L Simon

M Vincent

S Le Saux

V Lapinte

N Marcotte

Marie Morille

C Dorandeu

J M Devoisselle

S Bégu

Polyoxazolines based mixed micelles as PEG free formulations for an effective quercetin antioxidant topical delivery

Introduction

With a surface of 2 m², the skin is the most extended organ of the human body acting as barrier to mechanical and chemical aggressions [START_REF] Marks | 2 -Structure and Function of the Skin[END_REF]. It plays a key role in protecting the body from daily toxic aggressions due to environmental pollutants by generating reactive oxygen species (ROS) [START_REF] Valacchi | Cutaneous responses to environmental stressors[END_REF]. This overproduction of ROS unbalances the antioxidant defense system of the human body [START_REF] Poljšak | Antioxidant Effects of Quercetin and Catechin Encapsulated into PLGA Nanoparticles[END_REF] leading to oxidative stress that can create skin damage like premature skin aging [START_REF] Rinnerthaler | Oxidative stress in aging human skin[END_REF] but also skin cancer risks [START_REF] Baudouin | Environmental pollutants and skin cancer[END_REF]. Among the environmental sources responsible for skin damage [START_REF] Krutmann | The skin aging exposome[END_REF], UV radiations were unambiguously shown to cause skin cancers [START_REF] Valacchi | Cutaneous responses to environmental stressors[END_REF].

UVAs were more especially identified to induce DNA mutations in fine generating skin carcinogenesis. Other environmental pollutants, such as polycyclic aromatic hydrocarbons, cigarette smoke constituents and ozone, are also considered today as accountable for skin pathologies and cutaneous cancers [START_REF] Baudouin | Environmental pollutants and skin cancer[END_REF].

To avoid the deleterious excess of ROS, antioxidant can be administrated through the skin. This topical delivery strategy uses natural or synthetic exogeneous molecules, with low or high molecular weight, as antioxidants [START_REF] Andreia | Topical Delivery of Antioxidants[END_REF]. Among all the natural compounds to be delivered, the highly lipophilic polyphenolic molecules belonging to the flavonoids family are often preferred for their powerful antioxidant properties and multiple pathways of activities [START_REF] Nagula | Recent advances in topical delivery of flavonoids: A review[END_REF]. Quercetin is one of the flavonols of interest for topical delivery since it gathers the properties of antioxidant and anti-inflammatory activities, wound healing and skin aging retardation. However, its low water solubility, instability to light and poor skin permeability (Hatahet et al., 2016a) require its formulation for an efficient activity. Therefore, various formulations such as smart crystals (Hatahet et al., 2016b), lipid nanocapsules [START_REF] Hatahet | Dermal quercetin lipid nanocapsules: Influence of the formulation on antioxidant activity and cellular protection against hydrogen peroxide[END_REF], liposomes [START_REF] Hatahet | Liposomes, lipid nanocapsules and smartCrystals®: A comparative study for an effective quercetin delivery to the skin[END_REF], solid lipid nanosystems [START_REF] Bose | Formulation optimization and topical delivery of quercetin from solid lipid based nanosystems[END_REF], vesicles [START_REF] Chessa | Effect of Penetration Enhancer Containing Vesicles on the Percutaneous Delivery of Quercetin through New Born Pig Skin[END_REF], nanostructured lipid carriers [START_REF] Chen-Yu | Development of a Quercetin-loaded nanostructured lipid carrier formulation for topical delivery[END_REF], nanoparticles [START_REF] Tan | Preparation and evaluation of quercetin-loaded lecithinchitosan nanoparticles for topical delivery[END_REF], microemulsions [START_REF] Martena | Permeation and skin retention of quercetin from microemulsions containing Transcutol® P AU -Censi, Roberta[END_REF] and lipid-based nanosystems [START_REF] Caddeo | Topical Anti-Inflammatory Potential of Quercetin in Lipid-Based Nanosystems: In Vivo and In Vitro Evaluation[END_REF] have been developed to enhanced the topical delivery of quercetin. It should be noted that most of these formulations use poly(ethylene glycol) (PEG) as stabilizer or thickener because of its very 2 interesting properties including biocompatibility, low toxicity, high solubility in aqueous media and stealth behavior. This explains the PEG overuse for cosmetic and pharmaceutical applications [START_REF] Shen | 1 -PEGylated "stealth" nanoparticles and liposomes[END_REF] and its success as the "gold standard" for drug delivery. Recently, clinical awareness on PEG overuse for oral and parenteral delivery pointed out safety issues associated with an increase of PEG antibodies [START_REF] Lubich | The Mystery of Antibodies Against Polyethylene Glycol (PEG) -What do we Know?[END_REF] and the possible accumulation of the polymer in body tissues [START_REF] Rudmann | High Molecular Weight Polyethylene Glycol Cellular Distribution and PEG-associated Cytoplasmic Vacuolation Is Molecular Weight Dependent and Does Not Require Conjugation to Proteins[END_REF], [START_REF] Viegas | 6 -Poly(oxazolines)[END_REF].

Therefore, researchers as well as companies are trying to find a polymer alternative to PEG. In this context, the poly(2-R-2-oxazoline)s (POx)s are investigated as one of the most suitable candidates. This polyamide family can be regarded as analogous of poly(amino acid)s with pseudo-peptidic structure. The biomedical properties of POx have been largely described in recent publications [START_REF] Hoogenboom | Poly(2-oxazoline)s: A Polymer Class with Numerous Potential Applications[END_REF], [START_REF] Adams | Poly(2-oxazolines) in biological and biomedical application contexts[END_REF] or Lorson [START_REF] Lorson | Poly(2-oxazoline)s based biomaterials: A comprehensive and critical update[END_REF]. Its biocompatibility [START_REF] Goddard | Soluble polymeric carriers for drug delivery. Part 2. Preparation and in vivo behaviour of N-acylethylenimine copolymers[END_REF], excellent cytocompatibility [START_REF] Bauer | Poly(2ethyl-2-oxazoline) as Alternative for the Stealth Polymer Poly(ethylene glycol): Comparison of in vitro Cytotoxicity and Hemocompatibility[END_REF] and hemocompatiblity [START_REF] Leiske | Mission ImPOxable -or the unknown utilization of non-toxic poly(2oxazoline)s as cryoprotectants and surfactants at the same time[END_REF] as well as the absence of accumulation in tissues and the rapid clearance from the bloodstream [START_REF] Gaertner | Synthesis, biodistribution and excretion of radiolabeled poly(2-alkyl-2-oxazoline)s[END_REF] have been emphasized. Moreover, the stealth behavior with suppression of the interactions with proteins and the immune system was proved [START_REF] Zalipsky | Evaluation of Blood Clearance Rates and Biodistribution of Poly(2-oxazoline)-Grafted Liposomes §[END_REF] allowing an immunocamouflage [START_REF] Kyluik-Price | Comparative efficacy of blood cell immunocamouflage by membrane grafting of methoxypoly(ethylene glycol) and polyethyloxazoline[END_REF]. The most significant advance in biomedical POx concerns the recent results of Serina Therapeutics Inc. against Parkinson's disease [START_REF] Moreadith | Clinical development of a poly(2oxazoline) (POZ) polymer therapeutic for the treatment of Parkinson's disease -Proof of concept of POZ as a versatile polymer platform for drug development in multiple therapeutic indications[END_REF].

They validated phase I clinical trials on POx based rotigotine conjugates whereas multi-dose trials are currently in progress to phase II.

