequences which compose the VLPs or conjugated to them. We herein described the conjugation of a synthetic tetrasaccharide mimicking the Streptococcus pneumoniae serotype 14 capsular polysaccharide to recombinant adenoviral type 3 dodecahedron, formed by the self-assembling of twelve penton bases and investigated the induced immune response when administered subcutaneously (s.c.). Whether formulated in the form of a dodecahedron or disassembled, the glycoconjugate induced an anti-protein response after two and three immunizations equivalent to that observed when the native dodecahedron was administered. On the other hand, the glycoconjugate induced a weak anti-IgM response which diminishes after two doses but no IgM-to-IgG switch was observed in mice against the serotype 14 capsular polysaccharide. In definitive, the whole conjugation process preserved both particulate nature and immunogenicity of the adenoviral dodecahedron.

Further studies are needed to fully exploit adenoviral dodecahedron potential in terms of plasticity towards sequence engineering and of its capacity to stimulate the immune system via the intranasal route of administration as well as to shift the response to the carbohydrate antigen by playing both with the carbohydrate to protein ratio and the length of the synthetic carbohydrate antigen.

Introduction

Polysaccharides as expressed at bacterial cell surfaces have been for long time recognized as potential targets for humoral responses [START_REF] Heidelberger | THE SOLUBLE SPECIFIC SUBSTANCE OF PNEUMOCOCCUS[END_REF]. This has led to the advent of the polysaccharide vaccines and latter, to that of glycoconjugate vaccines [START_REF] Rappuoli | Glycoconjugate vaccines: Principles and mechanisms[END_REF]. Glycoconjugate vaccines consist in the covalent linking of a carbohydrate antigen to a protein. The protein referred to as carrier protein elicits a T-cell help which eventually evokes a carbohydrate-specific memory response [START_REF] Sun | Polysaccharide structure dictates mechanism of adaptive immune response to glycoconjugate vaccines[END_REF]. Although the carrier protein is essential in mounting the immune response, it often appears as the neglected valency in comparison with the carbohydrate antigen [START_REF] Bröker | Polysaccharide conjugate vaccine protein carriers as a "neglected valency" -Potential and limitations[END_REF], [START_REF] Micoli | Protein Carriers for Glycoconjugate Vaccines: History, Selection Criteria, Characterization and New Trends[END_REF]. Only a handful number of proteins have been approved for use as carriers in humans, typically bacterial toxoids such as tetanus toxoid (150kDa), diphtheria toxoid or CRM197 (62kDa) and Haemophilus protein D (42kDa) or the outer membrane protein complex of serogroup B meningococcus (OMPC) (36kDa). Using proteins from a pathogen playing the dual role of carrier as well as protective antigen has been envisaged to simplify multicomponent vaccine formulations and to reduce cost associated with the development of a new carrier [START_REF] Pinto | The development of an experimental multiple serogroups vaccine for Neisseria meningitidis[END_REF], [START_REF] Grandjean | Acid-detoxified Inaba lipopolysaccharide (pmLPS) is a superior cholera conjugate vaccine immunogen than hydrazine-detoxified lipopolysaccharide and induces vibriocidal and protective antibodies[END_REF]. However, such strategy may result in suboptimal response due to carbohydrate and protein B cell epitope competition for restricted T cell help [START_REF] Woodruff | B Cell Competition for Restricted T Cell Help Suppresses Rare-Epitope Responses[END_REF], [START_REF] Pillot | Site-Specific Conjugation for Fully Controlled Glycoconjugate Vaccine Preparation[END_REF], an issue which mitigates its scope. To satisfy the need for novel carriers, we have therefore thought to rely on VLPs as both an alternative and an attractive strategy. VLPs are self-assembled structural viral proteins forming nanoparticles. Particulate systems are inherently more immunogenic than soluble systems as nano-and microparticles mimics the size, geometry and properties that the immune system recognizes [START_REF] Gause | Immunological Principles Guiding the Rational Design of Particles for Vaccine Delivery[END_REF]. In a way VLPs are very similar to live viruses since they can drain freely into the lymphatic vessels, be taken up efficiently by the antigen-presenting cells and induce a strong immune response but they are not infectious. Thanks to these properties several VLP-based vaccines have been licensed for the prevention of viral diseases such as those caused by hepatitis B virus or papillomavirus [START_REF] Cimica | Adjuvant formulations for virus-like particle (VLP) based vaccines[END_REF], [START_REF] Yan | The application of virus-like particles as vaccines and biological vehicles[END_REF]. In addition, VLPs tolerate genetic or chemical conjugation to give rise to multivalent nanoplatform for exogenous antigen delivery [START_REF] Yan | The application of virus-like particles as vaccines and biological vehicles[END_REF].

VLPs engineered with exogenous antigens are usually more immunogenic than the antigen itself as they present a dense array of antigens which efficiently crosslinks B cell receptors thank to the VLPs' highly repetitive and symmetrical nature [START_REF] Marcandalli | Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus[END_REF], [START_REF] Powell | A single immunization with spike-functionalized ferritin vaccines elicits neutralizing antibody responses against SARS-CoV-2 in mice[END_REF]. These unique features combined with their protein nature suitable to support a T cell help design VLP as appealing platforms for glycoconjugate vaccines. However, few examples which make use of a VLP as a carrier for carbohydrate antigen have been reported. Hence tumor-associated carbohydrate antigen Tn [START_REF] Sungsuwan | Evaluation of Virus-Like Particle-Based Tumor-Associated Carbohydrate Immunogen in a Mouse Tumor Model[END_REF], oligomannosides [START_REF] Astronomo | Defining criteria for oligomannose immunogens for HIV using icosahedral virus capsid scaffolds[END_REF] or two synthetic tetrasaccharides representing one or two repeating units of the polysaccharide capsules of Streptococcus pneumoniae serotype 14 or 3, respectively, [START_REF] Polonskaya | T cells control the generation of nanomolar-affinity anti-glycan antibodies[END_REF] have been chemically attached to the isodecahedral capsid of bacteriophage Q. All resulting glycoconjugates induced an antibody response against the carbohydrate antigen. Of note, the pneumococcal glycoconjugate vaccines involved glycopeptide-specific T cells and were protective in a passive infectious model in mice [START_REF] Polonskaya | T cells control the generation of nanomolar-affinity anti-glycan antibodies[END_REF]. Keeping S. pneumoniae as a model, we decided to investigate the human adenoviral type 3 dodecahedron (Dd) as a VLPbased glycovaccine platform for its formidable capacity to infect human cells. Indeed, Dd is composed of twelve pentameric penton bases, responsible for adenovirus cell penetration at an extent of 200,000 to 300,000 Dd/cell in culture [START_REF] Fender | Adenovirus dodecahedron, a new vector for human gene transfer[END_REF]. Such penetration efficiency is explain by the presence of an RGD motif within a loop which favors its interaction with integrin receptor expressed at the target cell surfaces. In addition, Dd recognizes heparan sulfate proteoglycans expressed on mammalian cells surfaces which form a second entry door [START_REF] Vivès | Heparan sulfate proteoglycan mediates the selective attachment and internalization of serotype 3 human adenovirus dodecahedron[END_REF], [START_REF] Fender | Adenovirus dodecahedron cell attachment and entry are mediated by heparan sulfate and integrins and vary along the cell cycle[END_REF].

It has successfully been engineered to provide chimeric influenza [START_REF] Naskalska | Towards a novel influenza vaccine: engineering of hemagglutinin on a platform of adenovirus dodecahedron[END_REF], [START_REF] Szurgot | Self-adjuvanting influenza candidate vaccine presenting epitopes for cell-mediated immunity on a proteinaceous multivalent nanoplatform[END_REF] or Chikungunya vaccines [START_REF]Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display -PubMed[END_REF] meaning that it can be used as a vaccine platform but its use as carbohydrate antigen carrier remains unprecedented.

Material and methods

Reagents

Anhydrous solvents CH2Cl2, EtOH and MeOH were purchased from Acros Organics. Anhydrous sodium sulfate, sodium borohydride, boric acid and sulfuric acid were from VWR, nickel (II) chloride (NiCl2.6H2O) was from Acros Organics, 25% sodium methoxide solution in MeOH , ethylene diamine, glutaraldehyde (25% in H2O), amberlite IR120(H + ) and anthrone were purchased from Sigma-Aldrich.