Moreover, the versatility of POx can be noted with an easy additional functionalization when compared to PEG regarding the R pendent groups along the backbone [START_REF] Verbraeken | The chemistry of poly(2oxazoline)s[END_REF], (Guillerm et al., 2012b), (Guillerm et al., 2012a), [START_REF] Korchia | Photodimerization as an alternative to photocrosslinking of nanoparticles: proof of concept with amphiphilic linear polyoxazoline bearing coumarin unit[END_REF]. Consequently, hydrophilic POx can be designed with methyl or ethyl R groups by contrast to hydrophobic POx with longer alkyl chains. Concerning poly(2-methyl-2-oxazoline), its more hydrophilic character than PEG and poly(2-ethyl-2-oxazoline) [START_REF] Huber | Effect of end group polarity upon the lower critical solution temperature of poly(2-isopropyl-2-oxazoline)[END_REF] offers an opportunity to elaborate amphiphilic (co)polymers contrasting with the hydrophobic block. In this way, to generate amphiphilic polymers, POx were associated with various hydrophobic blocks such as saturated or unsaturated lipids including fatty alcohols [START_REF] El Asmar | Tuning the Thermo-Sensitivity of Micellar Systems through a Blending Approach[END_REF], fatty acids, triacyl glycerols [START_REF] Stemmelen | Synthesis and self-assembly of amphiphilic polymers based on polyoxazoline and vegetable oil derivatives[END_REF], [START_REF] Giardi | Nonionic polyoxazoline surfactants based on renewable source: Synthesis, surface and bulk properties[END_REF] or phospholipids [START_REF] Purrucker | Supported Membranes with Well-Defined Polymer Tethers-Incorporation of Cell Receptors[END_REF].

With the aim of protecting active pharmaceutical ingredients (API)s, several types of nanocarriers based on POx physically incorporating an API have been described. These formulations include solid dispersions, nanoparticles and micelles in which various APIs (paclitaxel, curcumin, doxorubicine) have been solubilized [START_REF] Lorson | Poly(2-oxazoline)s based biomaterials: A comprehensive and critical update[END_REF]. They were shown to be particularly interesting due to the limited number of steps required to synthesize the POx and the absence of necessity to tune the polymer towards the API as it is required for polymer-drug conjugates.

Regarding the association of POx and antioxidant API, little is reported in the literature. One can mention the conjugation of superoxide dismutase (SOD1) enzyme with POx to increase the release through the blood brain barrier and destroy superoxides [START_REF] Yang | Time Resolved UV-Vis Absorption Spectra of Quercetin Reacting with Various Concentrations of Sodium Hydroxide[END_REF]. A radical fullerene-POx trap making use of the hydrophilicity of POx to disperse the anti-radical C60 molecules was also reported [START_REF] Nukolova | Folate-decorated nanogels for targeted therapy of ovarian cancer[END_REF]. A tribloc copolymer (PMeOx-b-PDMS-b-PMeOx) in polymersomes was shown to entrap a photosensitizer (rose bengal dye conjugated to bovine serum albumin) generating increased level of ROS by photodynamic therapy [START_REF] Baumann | Cellular Trojan Horse Based Polymer Nanoreactors with Light-Sensitive Activity[END_REF], [START_REF] Lorson | Poly(2-oxazoline)s based biomaterials: A comprehensive and critical update[END_REF].

To our knowledge, no POx topical formulation of flavonoids has been developed yet. In this context, a mixed nanosystem of POx and phospholipids for topical delivery was considered in this paper. Indeed, this hybrid system combines the features of liposomes and polymersomes by mixing the two components, i.e. the lipids and the amphiphilic copolymers, in a single vesicle. Either called hybrid polymer/lipid vesicle (Le [START_REF] Meins | Hybrid polymer/lipid vesicles: state of the art and future perspectives[END_REF] or mixed micelles [START_REF] Almgren | Mixed micelles and other structures in the solubilization of bilayer lipid membranes by surfactants[END_REF], these systems have been studied over the past years and attracted attention for their versatile structure. It is notable that this mixture of lipid and polymer can lead to different nanostructures (D' Souza and Shegokar, 2016), such as core-shell of polymers and lipids [START_REF] Mandal | Core-shell-type lipid-polymer hybrid nanoparticles as a drug delivery platform[END_REF].

Pippa et al have already worked on such hybrid nanosystems with POx and phospholipids for drug delivery systems and proved their efficacy as nanocontainers for the incorporation of therapeutic molecules [START_REF] Pippa | DPPC:MPOx chimeric advanced Drug Delivery nano Systems (chi-aDDnSs): Physicochemical and structural characterization, stability and drug release studies[END_REF].

Herein, the investigated mixed-micelles were created based on phosphatidylcholine stabilized by an amphiphilic poly(2-methyl-2-oxazoline) bearing a long alkyl chain (Scheme 1). The stability and cell viability of these formulations were studied before and after being loaded with quercetin antioxidant. Then, the antioxidant activity of the loaded mixed micelles was measured. This work also aims to evaluate the potency of POx as an alternative to PEG in formulations.

Scheme 1. Schematic representation of the mixed-micelles loaded with quercetin.

Materials and methods

Materials 4

2-Methyl-2-oxazoline (MOx, Sigma Aldrich, 99.0%) was dried over CaH2, distilled at reduced pressure and stored under nitrogen atmosphere. Iodo-hexadecane (95.0 %), potassium hydroxide (KOH), quercetin (95% HPLC), phosphatidylcholine (L-α-Phosphatidylcholine from egg yolk ≈ 60%), Brij®58, DPPH (2.2-diphenyl-1picrylhydrazyl), phenazine methosulfate (PMS), tert-Butyl hydroperoxide solution (TBHP) 70 % in water, acetonitrile, diethyl ether, methanol, acetone, phosphoric acid and 2,7-dichlorofluorescin diacetate (DCFDA) were purchased from Sigma Aldrich (Sigma Aldrich, Germany). Tween 80 (polysorbate 80) was purchased from BASF (BASF, Germany) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, (MTS) from Promega (Promega, USA). MilliQ water was obtained from Milli-Q Gradient A10 (Merck Millipore, Germany) apparatus.

Chloroform (for HPLC stabilized with ethanol) was bought from Carlo-Erba (Carlo Erba Reagent, Spain). All the reagents were used without further purification. Spectra/Por 6 dialysis membranes pre-wetted RC tubing with 0.5-1 kDa MWCO were purchased from Spectrum Labs (Spectrum Labs, USA).

Methods

Amphiphilic polyoxazolines synthesis (POx)

POx synthesis was performed by cationic ring-opening polymerization of MOx. The polymerization was achieved under a nitrogen atmosphere after dissolving iodo-hexadecane (3.564 g, 10.1 mmol) and MOx (15 eq, 151.7 mmol) in dry acetonitrile (0.5 M). The solution was vigorously stirred at 80 °C for 8 hours and then quenched by addition of potassium hydroxide dissolved in methanol (5 eq, 50.5 mmol, 5 M). The solution was stirred at 40 °C for additional 8 hours. The resulting polymer was isolated by precipitation by dropwise addition in cold diethyl ether and then filtration. The POx was then dialyzed with the dialysis membrane to eliminate residual diethyl ether and salts.

Chemical characterization of POx

Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded in CDCl3 a Bruker 400 MHz spectrometer.

The POx exhibited the following shifts (δ) in reference to residual CHCl3 (δ=7.26 ppm), where (s) stands for singlet, (d) doublet, (t) triplet, (m) multiplet: δ = 3.7-3.2 (m, (4n +2)H, CH2 POx and CH2Nalkyl chain), 2.4-2.1 (m, 3n, CH3 POx), 1.3 (m, 20H, CH2 aliphatic), 0.9 (t, 3H, CH3 aliphatic).

Critical aggregation concentration determination

The critical aggregation concentration (CAC) of the POx was determined by surface tension measurements using the Wilhelmy plate method on K100 Krüss Processor Tensiometer from Krüss GmbH. A concentration range of polymer from 0.001 to 400 mg/L was prepared in MilliQ water. Prior to measurements, the solutions were kept under gentle stirring for 24 hours. Data were collected at 25 °C, a detection speed of 6 mm/min, detection sensitivity of 0.01 g and immersion depth of 2 mm using the Kruss software. A blank measurement was first performed using MilliQ water to ensure goodness of measure (γ= 69-73 mN).