Expression and purification of Dd

Dd was expressed in FlashBacGold (Oxford Expression Technologies) baculovirus expression system following the manufacturer's protocol. Virus stocks were prepared by infection of Spodoptera frugiparda 21 monolayer.

For Dd expression, Trichoplusia ni (HighFive) cells (2 × 10 6 cells/ml) grown in suspension were infected with the recombinant baculovirus at multiplicity of infection 4 and cultured for 48 h. Expressing cells were lysed and Dd was purified from the supernatant by size exclusion chromatography followed by anion exchange chromatography, as described previously [START_REF] Szurgot | Adenovirus Dodecahedron, a VLP, Can be Purified by Size Exclusion Chromatography Instead of Time-Consuming Sucrose Density Gradient Centrifugation[END_REF]. Dd preparation was concentrated using Amicon Ultra concentrators (Merck Millipore), protein concentration was determined by NanoDrop ND-200 UV-Vis Spectrophotometer (Thermo Scientific). The homogeneity of preparations were analyzed by SDS-PAGE 12%.

The assembly status of purified Dd was analyzed by electron microscopy.

Tetrasaccharide synthesis (Pn14TS)

To a solution of known 2-azidoethyl (2,3,4,6-

tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-β- D-galactopyranosyl)-(1→6)-[(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→4)]-2-deoxy-2-phtalimido-β-D-
glucopyranoside [START_REF] Prasanna | Semisynthetic glycoconjugate based on dual role protein/PsaA as a pneumococcal vaccine[END_REF] (29.6 mg, 0.023 mmol) in a mixture of CH2Cl2/EtOH (1.69:0.36 mL) were successively added NaBH4 (1.84 mg, 2.1 equiv) and a catalytic amount of NiCl2.6H2O at room temperature. The reaction mixture was stirred for 1h and then concentrated under reduced pressure. The crude residue was diluted in CH2Cl2 and washed with H2O (twice) and brine. The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to afford a peracetylated intermediate. To a solution of this intermediate (29 mg) in MeOH were added ethylene diamine (18 µL, 12 equiv) and MeONa in 25% solution in MeOH (10 µL).

The reaction mixture was stirred for 3 hour at room temperature, neutralized to pH 7 (paper) using Amberlite

Conjugate Synthesis

In a typical experiment, to a solution of Pn14TS (100 µg) in 100 μl of borate buffer (0.1M sodium borate, 150 mM NaCl, pH 9.6) was added 30 μl of glutaraldehyde (25% solution in H2O), then the solution is stirred for 4 min at 4 °C. Next, 1 ml of Dd solution at 1 mg / ml in 20 mM HEPES buffer (containing EDTA 2 mM, NaCl 150 mM pH 7.5) is added, and the reaction mixture is further stirred for 1 hour at 4 °C. Finally, NaBH4 (450 μg) is added and the reaction mixture stirred for 15 min at 4 °C. The solution was then dialyzed against 20 mM Tris, pH 7.5, containing 2 mM EDTA, 5% glycerol using Pierce Slide-A-Lyzer cassette (molecular weight cutoff, 20K) and stored at 4 °C. Bradford assay (BioRad protein assay) was used to determine the concentration of the protein. The anthrone/sulphuric acid method [START_REF] Herbert | Chapter III Chemical Analysis of Microbial Cells[END_REF] was used for the quantification of the sugar content using Pn14TS as a standard, as described previously [START_REF] Grandjean | Investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of Vibrio cholerae O1, serotype Inaba[END_REF]. The carbohydrate antigen protein ratio was determined by mass spectrometry.

Mass spectrometry

High-resolution mass spectrum was recorded in positive mode on Waters SYNAPT G2-Si HDMS with detection with a hybrid quadrupole time of flight (Q-TOF) detector. The compounds was dissolved in MeOH at a concentration of 1 mg/ml and then infused into the electrospray ion source at a flow rate of 10 μl•min-1 at 100 °C. The mass spectrometer was operated at 3 kV while scanning the magnet at a typical range of 4000-100 Da.

The mass spectrum was collected as continuum profile data. An accurate mass measurement was achieved based on every five-second auto-calibration using leucine-enkephalin ([M + H] + = 556.2771 m/z) as an internal standard.

The samples were analyzed by matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) MS. A sinapinic acid solution at 10 mg.mL -1 in H2O-CH3CN-TFA (50:50:0.3) was used as the MALDI matrix. The samples (75 pmol in 1 μL) were deposited and then covered by the matrix (1 μL) on a polished steel MALDI target plate. MALDI measurements were then performed on a rapifleX MALDI-TOF spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam 3D laser (355 nm, 10000 Hz) and controlled using the Flex Control 4.0 software package. The mass spectrometer was operated with positive polarity in linear mode. Spectra were acquired in the range of 15000-100000 m/z. The mass spectrometer was calibrated between 20 kDa and 70 kDa with the Protein Standard II (Bruker Daltonics, Bremen, Germany).

Electron microscopy

Negative Stain Mica-carbon Flotation Technique (MFT) was applied. Samples were absorbed to the clean side of a carbon film on mica, stained and transferred to a 400-mesh copper grid. The images were taken under low dose conditions (<10 e -/Å 2 ) with defocus values between 1.2 and 2.5 μm on a Tecnai 12 LaB6 electron microscope at 120 kV accelerating voltage using CCD Camera Gatan Orius 1000.

Immunization

The Dd and Pn14TS-Dd formulations were dialyzed against 10 mM PBS using Pierce Slide-A-Lyzer cassette (molecular weight cutoff, 10K). One batch of Pn14-Dd was freeze-dried in absence of cryo-protectant and then reconstituted with H2O. Actual concentration in protein of every formulation was about 400 µg/mL. The assembly status of the different formulation was analyzed by electron microscopy.

Groups of 5 male C57/BL6J (5 week old) mice were injected s.c. with PBS, Dd (64 µg protein/dose), Pn14TS-Dd conjugate or disassembled Pn14TS-Dd conjugate (64 µg protein/dose-1.9 µg tetrasaccharide/dose) diluted with 160 µL of RIBI adjuvant (Sigma) in PBS. The mice were immunized at day 0, 14, and 28. The sera were collected on days 21 and 35. Sera were stored at -80 °C.

ELISA

The Ab responses induced upon immunizations were assessed 1 week after the second and the third injections by ELISA. Dd and capsular polysaccharide serotype 14 (CP14) (Alliance Bio Expertise), were used as coated antigens to define the anti-Dd or anti-CP14 Ab titers. Dd (0.1 µg/well) in 10 mM PBS, pH 7.3 (100 µL/well), was coated on 96 wells microtiter plates Nunc Maxisorp (ThermoFisher Scientific) plates overnight at 4 °C.

CP14 (1 µg/well) was coated for 48 h at 4 °C in 10 mM PBS, pH 7.3 (100 µL/well). Plates were then washed with PBS 0.05% Tween 20 (3 × 200 µL), saturated using PBS containing 10% skimmed milk at 37 °C for 2 h, then washed using PBS Tween 20 (PBST, 50 mM Tris, 150 mM NaCl, 0,1% Tween 20) (3 × 200 µL). Series of dilution of sera in PBS containing 10% skimmed milk (100 µL/well), were incubated at 37 °C for 2 h. Plates were then washed with PBST (3 × 200 µL) and then incubated with goat anti-mouse IgG(H+L)-horse radish peroxidase-labeled conjugate (CliniSciences) used as secondary Ab at a dilution of 1/6,000, for 1 h at 37 °C and further washed with PBST (5 × 200 µL). The enzyme substrate, o-phenylenediamine dihydrochloride (100 µL at 0.4 mg mL-1) in 0.1 M sodium citrate (pH 5.2), containing 0.02% hydrogen peroxide, was added to each well and the plate incubated for 20 min at room temperature in the dark. The reaction was terminated by adding 3 M HCl (1,000 µL per well), and the A492 was read in an Infinite M1000 spectrophotometer (TECAN). The Ab titer was defined as the dilution of immune serum that gave an OD (405 nm) at least twice that observed with preimmune serum.