Preparation of quercetin loaded mixed micelles (Q-MM)

The mixed micelles (MM) were obtained by the thin film method developed by Bangham [START_REF] Bangham | Osmotic Properties and Water Permeability of Phospholipid Liquid Crystals[END_REF]. First phosphatidylcholine (PC), Polyoxazolines (POx) and quercetin (Q) were dissolved in a mixture of chloroform:acetone in 1:1 ratio, followed by the formation of thin film by evaporation of the solvent under vacuum. The film of mixed PC, POx and Q was hydrated by addition of filtered phosphate buffered saline (PBS, 150 mM, pH 7.4). Nanosized MM were obtained following a high pressure homogenizer process of 5 cycles at 10 000 PSI. Blank mixed micelles (B-MM) were prepared by the same method, except that quercetin was removed from the process.

Various molar ratios of Q: POx: PC were investigated from 3:1:10 to 7:1:50, after which the molar ratio 5:1:40 was selected. It was optimized to ensure the smallest and most stable MM, with the lowest POx amount and the highest drug loading of quercetin.

Photon correlation spectroscopy and electrophoretic mobility measurement

Hydrodynamic diameter and polydispersity index (PDI) were measured using a Zetasizer NanoZS apparatus (Malvern Instruments, UK) equipped with a He-Ne laser (wavelength: 632.8 nm) at a temperature of 20 °C and a scattering angle of 173 ° for detection. Zeta potential was measured with 1000 µL of mixed micelles in disposable capillary cell (Malvern Instruments, UK). All the results are the average of three independent measurements.

Stability of Q-MM

Blank and loaded MM samples were prepared and stored up to a month for stability study either at 4, 25 and 37 °C for B-MM and at 4 °C only for Q-MM. The stability was evaluated from particle size and PDI measurements (see section 2.5) at day 0, 7, 14, 21, 28 for each temperature. The measurements were performed without filtration before measurements.

The stability of loaded mixed micelles was also evaluated using the concept of in vitro release by studying the concentration of quercetin diffusing through a nitrocellulose dialysis membrane from Q-MM to a receptor medium. The dialysis membrane was a 12 -14 MWFC Spectra/Pore dialysis membrane (Spectrum laboratories, INC USA) and the receptor medium was composed of 40 mL PBS at pH 7.4 with 2 mass percent of Tween 80.

The Q-MM were pre-filtered through 0.2 µm syringe filter (Whatman) to remove aggregated quercetin and then 3 mL were incorporated into the membrane. The stability was determined at 25 °C after 1, 2, 3, 4, 5, 6 and 7 hours. Quercetin concentration was determined by HPLC (section 2.9).

Transmission electron microscopy

Transmission electron microscopy was performed on TEM Jeol 1400 PLUS (Jeol. Ltbd, Tokyo, Japan) associated with a camera Jeol 2K/2K. The samples previously diluted 10 times were deposited on grids type Cu formvar carbon and negatively stained with an aqueous uranyl acetate solution.

Differential Scanning Calorimetry (DSC)

The crude quercetin powder and lyophilized Q-MM were analyzed by DSC using a Perkin Elmer DSC400 apparatus (Perkin Elmer, USA).

The thermograms were recorded from 0 to 360 °C with a heating rate of 5 °C.min -1 .

HPLC analysis

High Pressure Liquid Chromatography (HPLC) analysis of quercetin was performed on LC6-2012HT apparatus (Shimadzu, Kyoto, Japan) using a Prontosil C18 column (120-5-C18 H5.0 µm, 250x4.0 mm) as stationary phase.

The mobile phase was composed by a 40/60 volume ratio of acetonitrile to phosphoric acid solution at 0.2% mass at pH=1.9. The injection volume was 10µL and the flow rate was kept constant at 1 mL.min -1 during the 10 minutes of analysis. The detection was achieved using a UV-vis detector, (Shimadzu, Kyoto, Japan) at 368 nm [START_REF] Yang | Time Resolved UV-Vis Absorption Spectra of Quercetin Reacting with Various Concentrations of Sodium Hydroxide[END_REF]. The calibration curve was performed with solutions of quercetin in methanol from 1 µg/mL to 100 µg/mL with a good linearity (r² = 0.9999).

The stability of quercetin in aqueous medium receptor (section 2.6) was established using a calibration curve of quercetin made in PBS buffer at pH 7.4 and Tween 80 (2% by mass) from 0.1 to 4 µg/mL (r² = 0.9996). The mobile phase, flow rate and detection wavelength remained unchanged.

Encapsulation efficacy (EE) and drug loading capacity (DL)

Quercetin loading capacity (DL) and encapsulation efficacy (EE) were determined by HPLC analysis as previously described (section 2.9) using 50 µL of the Q-MM sample solubilized in 950 µL methanol.

The amount of quercetin loaded (Eq.1) and the encapsulation efficacy (Eq.2) were calculated using the following equations:

% = [ ] [ ] x100 (Eq.1) # $ %& ' ()) $ $* % = [ + , ] [ - , ,]
x100 (Eq.2)

In vitro radical scavenging ability by 2.2-diphenyl-1-picrylhydrazyl (DPPH°)

The antioxidant activity of Q-MM loaded with quercetin was investigated by looking at the reactivity of quercetin with the free radical 2.2-diphenyl-1-picrylhydrazyl (DPPH°) [START_REF] Blois | Antioxidant Determinations by the Use of a Stable Free Radical[END_REF]. The antioxidant activity was determined by UV-visible spectroscopy from the decrease of the DPPH° radical absorbance at 517 nm using the following DPPH° scavenging equation (Eq.3):

../° &$ 1( % = 23 + 423 + + 23 +
x 100 (Eq.3)

In which absorption control corresponds to the absorbance of 100 µM DPPH° solution. For the antioxidant activity test, the concentration of quercetin in Q-MM was set well below that of DPPH° at 2.5 ; 5 ; 7.5 ; 10 ; 12.5 ; 15 µM.

Cell culture

Mice fibroblasts cell line (NIH3T3) was purchased from American Type Cell Culture organization (USA). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10 % fetal bovine serum (Life Technologies), 1 % L-glutamine and 1 % penicillin/streptomycin equivalent to final concentration of 2 nM for glutamine, 100 U.mL -1 for penicillin and 100 µg.mL -1 for streptomycin and incubated at 37 °C in humidified 5 % CO2.

Cell viability

NIH3T3 cells were seeded at 20,000 cells/cm² in 96 well plate and incubated for 24 hours at 37 °C, 5 % CO2 to allow cell adhesion. POx and Brij®58 at 40 g/L were solubilized in DMEM supplemented medium and filtered through 0.2 µm (no mass was lost through filtration).

Cells were treated or not (control) with POx or with the PEG reference (Brij ® 58) solutions at a concentration range from 0.01 to 20 g/L. After 24 hours of exposure, a CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega, USA), was used to evaluate the cell viability following the manufacturer's instructions. The absorbance was then recorded at 490 nm using Multiskan TM GO Microplate Spectrophotometer (Thermo Scientific TM , Waltham, Massachusetts, USA) and the background corresponding to wells free of cells (mean of 3) was subtracted from each triplicate mean. Values of treated cells were normalized to non-treated cells (representing the 100 % cell viability).

Influence of a 24 hours treatment with B-MM, crude quercetin, or Q-MM solutions was also performed following the same protocol with quercetin concentrations of 1, 5, 10, 25, 50 µg/mL. The absorbance background of the formulations without cells was subtracted to the cellular tests.