Results and discussion

Conjugate synthesis

We envisioned to use synthetic 2-aminoethyl (β-D-galactopyranosyl)-( 1→4 [START_REF] Polonskaya | T cells control the generation of nanomolar-affinity anti-glycan antibodies[END_REF] and Pn14TS-based semi-synthetic glyconjugates proved to be immunogen in mice in several laboratories including ours [START_REF] Safari | Identification of the smallest structure capable of evoking opsonophagocytic antibodies against Streptococcus pneumoniae type 14[END_REF], [START_REF] Kurbatova | Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa-and Octasaccharide Conjugates[END_REF], [START_REF] Prasanna | Semisynthetic glycoconjugate based on dual role protein/PsaA as a pneumococcal vaccine[END_REF]. Both carbohydrate antigen size/length and carbohydrate antigen/carrier protein ratio impact the anti-carbohydrate antigen response. Increasing the ratio usually counteracts the lower immunogenicity associated with short carbohydrate antigens compared to longer ones [START_REF] Pozsgay | Protein conjugates of synthetic saccharides elicit higher levels of serum IgG lipopolysaccharide antibodies in mice than do those of the O-specific polysaccharide from Shigella dysenteriae type 1[END_REF], [START_REF] Carboni | Evaluation of Immune Responses to Group B Streptococcus Type III Oligosaccharides Containing a Minimal Protective Epitope[END_REF]. This is also observed for Pn14TSconjugates [START_REF] Mawas | Immunogenicity in a mouse model of a conjugate vaccine made with a synthetic single repeating unit of type 14 pneumococcal polysaccharide coupled to CRM197[END_REF], [START_REF] Pillot | Site-Specific Conjugation for Fully Controlled Glycoconjugate Vaccine Preparation[END_REF]. Considering the huge molecular weight difference between Pn14TS (<1000 Da) and Dd (3,800,000 Da), we therefore elected to use homobifunctional glutaraldehyde as crosslinking reagent to attach the tetrasaccharide to the protein since we experienced that the actual loading was superior to other conjugation techniques likewise thiol/maleimide conjugation [START_REF] Milhomme | Synthesis and immunochemical evaluation of a non-methylated disaccharide analogue of the anthrax tetrasaccharide[END_REF].

Pn14TS was obtained from a 2-azidoethyl-functionalized peracetylated precursor [START_REF] Prasanna | Semisynthetic glycoconjugate based on dual role protein/PsaA as a pneumococcal vaccine[END_REF] in two steps comprising the nickel boride reduction [START_REF] Khurana | Synthetically Useful Reactions with Nickel Boride. a Review[END_REF] of the azide into amine followed by Zemplén deprotection of the acetates (Scheme 1). Then, Pn14TS was first reacted shortly with glutaraldehyde, and the resulting mixture further condensed with Dd at a 400:1 tetrasaccharide protein ratio, followed by sodium borohydride reduction of the imine linkages into amines to stabilize the conjugates.

Conjugate characterization

After dialysis the conjugates were analyzed for their protein and sugar contents according to colorimetric Bradford and anthrone/sulfuric acid method, respectively. Protein recovery was estimated equal to 70% while total sugar molar content was 180 times that of the protein. The MALDI-TOF mass spectrum of the conjugate preparation was next compared with that of Dd to determine the extent of derivatization (Fig. 1). The analysis can be performed on the penton base, the monomeric unit which composes Dd. The molecular weight of the penton base and the conjugated penton base differs from 2,086 mass unit which corresponds to 2.5 conjugated tetrasaccharides on average. Consequently a loading of 150 Pn14TS was achieved per particle on average. The penton base contains 25 lysine residues within its sequence among with K152, K166, K305, K306, K348, K349, K358, K361 and K390 appear readily surface accessible for conjugation as determined from adenovirus dodecahedron X-ray crystallography structure [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF], [START_REF] Szolajska | The structural basis for the integrity of adenovirus Ad3 dodecahedron[END_REF]. Vicinicity of most accessible lysine side-chain amines might contribute to mitigate the number of conjugated Pn14TS due to steric hindrance caused by the attachment of a first tetrasaccharide unit (Fig. 2).

The assembly status of Dd and Pn14TS-Dd was analyzed by electron microscopy (EM) prior to immunization (Fig. 3). Both Dd (Fig. 3a) and Pn14TS-Dd (Fig. 3b) formulations contain homogeneous particles meaning that neither sample preparation nor conjugation affect the particle assembly. Alternatively, in order to evaluate whether the nanoparticle nature of the glycoconjugate influences the level or the nature of the immune response, a batch of the conjugate was freeze-dried in absence of cryo-protectant and then reformulated in PBS. As seen in Fig. 3c, dodecahedra were not detected by EM suggesting that the freeze-drying step has led to the complete disruption of the complexes as expected. Finally, antigenicity of Pn14TS-Dd was assessed by Western-Blot experiment using polyclonal Abs contained in a hyperimmune mouse sera raised against a Pn14TS-based glycoconjugate [START_REF] Prasanna | Semisynthetic glycoconjugate based on dual role protein/PsaA as a pneumococcal vaccine[END_REF] and goat anti-mouse IgG(H+L)-Alexa680-labeled conjugate as secondary Ab. Neither conjugation and purification steps interfered with accessibility of Pn14TS which retained its antigenicity in comparison with purified serotype 14 capsular polysaccharide used as a positive control and PBS or native Dd (negative controls) (data not shown).

Immunization of mice and humoral response evaluation

Groups of C57BL/6 mice were immunized thrice s.c. at two weeks interval with Dd, the Pn14TS-Dd conjugate in both native (particle), and disassembled form and PBS as a negative control. Antibody responses against purified S. pneumoniae serotype 14 polysaccharide capsule (CP) and Dd was assessed one week after the second (D21) and the third (D35), immunizations.

The titers of anti-Dd IgG were first measured using Dd as a coating antigen (Fig. 4A and4B). Dd as well as both formulations of Pn14TS conjugates were able to induce anti-Dd IgGs while such response was not observed for PBS control. High level of Abs was detected after the second immunization (Day 28) (Fig. 4A) and maintained after the boost (Day 35), in the group of mice immunized with disassembled Pn14TS-Dd and increased for the groups immunized with either Dd or Pn14TS-Dd. Observed increase was only statistically significant (P<0.05), between the group immunized with Dd and that immunized with disassembled Pn14TS-Dd. The later conjugate corresponds to a conjugate whereby the tetrasaccharide has been conjugated to free adenoviral type 3 penton base. Recombinant penton base proteins are easily expressed in Escherichia coli and have sometimes been proposed as efficient surrogates for live-attenuated adenoviral vaccine [START_REF] Wang | Immune protection efficacy of FAdV-4 surface proteins fiber-1, fiber-2, hexon and penton base[END_REF]. However, true comparison with dodecahedra has not yet been reported to our knowledge. Of note, penton bases have been shown to be 1000 times less efficient than dodecahedra to infect cells [START_REF] Bal | Adenovirus type 7 penton purification of soluble pentamers from Escherichia coli and development of an integrin-dependent gene delivery system[END_REF], a feature which might explain observed differences in the IgG response between the two groups. In the present study, IgGs titers induced in mice by the Pn14TS-Dd conjugate is comprised between those induced by Dd and those induced by disassembled Pn14TS-Dd used as immunogens. Therefore, one cannot exclude that important B epitopes are masked by the attached tetrasaccharide, despite the low extent of derivatization and/or a response against unraveled ones, not detected by native Dd used as the coated antigen for ELISA assays.

We next turned our attention to the anti-CP response. The IgG response raised against CP by the conjugates was very low and not significant compared to Dd or the negative control group (data not shown) [START_REF]for positive controls -positive sera from previous immunizations -which are added in every tested ELISA plate[END_REF]. This response was equally low when we used Pn14TS as the coating antigen suggesting that the observed results were not due to a lack of recognition of CP by anti-Pn14TS Abs. Anti-CP IgM response was also determined after the second and the third immunizations. Significant titers of anti-CP IgM Abs could be measured in the sera of mice immunized with either native or disassembled Pn14TS-Dd in comparison with both Dd and PBS for which no anti-CP response was observed (Fig. 4C). Anti-CP response was however absent in third sera (data not shown).

Since the IgM response is not sustained between the second and the third immunization, this hyporesponsiveness, in particular the absence of IgM-to-IgG switch, unlikely derived from the presence of free Pn14TS (16%), which contaminates the formulations as suggested by the comparison of total sugar versus covalently bound sugar content as determined by colorimetric method and mass spectrometry (vide supra).