Antioxidant effect of quercetin on NIH3T3 cells

Statistical analysis

The statistical analysis of the data resulting from the cell viability and the antioxidant effect on cells was conducted with OriginPro software 8.1 (OriginLab, USA). A one-sample t-test with equal variance was performed to compare cell viability or antioxidant effect with formulations to viability of untreated cells (100 %). A two-sample t-test with unequal variances was carried out to compare cell viability of formulations two by two. The P value reflects the significance with * =P <0.05, ** =P <0.01 and *** =P < 0.001.

Results

Synthesis of amphiphilic POx

Amphiphilic POx considered as nonionic surfactants were synthesized from iodide initiators and potassium hydroxide as terminating agent (Scheme 2). The hydrophilic lipophilic balance (HLB) of the polymers depends on the length of the POx block predefined by the initial monomer/initiator molar ratio. In our case, 1iodohexadecane was employed to elaborate C16POx15 with narrow dispersity. The nonionic surfactant was well characterized by 1 H NMR spectroscopy with typical signals related to terminal CH3 unit of hydrophobic block at 0.85 ppm and the CH2 of POx block at 3.2-3.7 ppm (see Figure 1S). The integration of these signals corresponds

to the average degree of polymerization representing the length of the POx block, in our case 15, resulting in a molecular mass of the amphiphilic POx of 1520 g/mol and the measured critical aggregation concentration (CAC) of ~ 100 mg/L (66µM).

The chemistry of POx platform looks like the one of PEG using a ring-opening process of polymerization from cyclic monomer, 2-R-2-oxazoline for POx and ethylene oxide for PEG. However, the polymerization process differs for oxazolines with a cationic ring-opening polymerization (CROP) whereas for ethylene oxide an anionic one occurs. It should be noted that the high toxicity of ethylene oxide, gaseous at room temperature, required specific equipments and cautious handling which is not the case for oxazolines. To evaluate some of the POx surfactant properties, PEG surfactant named Brij ® 58 (reference) was selected based on its similar alkyl chain length alkyl chain (molar mass of 1125 g/mol) inducing an almost similar CAC (90 mg/L vs. 100mg/L) as it is the driving force of the self-assembly.

Scheme 2. Synthetic route of C16POx15 and structure of the Brij ® 58 used as a reference

The surfactant properties of amphiphilic polymers in water were estimated by tensiometry measurements using the Wilhelmy plate method [START_REF] Padday | The measurement of the surface tension of pure liquids and solutions[END_REF]. It allows determining the critical aggregation concentration (CAC) value of surfactive molecules from the inflexion point occurring between a rapid decrease of surface tension at low concentrations and an almost constant value of the surface tension at higher concentrations. The CAC of C16POx15 was thus estimated at 100 mg/L (65.9 µmol/L) (data not shown) at 25 °C.

POx toxicity on NIH3T3 cells in comparison with PEG reference

The influence of POx (C16POx15) versus PEG reference (Brij ® 58) for a 24 hour treatment on NIH3T3 cell viability was conducted by treating cells with increasing concentrations from 0.01 to 20 g/L (Figure 1). Results

were normalized to non treated cells (representing 100 %).

For concentrations ranging from 0.01 to 0.5 g/L no significant cell viability decrease was observed for both POx (C16POx15) and PEG reference (C16PEG20) By contrast, following a 0.1 g/L Brij ® 58 (C16PEG20) treatment, cell viability fell to 32 ± 3 % versus 112 ± 8 % for POx (C16POx15). A decrease of cell viability was observed for POx (C16POx15) to 60 ± 5 % only at 0.5 g/L. To summarize, following treatment from 0.1 to 0.2 g/L, POx has significantly (P < 0.001) less impact on cell viability than PEG reference. Interestingly, IC50 (cellular viability) of the PEG amphiphilic reference was estimated at 0.090 g/L versus 0.75 g/L for amphiphilic POx.

Particle size and surface charge of mixed micelles

MM composed of PC and POx, with or without quercetin, were produced by thin film method and a high pressure homogenizer process (HPH) (Barnadas and Sabes, 2003). Table 1 summarized the average particle size and zeta potential of B-MM and Q-MM. The loading of the MM with quercetin did not influence their diameter with a particle diameter of 19±2 nm (Q-MM) instead of 18±2 nm without quercetin (B-MM). It can be noted that, no light scattering from crude quercetin, which tends to aggregate from 2.5 µg/mL in PBS, was observed in Q-MM solutions. The zeta potential for both blank and loaded MM was measured at 0 mV, indicating that the formulations had no charge at the surface.

Table 1. Mixed micelles structure characteristics.

Morphology of mixed micelles

The morphology of MM was investigated by transmission electron microscopy. Figure 2 shows typical images for Q-MM. Both B-MM and Q-MM exhibit a spherical shape and a similar size of about 20-30 nm. This is in agreement with the diameter of particles obtained from DLS analysis (Table 1).

Figure 2 : TEM images for Q-MM

DSC analysis of Q-MM

The crystallinity of quercetin as powder and once formulated in Q-MM was evaluated by differential scanning calorimetry, as illustrated in Figure 3. Before the Q-MM measurement, the formulation was concentrated twice and lyophilized. The crude quercetin exhibited two peaks, one at 155 °C and 323 °C, which are consistent with the literature. The first one is due to water evaporation [START_REF] Poljšak | Antioxidant Effects of Quercetin and Catechin Encapsulated into PLGA Nanoparticles[END_REF] and the second one results from melting of the quercetin [START_REF] Sahoo | Preparation and Characterization of Quercetin Nanocrystals[END_REF]. The glass transition of POx only appears at 37 °C (Figure 2S). It is interesting to note that this glass transition was not observed on the Q-MM thermogram. More importantly, no peak for the quercetin crystallization could be observed. This indicates that nano-objects possess amorphous character, probably resulting from an homogeneous distribution of quercetin in the mixed micelle core. The difference of quercetin structure revealed by DSC was also observed on NIH3T3 mice fibroblasts cells 24 hours after addition of the Q-MM and the crude quercetin to the cells. The quercetin concentration was equivalent in both experiments (50 µg/mL). Optical microscopy clearly revealed the presence of needles on NIH3T3 with quercetin only (Figure 4). Those were missing with Q-MM, i.e. once quercetin has been loaded in mixed micelles (Figure 4). This argues in favor of an homogenous distribution of quercetin in MM.

Formulation stability studies of B-MM and Q-MM

14

The formulation stability of the B-MM was assessed over a month and evaluated every week at 4, 25 and 37 °C by analyzing the hydrodynamic diameter and polydispersity index (PDI) of MM using DLS experiments as shown in Figure 5. The B-MM remained stable over a month at each temperature, for example at 37 °C only a slight size increase from 24 nm to 31 nm with a PDI ≤ 0.3 was observed, thus indicating the good stability of the nano-formulations upon time and temperature.

As quercetin is very sensitive to thermal degradation, the Q-MM stability study was conducted at 4°C only. The particles were also stable over 14 days with no variation of the particle size from 26 nm on day 0 to 25 nm on day 28 with a PDI remaining at 0.3. The stability of the Q-MM has also been evaluated through the release of quercetin over time, which should happen after decomposition on skin before the formulation had penetrated in the epidermis. To this end, a suspension of Q-MM was dialyzed in PBS buffer and in presence of Tween 20 (2% by masss) for 7 hours. A slight release of quercetin of only 6% was observed after 7 hours of dialysis (Figure 3S), confirming the above mentioned stability of the quercetin encapsulated MM formulations.

Drug loading (DL ) and encapsulation efficacy (EE) of Q-MM

The drug loading (DL) and encapsulation efficiency (EE) for quercetin in Q-MM were calculated from HPLC analysis using the equations 1 and 2 described in section 2.10. DL reflects the amount of loaded quercetin relative to the total mass of the MM formulation, whereas EE represents the amount of quercetin experimentally encapsulated by comparison with the amount of quercetin added in solution. DL for this dermal delivery system was evaluated to 3.6±0.2 % corresponding to an EE of 94±4 %.

Mixed micelles cellular viability on NIH3T3

The cell viability test was performed on mice fibroblasts (NIH3T3) co-incubated with crude quercetin, B-MM and Q-MM at concentrations ranging from 1 to 50 µg/mL (Figure 6). Results were normalized to non-treated cells (representing 100 %). After 24 hours, crude quercetin treatment induced a decrease in cell viability reaching 72±9 % at quercetin concentration of 10 µg/mL. By contrast no significant decrease in cell viability was observed when cells were treated with B-MM, at all concentrations. Interestingly, the quercetin encapsulated in MM showed less toxicity than the crude antioxidant at equivalent concentration of quercetin. For example at 25µg/mL, the crude quercetin cell viability was determined at 33±17 % and the one for Q-MM was twice higher at 65±8 %. The IC50 (cellular viability), was determined at 17.5µg/mL for crude quercetin and 37.5µg/mL for Q-MM evidencing a lower impact of quercetin on cell viability once encapsulated in Q-MM. 3.9. Antioxidant effect 3.9.1. In vitro radical scavenging ability of Q-MM

The radical scavenging ability of the crude quercetin and Q-MM was investigated with in vitro test using 2.2diphenyl-1-picrylhydrazyl (DPPH°) as a radical source. The quercetin antioxidant reacts with the free radical located at the hydrazine position of the DPPH°, thus leading to the reduced hydrazine form (DPPHH) and decreasing the amount of radicals. The DPPH test allowed to determine the IC50 (DPPH° inhibition) value: the concentration of quercetin for which 50 % of the DPPH° reacted. Figure 7 illustrates the DPPH° inhibition with quercetin concentration ranging from 2.5 to 15 µM for the free quercetin and encapsulated in MM (Q-MM). The IC50 (DPPH° inhibition) values deduced from those measurements were 9 µM (3.6 µg/mL) for the free quercetin and 6.5 µM (2.6 µg/mL) for Q-MM. This indicates that the radical scavenging activity of quercetin was preserved upon encapsulation and even enhanced with respect to crude quercetin since its IC50 value was lower.

Cellular testing on NIH3T3 with TBHP

The antioxidant activity of the Q-MM was tested on mice fibroblasts (NIH3T3) in comparison with crude quercetin and B-MM (Figure 8). A quercetin concentration of 5 µg/mL was selected as no decrease in cell viability was observed regardless of the formulation.

Cells were first treated with formulations for 24 hours before being washed and labeled with 2,7dichlorofluorescin (DCFDA). The oxidative stress was then induced by tert-butyl hydroperoxide (TBHP) for 30

Discussion

Two aspects were investigated in this study: (i) the stability of mixed micelles formulated with POx as alternative to PEG and (ii) quercetin loading and quercetin antioxidant efficiency in these POx-formulations (Q-MM). Among all the previously developed formulations for topical delivery of quercetin [START_REF] Hatahet | Liposomes, lipid nanocapsules and smartCrystals®: A comparative study for an effective quercetin delivery to the skin[END_REF], mixed micelles were selected as the first proof of concept for antioxidant POx-formulations. These formulations were constituted of phosphatidylcholine and an amphiphilic poly(2-methyl-2-oxazoline) molecule bearing a long alkyl chain (POx, C16POx15).

The surfactant property of POx itself was first evaluated. It presents a critical aggregation concentration (CAC) of ~ 100 mg/L, which is similar to structural analogue Brij®58 used as a reference. In contrast, the impact of the two amphiphilic molecules on cell viability was very different. A better cell viability in presence of POx (Figure 1) was highlighted with a IC50 (cellular viability) of 0.75 g/L compared to 0.09 g/L for Brij®58. The POx concentration for formulation of mixed micelles was about 50 times under the IC50 (cellular viability) of POx, ensuring non toxic working conditions. Therefore, POx was proved to have significantly (P < 0.001) less impact on cell viability than its PEG equivalent. These results are consistent with the literature since the polyoxazolines polymer family is known to have an excellent cytocompatibility and hemocompatibility [START_REF] Lorson | Poly(2-oxazoline)s based biomaterials: A comprehensive and critical update[END_REF]. However, it is the first time that an amphiphilic POx with such architecture (C16POx15) was investigated. Then, this amphiphilic POx was associated with phospholipids to increase the solubility of hydrophobic antioxidant within the micelles.

The mixed micelles of phosphatidylcholine stabilized by C16POx15 (B-MM) were produced using thin film process. After a high pressure homogenization, the formulation presented a reduced homogeneous particle size of 18±2 nm (Figure 2). These DLS and CAC results confirmed that nanosized mixed-micelles were created and not POx micelles and liposomes coexisting.

The same process was used in the presence of quercetin, leading to an excellent encapsulation efficiency that reached up to 94±4 %. This allowed a particularly high concentration of antioxidant in the formulations to be loaded (3.6±0.17 %), which reached 188 µg/ml in Q-MM instead of 2.48 µg/ml in PBS. It was shown that neither the spherical shape nor the particle size of MM were modified upon loading with quercetin (Q-MM) (Table 1). This contrasts with the existing hybrid nanosystems DPPC : MPOx [START_REF] Pippa | DPPC:MPOx chimeric advanced Drug Delivery nano Systems (chi-aDDnSs): Physicochemical and structural characterization, stability and drug release studies[END_REF] for which the incorporation of indomethacin (with the same molar mass as quercetin and the same nature of polycyclic aromatic ring) induced a significant size increase. The incorporation efficiency was also significantly lower with a maximum of 17 %. Looking at some others lipid formulations for topical delivery of quercetin the encapsulation efficiency, drug loading and particle size reached with the Q-MM (94 %, 3.6 %, 19 nm), are more satisfactory than nanostructured lipid carriers (89.95 %, 3.05 %, 215.2 nm) (Chen-yu et al., 2012), phospholipids based vesicles (48-75 %, NV (no value), 80-220 nm) [START_REF] Chessa | Effect of Penetration Enhancer Containing Vesicles on the Percutaneous Delivery of Quercetin through New Born Pig Skin[END_REF], lecithin-chitosan nanoparticles (48.5 %, 2.45 %, 100 nm) [START_REF] Tan | Preparation and evaluation of quercetin-loaded lecithinchitosan nanoparticles for topical delivery[END_REF], and nanovesicles (81-91 %, NV, 80-110 nm) [START_REF] Manca | Improvement of quercetin protective effect against oxidative stress skin damages by incorporation in nanovesicles[END_REF].

Moreover, the nanometric size obtained for the MM formulation is particularly interesting since it is known to ensure enhanced penetration into skin [START_REF] Schäfer-Korting | Lipid nanoparticles for improved topical application of drugs for skin diseases[END_REF].

Concerning the quercetin dispersion in Q-MM, a thermal analysis performed by DSC corroborated by microscopy (Figure 4) indicated that quercetin was well dispersed in the core of Q-MM, losing its crystalline character in favor of an amorphous phase [START_REF] Poljšak | Antioxidant Effects of Quercetin and Catechin Encapsulated into PLGA Nanoparticles[END_REF], [START_REF] Sahoo | Preparation and Characterization of Quercetin Nanocrystals[END_REF]. This feature also indicates that once the Q-MM has penetrated into skin, quercetin should be easily released out of the formulation.