Along this line, to our knowledge, acceptable amount of free polysaccharide present in polysaccharide-protein conjugate vaccines is 20% of the total carbohydrate antigen content. Moreover suppression of the T-dependent immune response could not be determined for pneumococcal conjugates, which typically contain 10% of free polysaccharide [START_REF] Peeters | Immunogenicity of a Streptococcus pneumoniae type 4 polysaccharide--protein conjugate vaccine is decreased by admixture of high doses of free saccharide[END_REF], [START_REF] Rodriguez | Immunogenicity of Streptococcus pneumoniae type 6B and 14 polysaccharidetetanus toxoid conjugates and the effect of uncoupled polysaccharide on the antigen-specific immune response[END_REF]. On the other hand, Dd MHCII epitopes are functional since a strong anti-carrier IgG response is observed (Fig. 4A). This observation suggests that anti-CP IgG is delayed (in their study Polonskaya et al. carried out two immunizations at 6 weeks interval and analyzed the sera at weeks 5 and 8 [START_REF] Polonskaya | T cells control the generation of nanomolar-affinity anti-glycan antibodies[END_REF] while we carried out analyses at weeks 3 and 5), that Dd B cell epitopes are immunodominant compared to Pn14TS antigenic determinants, that conjugation took place too far from Dd MHC II epitopes to benefit of T cell help or to recruit Tcarb [START_REF] Ma | Peptide adjacent to glycosylation sites impacts immunogenicity of glycoconjugate vaccine[END_REF], [START_REF] Pillot | Site-Specific Conjugation for Fully Controlled Glycoconjugate Vaccine Preparation[END_REF], [START_REF] Sun | Polysaccharide structure dictates mechanism of adaptive immune response to glycoconjugate vaccines[END_REF] or that Pn14TS loading or density is too weak [START_REF] Marcandalli | Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus[END_REF]. Anti-Pn14TS response strongly depends on both dose and Pn14TS/carrier protein ratio. For example, it has been reported that levels of anti-Pn14TS and anti-CRM197 (used as a carrier) IgG correlate positively and negatively, respectively, with Pn14TS loading at a 2.5 µg Pn14TS/mouse/dose [START_REF] Mawas | Immunogenicity in a mouse model of a conjugate vaccine made with a synthetic single repeating unit of type 14 pneumococcal polysaccharide coupled to CRM197[END_REF]. Significant IgG level were raised against CRM197 and, conversely moderate anti-Pn14TS IgG response, even at a 5:1 Pn14TS/CRM197 ratio whilst CRM197 is known to induce low anti-carrier Abs and is therefore considered as the best protein carrier for carbohydrate antigens to date [START_REF] Pecetta | Carrier priming with CRM 197 or diphtheria toxoid has a different impact on the immunogenicity of the respective glycoconjugates: biophysical and immunochemical interpretation[END_REF]. This situation should be stressed herein whereby Pn14TS has been administered at a 1.9 µg dose at a 2.5:1 Pn14TS/Penton base (which has same molecular weight as CRM197) molar ratio or at a 150:1 Pn14TS/Dd molar ratio which corresponds to a 1.9/64 µg ratio (0.03 weight ratio). In the past, IgG responses have been observed at Pn14TS dose of 1.1 µg [START_REF] Kurbatova | Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa-and Octasaccharide Conjugates[END_REF] and we obtained a significant response using as less as 0.5 µg of Pn14TS with pneumococcal surface protein A as the carrier protein (unpublished results) but at higher 5. was not elicited by the former conjugate while the later elicited some variability in both the primary and the secondary responses, observations which are in agreement with our own results. It is thus possible that the threshold required to induce a strong anti-Pn14TS was not reached by Pn14TS-Dd.

Conclusion

Glycoconjugate vaccines are very efficient and well tolerated in humans. Future glycoconjugate vaccine improvements consist in increasing the number of carrier protein available to avoid carrier epitope suppression phenomena as well as in mobilizing broader immune cell population subsets such as T follicular helper-like cells [START_REF] Vinuesa | Follicular Helper T Cells[END_REF]. For example, it has been shown that recruitment of T follicular helper cells strongly correlates with enhanced oposonophagocytic antibody response induced by conjugated pneumococcal vaccines in young compare with elderly population. This observation suggests that elicitation and boosting of T follicular helper cells among the later population is a valuable strategy for the generation of durable antibody responses to optimize current glycoconjugate vaccines [START_REF] Sterrett | Peripheral CD4 T follicular cells induced by a conjugated pneumococcal vaccine correlate with enhanced opsonophagocytic antibody responses in younger individuals[END_REF]. Virus-like nanoparticles, due to their large surface area to volume ratio and mutivalency which resembles the natural features of viruses can fulfil these goals: B cells specific for Q-derived VLP used as antigen are sufficient to induce T follicular helper cell development in absence of antigen presenting cells [START_REF] Hong | B Cells Are the Dominant Antigen-Presenting Cells that Activate Naive CD4+ T Cells upon Immunization with a Virus-Derived Nanoparticle Antigen[END_REF]. Therefore, VLPs are expected to favor follicular lymph nodes processing of glycoconjugates. Along this line, adenoviral type 3 Dd has been herein proposed as a novel nanoparticle carrier for glycoconjugate vaccine using a synthetic tetrasaccharide mimic of the S. pneumoniae serotype 14 capsular polysaccharide. Resulting VLP-based glycoconjugate proved robust to sequential synthetic and purification steps and easily characterized. Noticeably, conjugation of the synthetic tetrasaccharide to Dd particles was carried out without impacting their assembly. Glutaraldehyde was selected as bifunctional immuno-silent linker for the conjugation step as it was anticipated that a high carbohydrate antigen/carrier ratio would be necessary to induce an anti-carbohydrate antigen response. Despite a 150:1 ratio, a weak, early anti-carbohydrate antigen IgM and no IgG response have been determined in mice following s.c. immunizations. Contrasting with these observations the Dd carrier was strongly immunogenic and induced an anti-Dd IgG response. Further studies will thus be needed to validate adenoviral type 3 Dd as a carrier. Efforts will focus on increasing length of the linker to make the carbohydrate antigen more accessible to the immune system, or increasing the length of carbohydrate antigen itself and reaching higher extent of derivatization while exploiting its tolerance towards sequence engineering, the opportunity to encapsulate molecules e.g. Toll-like receptor agonists and exploring mucosal administration, the natural route of infection of adenoviruses, and GIS-IBiSA. Finally, We also thank the mass spectrometry core facility of CRNH for exact mass determination.

Compliance with ethical standards

Conflicts of interest The authors declare that they have no conflicts of interest.

Ethical approval

The animal study was reviewed and approved by ethical permit number from Comité d'Ethique en Expérimentation Animale (CEEA): 7897.

FIGURE LEGENDS

Scheme 1 Synthesis of the conjugates. Preparation of Pn14TS from peracetylated precursor followed by conjugation with Dd (represented using PyMol from pdb structure 2C9G [START_REF] Fuschiotti | Structure of the dodecahedral penton particle from human adenovirus type 3[END_REF])using glutaraldehyde to give Pn14TS-Dd.

  IR120(H + ), filtered and concentrated under reduced pressure to provide 2-aminoethyl (β-D-galactopyranosyl)-(1→4)-(β-D-galactopyranosyl)-(1→6)-[(β-D-galactopyranosyl)-(1→4)]-2-deoxy-2-acetamido-β-Dglucopyranoside (Pn14TS) (17 mg, quantitative); HR-ESI-MS: m/z Calcd for C28H50N2O21 [M + H]+ 751.2984, found 751.2949.

  )-(β-D-galactopyranosyl)-(1→6)-[(β-D-galactopyranosyl)-(1→4)]-2-deoxy-2-acetamido-β-D-glucopyranoside tetrasaccharide (Pn14TS, Scheme 1) as a model carbohydrate antigen to test Dd as a carrier protein in glycoconjugate vaccine formulation. Indeed, Pn14TS is readily accessible by chemical synthesis from common sugars. Moreover, this tetrasaccharide was previously selected by Polonskaya et al. to develop Q-based VLP glycoconjugates

4 : 1

 41 Pn14Ts/carrier protein ratio. In their study, Polonskaya et al. used a different VLP carrier, bacteriophage QMice were immunized with µg of conjugates bearing on average 20, 40, 80 or 200 Pn14TS per particle i.e. roughly 1.4, 2.7, 5.4 and 13.4 µg of Pn14TS, respectively. The herein described Pn14TS-Dd conjugate is similar to the 20:1 (0.01 weight ratio) and 40:1 (0.03 weight ratio) Pn14TS-Q conjugates. Noticeably, anti-Pn14TS IgG response

Figure 1

 1 Figure 1 MALDI-TOF-MS spectra of Dd and of Pn14TS-Dd. Zoom of the m/z 55 000-73 000 region (Insert panel). Penton base protein of Dd (Top) and penton base protein of Dd conjugated to Pn14TS (Bottom); 60 copies of penton base protein make up 1 Dd.