To evaluate POx formulation stability, the size and PDI of B-MM and Q-MM were measured every week at various temperatures (4, 25 and 37 °C) for 28 days. B-MM remained stable at all 3 temperatures reflecting the high stabilizing capacity of POx. Regarding Q-MM, since quercetin is very sensitive to thermal degradation [START_REF] Wang | Degradation kinetics of fisetin and quercetin in solutions affected by medium pH, temperature and co-existing proteins[END_REF] even at 25 and 37 °C leading overtime to antioxidant degradation into intermediate products, the stability study was only conducted at storage condition 4°C, as also performed by Bose et al [START_REF] Bose | Formulation optimization and topical delivery of quercetin from solid lipid based nanosystems[END_REF]. The size of Q-MM remained around 26 nm over 14 days with a PDI under 0.3. The PDI increase after 14 days might be due to the quercetin loading as it was not the case for B-MM. This formulation could be more stable once loaded with another active compound. MM thus represents a quite stable quercetin formulation as a low quantity of quercetin (less than 6%) was released in vitro from the formulation over 7 hours. All the results demonstrated the efficiency of POx to stabilize MM formulation and its ability to load a large amount of antioxidants such as quercetin, indicating this formulation is promising for topical application. Indeed, the lipid composition of the MM along with the presence of POx could facilitate the delivery of quercetin in the skin epidermis [START_REF] Ashtikar | Transdermal delivery from liposomal formulations -Evolution of the technology over the last three decades[END_REF], [START_REF] Casiraghi | Nonionic Surfactants: Polyethylene Glycol (PEG) Ethers and Fatty Acid Esters as Penetration Enhancers[END_REF].

The cellular viability of crude quercetin, B-MM and Q-MM was evaluated on mice fibroblasts after 24 hours of exposure. With a quercetin concentration from 10 to 25 µg/mL, cell viability dropped from 72±9 % to 33±17 % for crude quercetin whereas a slightler decrease for Q-MM (from 82±10 % to 62±8 %) was observed (Figure 6).

This evidenced the interest of quercetin in association with MM. This statement is also highlighted by difference in IC50 (cellular viability) values, as crude quercetin has a value twice higher than Q-MM (17.5 µg/mL versus 37.5 µg/mL respectively). Moreover, it has been confirmed that the effect of Q-MM was only due to the quercetin itself as B-MM had no impact on cell viability. These results confirmed the cell viability study of polyoxazolines (Figure 1) in which POx had no impact on cell viability over a broad concentration range including the one used for MM formulation (about 15 mg/L). MM formulations were concentrated enough in quercetin so their antioxidant efficacy could be evaluated.

The antioxidant activity of Q-MM related to crude quercetin was first evaluated in vitro using DPPH assay, to ensure that the radical scavenging activity of quercetin was preserved and was even enhanced once encapsulated.

The IC50 (DPPH° inhibition) value of Q-MM compared to crude quercetin decreased from 9 to 6.5 µM (equivalent to 3.6 to 2.6 µg/mL). The Q-MM antioxidant capacity also relies on POx being more stable regarding oxidative degradation compared to PEG: the polyether backbone is more prone to oxidative degradation [START_REF] Ostuni | A Survey of Structure-Property RelaUonships of Surfaces that Resist the AdsorpUon of Protein[END_REF]. To evaluate the ability of quercetin to counteract the oxidative stress induced by ROS, mice fibroblasts were used as a cellular model with an oxidative stress induced by TBHP. The antioxidant efficacy of the quercetin was maintained once encapsulated in Q-MM. By comparison, crude quercetin was able to reduced the ROS generated by TBHP by 36 % whereas Q-MM decreased them by 42 % (Figure 8). Moreover, Q-MM was able to scavenge the excess of ROS allowing a recovery to basal ROS concentration values (about 75% as measured with untreated cells). Since the toxicity of Q-MM was lower than crude quercetin, the concentration for the antioxidant test could be increased for a potentially even higher efficacy on cells.

Conclusion

This study on amphiphilic polyoxazolines (C16POx15) provides insight into the great promise this polymer family holds for the development of pharmaceutical and cosmetic formulations. The higher cell viability (at a similar CAC value) compared to Brij®58, a worldwide used industrial nonionic surfactant, demonstrates the potential of this POx family in the formulation of API. The ability of the amphiphilic POx to produce stable formulations was investigated for the first time with mixed micelles of POx and phosphatidylcholine using thin film and HPH process. DLS and TEM measurements showed spherical micelles of around 18 nm without API (B-MM) and no size increase once loaded in a natural antioxidant quercetin (Q-MM). These mixed-micelles were stable over 28 days and present a long-lasting encapsulation with less than 6% release over 7 hours. Moreover, a good quercetin encapsulation (94±4 %) efficiency was measured with these nanosystems allowing its homogeneous solubilization in the core under amorphous character. Therefore, Q-MM can be used as a reservoir of quercetin.

Once loaded in the MM, the quercetin impact on cell viability was twice reduced while the quercetin antioxidant efficacy was preserved. Q-MM was able to reduce by 42 % of the ROS generated by TBHP allowing a recovery to basal ROS concentration values.

activity are brought together in the mixed micelles, a very promising formulation for an effective topical delivery to the deepest layer of the epidermis. Future studies will focus on the evaluation of the penetration capacity of amphiphilic polyoxazolines and Q-MM and the topical delivery of quercetin in in vivo testing.

  NIH3T3 cells were seeded at 20,000 cells/cm² in a 96 well black plate with clear bottom (Corning® Massachusetts, USA) and incubated 24 hours at 37 °C, 5 % CO2 to allow cell adhesion. Then B-MM, crude quercetin, or Q-MM at a quercetin concentration of 5 µg/mL were added and incubated for another 24 hours in supplemented DMEM. Cells were washed twice with PBS before addition of 200 µL of 2,7-dichlorofluorescin diacetate (DCFDA) reagent (20 µM in DMEM without phenol red). A serum free media was used to avoid deacetylated the DCFDA from serum esterases into a non fluorescent compound, which can later be oxidized by ROS into DCF resulting in erroneous data.After 30 minutes incubation at 37 °C, 5 % CO2 , cells were washed with PBS and placed in 200 µL DMEM without phenol red. A solution of tert-Butyl hydroperoxide (TBHP) (500 µM in PBS) was then added and the cells were incubated for 30 additional minutes. The fluorescent signal of dichlorofluorescein (DCF) generated by the reaction of DCFDA reagent with ROS was then measured (λexc 485 nm, λem 535 nm) using Tristar LB941 Spectrofluorimeter (Berthold Technologies, Germany). Values of treated cells were normalized to non-treated cells (representing the cells with 100 % ROS intensity).

Figure 1 .

 1 Figure 1. Cell viability of mice fibroblast NIH3T3 co-incubated with increasing concentration of amphiphilic POx (C16POx15) compared to amphiphilic PEG surfactant Brij® 58 (C16PEG20) for 24 h (n=3). Statistical analysis was performed by a two-sample t-test between POx and PEG values (*: P < 0.05, **: P < 0.01 and ***: P < 0.001).

Figure 3 :

 3 Figure 3: Thermograms properties of crude quercetin and Q-MM

Figure 4 :

 4 Figure 4: Optical microscopy images of crude quercetin and Q-MM on NIH3T3 cells.

Figure 5 .

 5 Figure 5. Particle size (column) and PDI (symbols) of B-MM at 4, 25, 37 °C (left) and Q-MM at 4 °C (right) over a month (n=3).

Figure 6 .

 6 Figure 6. Cell viability of mice fibroblasts (NIH3T3) co-incubated with crude quercetin, B-MM and Q-MM for 24 h (n=3). Statistical analysis was performed by a two-sample t-test between crude quercetin and B-MM, Q-MM (*: P < 0.05, **: P < 0.01).

Figure 7 .

 7 Figure 7. DPPH° inhibition of free quercetin and Q-MM with quercetin concentration

  minutes before measuring the fluorescence intensity signal. A serum free medium was used to avoid deacetylated the DCFDA from serum esterases into a non fluorescent compound, which can later be oxidized by ROS into DCF resulting in erroneous data.The results were normalized to TBHP treated cells (positive control) and the untreated cells were set as negative control. The addition of TBHP increased the ROS generated by 25±2 % (P < 0.001) compared to negative control. The crude quercetin and Q-MM formulation significantly reduced generated ROS by 36±3 % and by 42±2 % respectively compared to cells treated with TBHP. The antioxidant activity of the quercetin was maintained once formulated in MM. No significant difference in the antioxidant activity of crude quercetin versus Q-MM was evidenced.Therefore Q-MM was able to scavenge the excess of ROS allowing a recovery to basal ROS concentration values. The antioxidant effect of the Q-MM seems to be only due to the quercetin encapsulated in the formulation as the ability of B-MM to reduce ROS was very low.