Figure 2

 2 Figure 2 Lysine residues (salmon) with amine side chain groups (green) exposed on the surface of the Dd structure (PDB ID: 4AQQ), as seen in penton base protein monomer (a), pentameric penton base (blue) in the context of whole dodecahedron (gray) (b), and dodecahedron (c). The K337 and K339 are present in the part of RGD loop (from L315-A344) invisible in Dd crystallographic structure (indicated by arrows). Structures rendered in Chimera software.[START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF]

Figure 3

 3 Figure 3 Negative-stain electron microscopy of Dd (a) Pn14TS-Dd conjugate (b) and disassembled Pn14TS-Dd conjugate (c). Stain Acetate d'uranyle (AcUr) UO₂(CH₃COO)₂•2H₂O at 2% in distilled water (pH4,2-4,5) Magnification ×23,000; bar represents 100 nm.

Figure 4 Figure 4

 44 Figure 4 Titers of anti-Dd (coated on microtiter plates) IgG Abs of mice immunized with Dd, Pn14TS-Dd, disassembled Pn14TS-Dd and PBS after the second (aA) and the third immunization (bB); Titers of anti-CP (coated on microtiter plates) IgM Abs of mice immunized with Dd, Pn14TS-Dd, disassembled Pn14TS-Dd and PBS after the second immunization (cC). The serum samples data presented as geometric mean titer of five mice per group. Statistical analysis was performed using one-way ANOVA with Tukey analysis for multiple comparisons. Statistical difference between the groups is *P < 0.05, **P<0.01, ***P<0.001.

Acknowledgments

MP acknowledges doctoral fellowship from the European Commission, Education, Audiovisual and Culture Executive Agency (EACEA), under the Erasmus Mundus program, NanoFar. NR thanks the Région Pays de la Loire, International Strategy Program "NanoFar + " for a travel grant. The authors gratefully acknowledge Anthony Piticco for its technical assistance, Dorian Caudal and Aude Lafoux from platform Therassey (Nantes) for animal experiments and Dr Jadwiga Chroboczek for helpful discussion. This work used the EM facilities at the Grenoble Instruct-ERIC Center (ISBG; UMS 3518 CNRS CEA-UGA-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) within the Grenoble Partnership for Structural Biology. The IBS Electron Microscope facility is supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


Genki Katata

Haruyasu Nagai

Leiming Zhang

Andreas Held

Dominique Serça

Otto Klemm

Development of an atmosphere-soil-vegetation model for investigation of radioactive materials transport in the terrestrial biosphere

Keywords: numerical model, atmosphere, soil, vegetation, gas deposition, particle deposition, radionuclides transport

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

I. Introduction 1

The environmental modeling of radionuclides transport to vegetation is important when assessing the impact of radionuclides on terrestrial ecosystems. From the perspective of impacts of the nuclear fuel cycle or nuclear accidents, many process-oriented models have been developed and tested in international programs such as BIOMASS (BIOsphere Modelling and ASSessment [START_REF] Klemm | Modelling the environmental transport of tritium in the vicinity of long term atmospheric and sub-surface sources[END_REF] ) and EMRAS (Environmental Modelling for RAdiation Safety 2) ). It is however well recognized that model predictions for environmental contamination are still associated with high uncertainties. This raises the need for development of sophisticated process-based model, as well as for an increase of appropriate data for verification of these models.

Dry and wet deposition of airborne pollutants onto the vegetation is key processes in such environmental models. The current dispersion (chemistry-transport) models used for radionuclides strongly rely on parameterizations for the processes. [START_REF] Sportisse | A review of parameterizations for modelling dry deposition and scavenging of radionuclides[END_REF] In particular, an accurate estimation of dry deposition of gases and particles by the model is difficult as compared with the estimate of wet deposition. The process of dry deposition is driven by a variety of physical, chemical and biological factors and processes that can cause large variations in deposition on both small and large temporal *Corresponding author, E-mail: katata.genki@jaea.go.jp and spatial scales. [START_REF] Ould-Dada | Effect of forest edges on deposition of radioactive aerosols[END_REF] Further complications arise from the fact that radionuclides, when released into the atmosphere, exist in various chemical and physical states. For instance, it is usually assumed that most of cesium released to the atmosphere in nuclear accidents is incorporated into particles, while iodine is either bound to particles or in gaseous form (elemental form and organic iodine CH 3 I). Radionuclides such as tritiated water vapor (HTO), [START_REF] Katata | Development of a land surface model including cloud water deposition on vegetation[END_REF] CO 2 and 14 CH 4 (carbon-14) that originate in the nuclear fuel cycle are also released to the atmosphere in gaseous form. A detailed process-based model that can estimate the dry deposition of both gases and particles is needed to assess the impact of radionuclides on the terrestrial biosphere.

The authors have developed a one-dimensional multi-layer numerical model that predicts the transfer of water, heat, and CO 2 in the atmosphere-soil-vegetation system named SOLVEG. [START_REF] Nagai | Atmosphere-soil-vegetation model including CO 2 exchange processes: SOLVEG2[END_REF][START_REF] Katata | Improvement of a land surface model for accurate prediction of surface energy and water balances[END_REF] Though SOLVEG is similar to other land surface models such as MATSIRO, [START_REF] Takata | Development of the minimal advanced treatments of surface interaction and runoff[END_REF] SiB2, [START_REF] Sellers | A revised land surface parameterization (SiB2) for atmospheric GCMS. Part I: Model formulation[END_REF] and CASA, [START_REF] Potter | Terrestrial ecosystem production: A process model based on global satellite and surface data[END_REF] the model has unique in representing atmosphere-soil-vegetation system as a multi-layer structure which enables to predict vertical profiles of each predicted variables. SOLVEG has been used to examine the heat and water vapor exchange of short and tall vegetation, as well as semi-arid bare soil areas. [START_REF] Nagai | Validation and sensitivity analysis of a new atmosphere-soil-vegetation model[END_REF][START_REF] Nagai | Validation and sensitivity analysis of a new atmosphere-soil-vegetation model. Part II: Impacts on in-canopy latent heat flux over a winter wheat field determined by detailed calculation of canopy radiation transmission and stomatal resistance[END_REF][START_REF] Nagai | Incorporation of CO 2 exchange processes into a multilayer atmosphere-soil-vegetation model[END_REF][START_REF] Katata | Development of a land surface model including evaporation and adsorption processes in the soil for the land-air exchange in arid regions[END_REF][START_REF] Katata | Development of a land surface model including cloud water deposition on vegetation[END_REF][START_REF] Katata | Numerical study of fog deposition on vegetation for atmosphere-land interactions in semi-arid and arid regions[END_REF] In the present study, new schemes for calculations of dry deposition of gaseous and particulate materials onto vegetation were developed and incorporated into the model. Performance tests of SOLVEG are carried out through comparisons using field datasets of fluxes of gases and particles over vegetative surfaces.

II. Numerical Model 1. Model Overview

SOLVEG is a one-dimensional multi-layer model that consists of four sub-models for the atmosphere near the surface, the soil, the vegetation, and the radiation within the vegetation canopy. Figure 1 illustrates all the processes modeled in SOLVEG. Details of the model have been described by References 5 and 6.

The atmosphere sub-model calculates variables at each atmospheric layer by numerically solving one-dimensional diffusion equations for the horizontal wind speed components, potential temperature, specific humidity, liquid water content of fog, turbulent kinetic energy and length scale, and gas and particle number concentrations. By using  for these variables, one-dimensional diffusion equations are described in the same form as

       F z K z t z   , ( 1
)
where K z is the vertical turbulence diffusivity calculated by the turbulence closure model by Yamada. [START_REF] Yamada | A numerical model study of turbulent airflow in and above a forest canopy[END_REF] The last term F  is a forcing term which includes the exchange between the vegetation and canopy air as the volume source/sink for each atmospheric variable. The top boundary conditions are provided as input data. The soil surface boundary conditions are the momentum, heat, and water vapor fluxes calculated using bulk transfer equations of wind speed, potential temperature, and specific humidity at the lowest air layer and the soil surface temperature and specific humidity, which are determined with the soil sub-model.