Figure 8 .

 8 Figure 8. ROS relative intensity for crude quercetin, B-MM and Q-MM (quercetin concentration 5 µg/mL) (n=3). Negative control was untreated cells (without TBHP or formulations) and positive control was cells treated with TBHP without formulation. Fluorescent intensity was normalized to TBHP treated cells (positive control). A one sample t-test relative to positive control cells was performed (*: P < 0.05, **: P < 0.01 and ***: P < 0.001).

Supplementary information


Stephen Valas
email: stephen.valas@anses.fr

Isabelle Brémaud

Sophie Stourm

Benoit Croisé

Sophie Mémeteau

David Ngwa-Mbot

Marc Tabouret

Anses

Improvement of eradication program for infectious bovine rhinotracheitis in France inferred by serological monitoring of singleton reactors in certified BoHV1-free herds

Keywords: Surveillance plan, IBR, BoHV1, diagnostic tools, cross-reactions

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

Introduction

Bovine alphaherpesvirus 1 (BoHV1) is recognized as an important pathogen of livestock industry causing infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis/balanoposthitis (IPV/IPB). The virus belongs to the genus Varicellovirus in the Alphaherpesvirinae subfamily and has been reported worldwide. Although BoHV1 infections are often subclinical, symptoms in the upper respiratory tract and conjunctivitis are the most frequent clinical manifestations. Following acute infection of the respiratory or genital tract, BoHV1 undergoes cycles of latency to reactivation in local sensory neurons, leading to shedding and transmission of the virus via the respiratory, ocular or genital secretions as well as the semen from infected bulls. Reactivation may be triggered by stressful events (parturition, diet, dramatic whether conditions, transport and animal mixing, etc.), immunosuppression or treatments with corticosteroids [START_REF] Thiry | Parturition as a stimulus of IBR virus reactivation[END_REF][START_REF] Thiry | Reactivation of infectious bovine rhinotracheitis virus by transport[END_REF][START_REF] Turin | BHV-1: new molecular approaches to control a common and widespread infection[END_REF][START_REF] Winkler | Persistence and reactivation of bovine herpesvirus 1 in the tonsils of latently infected calves[END_REF]Akermann and Engels, 2006;van Drunen Little-van den Hurk, 2006;[START_REF] Jones | Bovine herpesvirus type 1 (BHV-1) is an important cofactor in the bovine respiratory disease complex[END_REF].

BoHV1 infection can induce direct production losses through reduced milk yield, weight loss, abortions, infertility, and higher susceptibility to secondary bacterial infections [START_REF] Hage | Milk production and reproduction during a subclinical bovine herpesvirus 1 infection on a dairy farm[END_REF][START_REF] Nandi | Bovine herpes virus infections in cattle[END_REF]. However, the economic impact of BoHV1 mainly results from restrictions in livestock trade. Indeed, IBR is included into the Terrestrial Animal Health Code of the World Organization for Animal Health (OIE) and is associated with additional guarantees at the European level. Several member states of the European Union (EU) have either successfully eradicated IBR (Austria, Denmark, Germany, Finland, Sweden, Switzerland) or implemented an EU-approved compulsory program at the national or regional scales (Belgium, Czech republic, Italy). In France, a voluntary eradication program based on the test-and-removal strategy without vaccination started in 1996. Concomitantly, a mandatory control plan based on serological testing together with vaccination and/or removal of positive animals was implemented in 2006. A steady increasing number of certified BoHV1-free herds was observed at the national level (from 49% in 2006 to 69% in 2016), leading the French Ministry for agriculture, food and forestry to approve an unique compulsory program since May 2016 in order to speed up the eradication process. The monitoring of certified-free status of herds is performed by whole-virus indirect ELISA test on bulk tank milk every 6 months or by annual testing of blood samples from animals older than 24 months. Samples with non-negative results are retested by a glycoprotein B (gB) blocking ELISA test used as gold standard.

Results of the voluntary program in mainland France revealed that 65.9% of farms (123,070 herds) were certified as free of IBR in 2014 [START_REF] Gache | Report on regulatory and voluntary surveillance of infectious bovine rhinotracheitis in 2013/2014: a stable situation and new opportunities[END_REF]. Seropositive animals were found in 1.1% of farms (1,303 herds) which had a certified-free status at the start of the campaign. However, no more than two positive animals were detected for 93% of herds. In most of these cases, epidemiological investigations never provided evidence for BoHV1 infection, which was consistent with the fact that BoHV1 infection of naïve herds mainly results in an intense spread of virus and a high within-herd seroprevalence. Such epidemiological non-feasible singleton reactors were also reported in Germany and The Netherlands [START_REF] Mars | Presence of bovine herpesvirus 1 gB-seropositive but gE-seronegative Dutch cattle with no apparent virus exposure[END_REF][START_REF] Isa | Comparative studies with three different bovine blood sample BHV-1 ELISA tests: indirect ELISA and bG-blocking-ELISA[END_REF]. Further characterization of unexplained gBpositive reactors from these countries revealed that they were consistently found negative in gE blocking ELISA and that no virus could be recovered following treatment with corticosteroids, ruling out infection with BoHV1 [START_REF] Mars | Presence of bovine herpesvirus 1 gB-seropositive but gE-seronegative Dutch cattle with no apparent virus exposure[END_REF]Böttcher et al., 2012).

In view of the eradication program, the understanding of the causes for epidemiological non-feasible gB-positive reactivity is crucial. We hypothesized two main reasons for the occurrence of nonspecific reactivity in gB ELISAs. Firstly, only few studies aimed to evaluate the diagnostic performances of these tests [START_REF] Kramps | Evaluation of tests for antibodies against bovine herpesvirus 1 performed in national reference laboratories in Europe[END_REF]. Thus, false-positive reactions due to suboptimal specificity of gB ELISA tests might be responsible.

Secondly, an epidemiological study revealed that cross-reactivity due to infection with bovine alphaherpesvirus 2 (BoHV2) was one reason for singleton reactors in German cattle herds (Böttcher et al., 2012), highlighting that circulation of BoHV1-related herpesviruses may confound the serological diagnosis of IBR. In this study, evaluation of the specificity and sensitivity of BoHV1 gB ELISA kits was performed using large panels of field sera.

Additionally, different monitoring schemes were applied during the two successive surveillance campaigns 2014-15 and 2015-2016 in France in order to characterize singleton reactors from certified BoHV1-free farms.

Materials and methods

Herds and samples

A collection of field sera was used to evaluate the diagnostic specificity and sensitivity of gB blocking ELISAs as well as the between-herd seroprevalence for BoHV2 in French cattle herds. All samples were collected from animals older than 24 months in herds from various geographical origins, covering 33 departments in mainland France. An average number of 20 animals were sampled per herd, ranging from 10 to 30 animals. The 716 negative sera were originated from 35 herds with a BoHV1-free status for at least 3 years. The 281 positive sera were collected before vaccination in 15 herds previously classified as BoHV1-free and for which a massive seroconversion for BoHV1 was detected during the surveillance control testing.