The soil sub-model calculates the soil temperature, volumetric soil water content, and specific humidity of air in the soil pore space using a heat conduction equation, mass balance equation for liquid water, and the diffusion equation for water vapor, respectively. These three equations are closely related to each other through the phase change in water that takes place as the term of evaporation rate in the soil. The uptake of soil water by roots is derived from the rate of transpiration to the atmosphere calculated in the vegetation sub-model. At the soil surface boundary the soil sub-model can then be coupled with the atmosphere sub-model by solving the soil surface heat and water budget equations. In the soil CO 2 model that is included in the soil sub-model, the diffusive and convective transport of CO 2 in both aqueous and gas phases are considered in the mass conservation of CO 2 for unsaturated soil.

The vegetation sub-model calculates the leaf temperature and water on the surface of leaves (leaf surface water) for each canopy layer, and the vertical liquid water flux in the entire canopy. The leaf temperature is calculated by solving the heat budget equation at the leaf surface using the leaf temperature and other variables from the atmosphere sub-model in addition to the radiation scheme. For the ver-tical liquid water flux in the canopy, the input precipitation intensity becomes the boundary value at the top of the canopy, with the value calculated for the bottom of the canopy then being available for use in the surface water budget calculation in the soil sub-model. In the vegetation sub-model, the photosynthesis is also incorporated for calculating the CO 2 assimilation rate based on formulations for the relationship between the stomatal resistance and net CO 2 assimilation rate.

The radiation sub-model separately calculates the direct and diffuse downward and upward fluxes of solar and long-wave radiation in the canopy and provides the radiation energy input for heat budget calculations at the soil surface and canopy layers. The leaves of each canopy layer are separated into two fractions: sunlit and shaded. The stomatal resistance and energy budget are then independently calculated for each fraction. The above-mentioned sub-models are closely related to each other, with iterative calculations being required to solve some of the equations numerically. A small value (such as several seconds) of a time step adapted for each simulation scenario is therefore used to reduce the iterations in the sub-models.

Formulations for Dry Deposition Calculation

(1) Water Vapor and CO 2 Exchange over Vegetative Surfaces Dry deposition of atmospheric gases can be formulated based on photosynthesis at plant's leaves similar to water vapor and CO 2 exchanges. The modeled source/sink terms F  in Eq. ( 1) for water vapor and CO 2 are described in the present section. A full description of formulations for photosynthesis can be found in Reference 5.

The source/sink term for CO 2 , F co2 , is represented as the aggregation of the net CO 2 assimilation rate A n of leaves at each canopy layer as

 / 2 n a co aA M F   , (2)
where M a is the molecular weight of air, a the leaf area density, and  the air density.

In SOLVEG, the CO 2 assimilation rate and stomatal resistance are calculated based on photosynthetic process. The CO 2 assimilation rate A n , the stomatal resistance r s , and the CO 2 partial pressure of the leaf interior, c i , are calculated by the following formulations as

  d s e c n R w w w A   , , min , (3)
a s i s a lb s a n p r c c p r c c A 65 . 1 37 . 1     , (4)
where w c , w e , and w s are the potential rate of assimilation under unlimited conditions by the photosynthetic enzyme system's (Rubisco's) efficiency, the absorbed PAR (Photosynthetically Active Radiation), and the capacity of the leaves to export the products of photosynthesis, respectively, R d the leaf respiration rate, c a , c s , e s , e sat (T c ), and p a the CO 2 partial pressure of canopy air and that at leaf surface, vapor pressure at leaf surface, saturated vapor pressure for leaf temperature T c , and atmospheric pressure, respectively, e i the vapor pressures of the leaf interior, and e a the canopy air.

The coefficient m and minimum stomatal conductance g smin are empirically determined from observations. w c and w s are calculated using the parameter of V m25 in Table 1 depending on vegetation type. [START_REF] Nagai | Atmosphere-soil-vegetation model including CO 2 exchange processes: SOLVEG2[END_REF] r lb is the leaf boundary layer resistance for CO 2 exchange between leaf and canopy air, which is determined from the wind speed of each canopy layer calculated by the atmosphere sub-model. Eqs. ( 3) and ( 5) are based on the formulations by Farquhar et al. [START_REF] Farquhar | On the gaseous exchange of ammonia between leaves and the environment: determination of the ammonia compensation point[END_REF] and Collatz et al., [START_REF] Collatz | Physiological and environmental regulation of stomatal conductance, photosynthesis and transpiration: a model that includes a laminar boundary layer[END_REF][START_REF] Collatz | Coupled photosynthesis-stomatal conductance model for leaves of C4 plants[END_REF] respectively. Eqs. ( 4) and ( 6) are derived from resistance models for CO 2 and water vapor pressure, respectively. Equations ( 3) to ( 6) are solved iteratively to determine r s and A n for each integration time step. At first, A n is calculated by Eq. (3) using a initial value of c i , and this value and c a are used to determine c s by Eq. ( 4). Then, r s and e s are determined by solving Eqs. ( 5) and ( 6) simultaneously. With the derived values of A n and r s , c i is again calculated by Eq. ( 4). Using the calculated c i , A n is recalculated by Eq. ( 3). This procedure is repeated until the value of c i converges below a specified threshold.

When the plant's leaves are not wet, the source/sink term for water vapor, F q , is represented as the aggregation of the evaporation rate of leaf surface water, E d , and the transpiration rate, E s , at each canopy layer as

   s d q E E a F   , ( 7
)    a c sat d s q T q R r E     , ( 8
)    a c sat s d q T q R r E     , (9)
where r a , r d , and r s are the resistances of leaf boundary layer, of evaporation of leaf surface water, and of stomata, respectively, R' = r a r s + r a r d + r s r d , q sat (T c ) the saturated specific humidity for T c , and q a the specific humidity of air, respectively.

(2) Gas Deposition onto Vegetation Atmospheric gases are vertically transported by turbulent eddy diffusion and mainly absorbed by plants due to stomata uptake at plants' leaves. Based on the analogy of evaporation rate represented as Eq. ( 9), the gas deposition rate, F g , at each canopy layer is modeled using the stomatal resitance (r s ) calculated by Eq. ( 5) and quasi-laminar resistance over the leaves (r a ) as

     gs ga s a w g g c c r r D D a F    1 / , ( 10
)
where D g and D w are the diffusivity of trace gas and water vapor, and c ga and c gs the gas concentration around the leaf surface and within the stomata, respectively. For simplicity, the gas concentration in sub-stomatal cabity is assumed to be zero, i.e., c gs = 0. Some of trace gases studied here, such as O 3 and SO 2 , have also other transfer pathways to the vegetative surfaces: for example, the cuticle and wetted surfaces of leaves (especially for water-soluble compounds), the branches, the trunks, and the soil exposed to atmosphere. All of these pathways, which are often summarized as the so-called 'non-stomatal deposition', have become recognized as a sink of gases at the terrestrial surface, [START_REF] Fowler | Measurements of ozone deposition to vegetation quantifying the flux, the stomatal and non-stomatal components[END_REF][START_REF] Coyle | Measurements of ozone deposition to a potato canopy[END_REF] but its impact on deposition onto vegetation is still unclear.

The non-stomatal deposition is so far mainly formulated based on empirical and site-specific parameterizations in current models and it is not straightforward to include it to SOLVEG mechanistically. Thus, we incorporated only the stomatal deposition into SOLVEG and evaluate its impact by comparing its calculation result with that of the commonly used model proposed by Zhang et al. [START_REF] Zhang | A revised parameterization for gaseous dry deposition in air-quality models[END_REF] which includes both stomatal and non-stomatal deposition.

(3) Particle Deposition onto Vegetation

The atmosphere and vegetation sub-models include the module for the calculation of fog deposition onto the leaves based on the processes of inertial impaction and gravitational settling of particles at each vegetation layer. [START_REF] Katata | Development of a land surface model including cloud water deposition on vegetation[END_REF] In the present study, a novel scheme of the collection rates due to Brownian diffusion and interception, which can affect fine particles typically smaller than 0.1 μm in diameter, is developed. Both processes are formulated based on semi-empirical equations obtained by wind-tunnel studies for packed fibres of a filter.

The particle deposition rate, F p , in each canopy layer is represented as

 p p aE F  , ( 11
)
p f p N F E u   , ( 12
)
where E p is the capture of particles by leaves,  the total capture efficiency of the plants' leaves for particles, F f the shielding coefficient for particles in horizontal direction, |u| the horizontal wind speed at each canopy layer, and N p the number concentration of particles.

Assuming that each collection mechanism acts in series, the total capture efficiency  can be expressed as

      x x   1 1 , ( 13
)
where x is the collection mechanism of inertial impaction, gravitational settling, Brownian diffusion, and interception.

Since the formulations of  for inertial impaction  imp and gravitational settling  grv have been described in Reference 14 only collection efficiencies due to Brownian diffusion and interception are described below. Fine particles smaller than some 0.1 m diffuse toward the foliar surface (Brownian diffusion), when moving along the streamline around the leaves under forced convection.

The collection efficiency due to Brownian diffusion,  df , is described by the following formula based on Kirsch and Fuchs [START_REF] Kirsch | Studies on fibrous aerosol filters. III . Diffusional deposition of aerosols in fibrous filters[END_REF]

as 3 / 2 7 . 2   Pe df  , (14)
  p B trg d D d Pe u  , ( 15
)
where Pe is the Peclet number, which is the product of Schmidt (Sc) and Reynolds number (Re), d trg the characteristic length of vegetation element (e.g., needle diameter for coniferous forest), and D B (d p ) the Brownian diffusion coefficient as a function of particle diameter d p . The value of 2.7 in Eq. ( 14) is an empirically determined number based on experimental measurements of filter efficiencies. Equation ( 14) includes both effects of convection (Re) and particle diffusion (Sc) and is physically more reasonable than the prior parameterizations using only Sc in the commonly used model such as Slinn [START_REF] Slinn | Predictions for particle deposition to vegetative canopies[END_REF] and Zhang et al. [START_REF] Zhang | A size-segregated particle dry deposition scheme for an atmospheric aerosol module[END_REF] . When small particles perfectly follow a streamline that happens to come within one particle radius of the foliar surface, the particle hits the leaf and is captured because of its finite size (interception). The collection efficiency due to interception,  in , is expressed according to the formulation by Fuchs [START_REF] Fuchs | The Mechanics of Aerosols[END_REF])

    1 1 1      R R in  , ( 16
) trg p d d R  , ( 17
)
where R is the dimensionless parameter of interception. Equation ( 16) is associated with theoretical solution of the particle motion equation in a potential flow assumed when any other deposition mechanisms are negligible.

Numerical Scheme

The prognostic equations for all predicted variables in the model are solved at each time step using a finite difference scheme. The basic equations of SOLVEG are based on one-dimensional advection diffusion equations, which can be written in a generalized form as

F A z K z z w t                  . ( 18
)
The model numerically solves the equation in two steps: one for the advection term with an explicit method, and the other for the rest terms with a semi-implicit method except for the forcing term,

  t t adv t       * , ( 19
)   . 1 * * * t t t t t t t F A z K z A z K z t                                                           (20)
A usual center-in-space differentiation is used for the diffusion terms with fully implicit scheme. The advection terms in basic equations are spatially discretized with the low-numerical diffusion scheme HIFI, [START_REF] Yamazawa | Development of a numerical solution method for advection terms and its application to the atmospheric dynamic model, PHYSIC[END_REF] which is a hybrid scheme of FI interpolation, the first-order upwind, and second-order upwind schemes. The FI interpolation method is designed so as to maximize the accuracy without loosing the stability through the use of linear analysis. [START_REF] Yamazawa | Development of a numerical solution method for advection terms and its application to the atmospheric dynamic model, PHYSIC[END_REF]) Detailed descriptions of the numerical solution method can be found in References 28 and 29.

Simulation Conditions

In order to run SOLVEG, hourly data of meteorology (horizontal wind speed, air temperature and humidity, short and long wave radiation, and precipitation) and atmospheric concentrations of gases and/or particles are required to determine its upper boundary conditions. Due to the lack of the above datasets with radionuclides, comprehensive datasets including fluxes of gases (water vapor, CO 2 , O 3 , and SO 2 ) over a maize field in France (Meyrargues) and a deciduous

V m25 [mol m -2 s -1 ] m g smin [mol m -2 s -1 ]

III. Results and Discussions

Gas Deposition onto the Vegetation

Calculations by the model are compared with measurements 30) of latent heat and CO 2 fluxes by the eddy covariance method and deposition velocity of O 3 gas measured by the gradient method over the maize field in Fig. 2. It is shown that SOLVEG predicted measured daytime upward and downward fluxes of water vapor and CO 2 successfully (Figs. 2(a) and(b)). This indicates that SOLVEG can be used to simulate water vapor and CO 2 fluxes predominantly originated from photosynthesis over a maize (C4 plant) field. A good performance of SOLVEG in predicting these fluxes has also been confirmed over the C3 grassland. 12) SOLVEG also simulated temporal changes in the measured O 3 flux (Fig. 2(c)). From June 23 to 29, SOLVEG underestimated the measurements of daytime CO 2 and O 3 fluxes to some extent, which may be caused by the uncertainty to set the parameters of soil (e.g., unsaturated hydraulic conductivity) and vegetation (e.g., V m25 and m) used in the simulation. O 3 uptake during the daytime by the vegetation coincided with downward CO 2 flux (Figs. 2(b) and(c)). This indicates that the modeled scheme for gas absorption by stomata based on photosynthesis is reasonable and can be used to predict the gas deposition onto the crop field.

Figure 3 illustrates the temporal changes of calculations and measurements [START_REF] Finkelstein | Ozone and sulfur dioxide dry deposition to forests: Observations and model evaluation[END_REF] in CO 2 flux and deposition velocity of O 3 and SO 2 over the deciduous broad-leaved forest. While SOLVEG in general predicted the measured diurnal changes in gas fluxes, it underestimated measurements during the daytime for both O 3 and SO 2 several times. Similar to the maize field case, this discrepancy between calculations and measurements is mainly caused by the uncertainties in the model settings of soil and vegetation parameters. In addition to this, the non-stomatal deposition can affect the smaller predicted values using SOLVEG than observations in deposition velocity. In Fig. 3b andc, the calculations using the model of Zhang et al. [START_REF] Zhang | A revised parameterization for gaseous dry deposition in air-quality models[END_REF] which consists of a simpler structure than SOLVEG but includes non-stomatal deposition, are plotted. It predicts a higher deposition velocity of both O 3 and SO 2 (Fig. 3, blue dots), which are often closer to the observations. This indicates that other pathways of gas deposition in addition to stomatal absorption may cause an under prediction of deposition velocities in SOLVEG.

Similar tendencies can be seen in averaged diurnal cycles of flux and deposition velocity of gases as shown in Fig. 4. The results again show that SOLVEG reproduced diurnal variations of deposition velocity at both maize and forest sites. However, SOLVEG underestimated the deposition velocity of both O 3 and SO 2 gases during daytime at the Kane forest site. The discrepancy is explained by that the model does not include non-stomatal deposition and is not optimized with vegetation parameters, as is the case of the maize field. Further, the following processes may cause the differences between observations and model predictions. For O 3 , there may be an increase of flux over the forest caused by the chemical reaction as NO + O 3 → NO 2 + O 2 under the forest canopy when there is the emission of NO gas from the forest floor. This reaction can reduce the O 3 concentration within the forest, resulting in an increase of O 3 flux. [START_REF] Cape | Reactive uptake of ozone at simulated leaf surfaces: Implications for 'non-stomatal' ozone flux[END_REF] For SO 2 , the presence of water at foliar surfaces due to rain may increase its deposition velocity since SO 2 can be readily dissolved with the water surface. It is known that deposition velocity of the studied gases onto the wet canopy is large compared with the dry canopy at the study area. [START_REF] Zhang | On ozone dry deposition-with emphasis on non-stomatal uptake and wet canopies[END_REF] The effect of non-stomatal deposition should be investigated by further model simulations.

Particle Deposition onto the Vegetation

Calculations of turbulent fluxes of fog droplets (d p > 5 m) and fine particles (11 nm < d p ≤ 0.9 m and 3 nm < d p ≤ 11 nm) over the coniferous forest by SOLVEG are compared with measurements [START_REF] Held | Direct measurement of turbulent particle exchange with a twin CPC eddy covariance system[END_REF][START_REF] Held | Field measurements and size-resolved model simulations of turbulent particle transport to a forest canopy[END_REF][START_REF] Klemm | Fog deposition fluxes of water and ions to a mountainous site in Central Europe[END_REF] by the eddy covariance method in Fig. 5. The fluxes calculated by the size-resolved particle deposition model of Zhang et al. [START_REF] Zhang | A size-segregated particle dry deposition scheme for an atmospheric aerosol module[END_REF] were larger than the measured ones. In contrast, computations by SOLVEG agreed with the observations of these fluxes. This indicates that the formulation of collection efficiency used in this study is reasonable in prediction of particle deposition onto the vegetation.

Feeding SOLVEG with a hypothetical uniform number concentration over the entire particle size range, the deposition velocities of particles over the coniferous forest in Germany was calculated with SOLVEG for each size bin. Figure 6 shows the comparisons of size-resolved deposition velocities calculated with SOLVEG and observations from the literature obtained at various coniferous forests. The model predictions by SOLVEG agreed better with measurements of deposition velocity for the large (> 1 μm) and small particles (< 0.1 μm) for some datasets than other particle deposition models.

For particles with a diameter ranging from 0.01-1 μm, the observed values of deposition velocity have a large uncertainty associated with the difference of environmental factors from various datasets. This makes it difficult to evaluate the model in the size range. Nevertheless, the calculations of deposition velocity seem to be significantly smaller than the measured values. This particle size range includes the crucial radionuclides such as cesium and particulate iodine, [START_REF] Brandt | Modelling transport and deposition of caesium and iodine from the Chernobyl accident using the DREAM model[END_REF][START_REF] Chamberlain | Radioactive Aerosols[END_REF] as well as the major atmospheric organic (e.g., black carbon) and inorganic compounds (e.g., sulfates and nitrates). As suggested by Reference 40. the processes of particle collection, such as hygroscopic particle growth, electrostatic forces, and thermo-and diffusio-phoresis, may affect the deposition velocity at this range. On the other hand, it has been hypothesized that the large measured deposition velocities in this size range may have been caused by heterogeneous processes between gases and particles as NH 4 NO 3 (aerosol) ↔ HNO 3 (gas) + NH 3 (gas), 41) which are not included in any of the particle deposition models. These effects should be incorporated into the model and evaluated in the future.

IV. Conclusion

New schemes for calculations of the dry deposition of gaseous and particulate materials onto vegetation were developed and incorporated into a one-dimensional multi-layer atmosphere-soil-vegetation model (SOLVEG). Dry deposition of atmospheric gases was formulated based on the schemes that calculate photosynthesis of plants and root uptake from the soil. For particle deposition, the scheme of collection efficiencies of plants' leaves due to Brownian diffusion and interception was developed to calculate the dry deposition of large and small particles. SOLVEG modified to include the schemes of gases and particle deposition was tested using comprehensive datasets including flux of gases and particles obtained over a maize field and forests at three observational sites. A good agreement in fluxes of water vapor, CO 2 , and O 3 was shown between the model calcula- tions and observations over the vegetation. SOLVEG also predicted the measured fluxes from nano-to micrometer-sized particles over the forest. The results suggest the effects of wet canopy, hygroscopic particle growth, electrostatic forces, thermo-and diffusio-phoresis, and heterogeneous chemical reactions on dry deposition should be investigated.

Fig. 6 Deposition velocity of aerosols and fog droplets onto coniferous forest canopies against the particle diameter, as given by measurements obtained by eddy covariance, throughfall, and gradient methods, and by SOLVEG and three other particle deposition models.

Fig. 1

 1 Fig. 1 Schematic illustration of modeled processes in the atmosphere-soil-vegetation system. Underlined words in boldface print are newly modeled processes in the present study.

  forest in USA (Kane Experimental Forest), and those of fog droplets and fine particles over a coniferous forest in Germany (Waldstein) are used to validate the newly incorporated schemes for calculations of dry deposition. The soil temperature and the volumetric soil water content were set to be constant values as initial and lower boundary conditions. The boundaries of the atmosphere are set as heights from 0 to 3 m with an increment of 0.1 m for the maize field and from 0 to 34 m with an increment of 1 m for the forests. The boundary depths of soil are 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0 m for all vegetations.The parameters used in SOLVEG are summarized in Table 1. Other parameters for each landuse category are given by Reference 5.

Fig. 3

 3 Fig. 3 Time series of calculated and observed (a) CO 2 flux and deposition velocity of (b) O 3 and (c) SO 2 at decidous broad-leaved forest in USA on September 1997. Calculations by the model of Zhang et al. 22) are also plotted in the graph.

Fig. 2

 2 Fig. 2 Time series of calculated and observed (a) latent heat (i.e., water vapor) and (b) CO 2 , and (c) O 3 fluxes over a maize field in France in June 2001.

Fig. 5

 5 Fig. 5 Time series of calculated and observed (a) fog water flux and (b) aerosol number fluxes with particle diameters from 11 nm to 0.9 μm and (c) from 3 to 11 nm over a coniferous canopy in Germany in July 2001. Calculations by the model of Zhang et al. 25) are also plotted in the graph.

Fig. 4

 4 Fig. 4 Averaged diurnal cycles for the entire simulation periods of predicted and observed latent heat and CO 2 fluxes and deposition velocity of O 3 and SO 2 at the maize and deciduous broad-leaved forest. Calculations by the model of Zhang et al.[START_REF] Zhang | A revised parameterization for gaseous dry deposition in air-quality models[END_REF] are also plotted in the graph.

Table 1

 1 Vegetation parameters used in SOLVEG calculations. The parameter of V m25 depends on vegetation type and is used to calculate w c and w s in Eq. (3).

	Items	For gases	For par-
				ticles
	Site names	Meyrargues 30)	Kane 31)	Waldstei
				n 32-34)
	Simulation periods	Jun. 9~28,	Apr. 29~	Jul. 9~
		2001	Oct. 23,	20, 2001
			1997
	Soil texture	Silty-clay-loam	Clay-loam	Loam
	Plant type	C4 grass (ma-	Deciduous	Needle
		ize)	broad-leav
			ed
	Canopy height [m]	0.4~1.0	22.0	19.0
	Leaf Area Index	0.8~2.7	1.7~6.88	6.4
	(LAI) [m 2 m -2 ]
	For CO 2 exchange

Acknowledgment

The authors wish to express their gratitude to two reviewers of the paper. The present study was partially supported by the Ministry of Education, Culture, Sports, Science and Technology, the Grant-in-Aid for Scientific Research on Innovative Areas (21120512) and for Young Scientists B (21710035).


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PROJECT AIMS

Water is a fundamental resource for human and---economical welfare and modern society depends on complex, interconnected infrastructures to provide safe water to consumers. Water Distribution Systems (WDSs) are exposed to deliberate or accidental contaminations or may undergo a partially or full system collapse.

The project ResiWater aims to develop tools to prepare water utilities for crisis management and enhance their resilience with regards to three specific case studies: collapse of WDS, water quality deterioration and cascade effects between water, energy and IT infrastructures.

ResiWater

  Olivier Piller, Denis Gilbert. ResiWater: An Innovative Secure Sensor Networks and Model-based Assessment Tools for Increased Resilience of Water Infrastructures. WISG2016, Workshop interdisciplinaire sur la Sécurité Globale, Feb 2016, Troyes, France. hal-03313340 the german water center

  Photos: TZW, Reik Nitsche

Steps Tools Natural disaster, terrorist attack, cascade effect Collapse of system Deterioration of water quality Cascade effect: IT/Energy

  Innovative Secure Sensor Networks and Model-based Assessment Tools for Increased Resilience of Water Infrastructures Contacts : Olivier PILLER 33 (0)5 57 89 08 27 -Denis GILBERT 33 (0)5 57 89 08 24 olivier.piller@irstea.fr -denis.gilbert@irstea.fr IRSTEA 50 avenue de Verdun 33612 CESTAS Cedex

	Low Level
	Medium Level
	High Level
			Level of preparedness	Level of preparedness	Level of preparedness
			Actual State Project goal Actual State	Project goal	Actual State Project goal
		Sensors	n.u.n.		WP2
		Hydraulic models	n.u.n.			WP4
	1. Detection +	Alarm generation modules	WP3		WP3	WP3
	Identification	Self learning monitoring	WP3		WP3	WP3
		Uncertainty analysis	WP4		WP4	WP4
		Vulnerability analysis	WP5		WP5	WP5
	2. Planning	Hydraulic model Training simulator	WP4 WP4		WP4	WP4
	3. Action	Decision making-aid tool	WP5		WP5	WP5
	n.u.n: no update necessary