The numbers of herds and animals which were further analyzed in this study during the two successive surveillance campaigns 2014-15 and 2015-16 are given in the respective results sections. All of these animals were originated from certified BoHV1-free herds and tested positive by indirect and gB blocking ELISA tests during the compulsory serological control carried out in official laboratories for IBR diagnosis. They pertained to the category of singleton reactors defined as followed: no more than one, two or three positive animals for a maximum herd size of 20, 200 or more animals older than 24 months, respectively. Virus neutralization test (VNT) using BoHV1 strain Los Angeles and BoHV2 strain RVB-0064 was performed on Madin-Darby bovine kidney (MDBK) cells according to the protocol described in the OIE manual (OIE, 2018). The BoHV2 strain was kindly provided by Patricia König (Friedrich-Loeffler-Institut, Reims).

ELISA kits and virus neutralization test

Statistical analysis

The Receiver-Operator-Characteristic (ROC) analysis was performed using R statistical software. Correlation between gB ELISA reactivity and serological status for BoHV2 was statistically evaluated by Mann-Whitney U-test using Statistica software (StatSoft France, version 10). Confidence intervals were calculated by using Exact Binomial test using the online Epitools epidemiological calculators (http://epitools.ausvet.com.au).

Results

Diagnostic sensitivity and specificity of gB ELISAs

The performances of the three commercial gB blocking ELISA kits (from Idexx, IDVet and Biosellal) which were carried out in official laboratories (OL) for BoHV1 diagnosis in France were evaluated using a panel of 997 field sera. The sensitivity was estimated using 281 positive sera from 15 infected herds. The specificity was evaluated using 716 negative sera from 35 certified BoHV1-free herds. The status of all sera was verified with two other sets of commercially available BoHV1 serological tests, including two whole-virus indirect ELISAs and two gE blocking ELISAs, and by VNT for sera with discordant results between ELISA tests. Analysis of the 281 positive sera with concordant results between the two sets of indirect and gE ELISA tests revealed a sensitivity of 100% (CI95%: 98.7-100.0%) for the three gB ELISAs (Table 1). Among the 716 negative sera, 56 sera (8%) were found either doubtful or positive with at least one gB ELISA, seven out of them being also positive in at least one indirect ELISA. In contrast, all sera tested negative in gE ELISA, and sera with discordant ELISA results were found negative in VNT. Considering equivocal reactions as positive, the specificity of gB-Idexx, gB-IDVet and gB-Biosellal ELISAs was estimated at 97.5% (CI95%: 96.1-98.4%), 93.2% (CI95%: 91.1-94.8%) and 99.2% (CI95%: 98.2-99.6%), respectively (Table 1). Reactions above the positive threshold in gB ELISAs were found in 16/35 (46%) BoHV1-free herds, 63% of them with singleton reactors only. Since antigenic cross-reactions with BoHV2 could be responsible for singleton reactors in BoHV1 ELISA tests, the entire panel of sera was screened with a commercially available BoHV2 indirect ELISA (IDVet). Antibodies against BoHV2 were detected in 27/50 (54%) herds with a within-herd prevalence ranging from 4% to 92% in positive herds (median of 40%).

Seropositivity towards BoHV2 in these 27 herds was confirmed by VNT using BoHV2 as challenging virus. Among sera from herds with BoHV1-negative/BoHV2-positive status, the reactivity by three gB ELISAs was significantly increased for sera with BoHV2 antibodies (p < 0.001), leading to a higher number of false-positive reactions in BoHV2 positive herds, compared with the negative ones (Fig. 1). Furthermore, the number of false-positive reactions by gB-IDvet remained relatively high in BoHV2 negative herds (17 out of 339 sera), compared with the two others gB ELISAs (two sera for gB-Idexx and one serum for gB-Biosellal), accounting for the lowest specificity observed for this kit. In an attempt to increase the performances of gB ELISAs, a Receiver Operator Characteristic (ROC) analysis was applied to the same panels of sera. A specificity of at least 99.9% and a sensitivity ranging from 99.3% to 100% were achieved for cut-off values clearly distinct from those defined in the manufacturer's instructions (Table 1).

Surveillance campaign 2014-2015

Assuming that both suboptimal specificity and cross-reactions in gB ELISAs likely accounted for false-positive, singleton reactors in BoHV1-free herds, a new analytical scheme for IBR surveillance was carried out at the national level during the campaign 2014-2015 (Fig. 2A). gB-positive sera from singleton reactors were transferred from OL to the NRL for further analysis using a distinct gB ELISA (gB-Idexx or gB-IDVet). Sera were classified as weak or strong positive samples according to threshold levels distant by 5% OD from those defined by the ROC analysis (cut-off values of 70% and 25% for gB-Idexx and gB-IDVet, respectively), in order to take into account the interlaboratory reproducibility of gB ELISAs. This parameter was estimated through two consecutive proficiency tests including 67 and 47 OL. Singleton reactors with strong reactivity in gB-ELISA were vaccinated and/or removed, while those found negative or classified as weak positive at the NRL were analyzed at least 6 weeks later in a same manner. Comparative analysis of data between OL and the NRL is summarized in Table 2. Only sera with gB-Idexx or gB-IDVet results were included in this study because they accounted for more than 92% of samples submitted to the NRL, which represented 728 gB-positive sera from 656 herds. On the basis of data from 42 OL, 459 (63%) sera were classified as weak positive. Sixty-four percent of these sera were similarly classified at the NRL, while the remaining sera were found negative (16%) or classified as strong positive (20%). Among the 269 strong positive sera send by OL, 87% sera were confirmed as strong positive at the NRL. Overall, 83% of samples were similarly classified in both ELISAs.

To test whether weak positive reactions corresponded to animals at early stage of infection, new blood samples of 384 (95%) singleton reactors which were found negative or weak positive at the NRL were collected at least 6 weeks later and characterized as described above. As shown in Table 2, results of gB ELISA performed by OL revealed 63% negative sera, 34% weak positive, and only 3% strong positive. Analysis of the 142 non-negative sera at the NRL confirmed that most of them (87%) showed weak or no reactivity in gB ELISA.

Finally, the 121 samples which were found positive at the NRL were analyzed by VNT as confirmatory test. Only three sera tested positive, with neutralizing antibody titers ≤ 6.

However, these three sera tested negative by gE ELISA. Altogether, these results indicated that a great number of singleton reactors from certified herds showed weak gB-positive reactions that did not increase with time and tested negative by confirmatory tests such as VNT or gE ELISA, ruling out putative infection with BoHV1.

Surveillance campaign 2015-2016

Taken into account the results of IBR surveillance in 2014-2015, new measures were adopted for the next compulsory campaign in certified BoHV1-free herds (Fig. 2B). Since the classification of sera as weak or strong positive in gB ELISA was strongly concordant antibodies could be responsible for serological drawback in BoHV1 diagnosis, a panel of 287 gB-positive/gE-negative singleton reactors corresponding to 215 herds from 48 departments was screened with the BoHV2 indirect ELISA (IDVet). Remarkably, antibodies against BoHV2 were detected in 85.7% of animals, leading to a calculated between-herd seroprevalence of 89.8% (CI95%:85-93.1%).

Discussion

Several studies were designed to evaluate the specificity and sensitivity of markerindependent BoHV1 ELISA tests. The results of an international ring test based on a limited number of samples indicated that indirect ELISAs were suitable for the detection of antibodies in both sera and milk samples, and that gB ELISAs exhibited the best performances for testing serum samples, with a sensitivity of 96% and a specificity of 99% [START_REF] Kramps | Evaluation of tests for antibodies against bovine herpesvirus 1 performed in national reference laboratories in Europe[END_REF]. In contrast, another study using a large field panel of 2754 sera revealed a relative low specificity of gB ELISAs which could potentially increase the number of BoHV1 positive farms with up to 20% [START_REF] Isa | Comparative studies with three different bovine blood sample BHV-1 ELISA tests: indirect ELISA and bG-blocking-ELISA[END_REF]. In the present study, we showed that the gB ELISA kits currently used for BoHV1 surveillance in France were highly sensitive whereas their specificity was lower than expected (specificity of 97.5% and 93.2% for the gB-Idexx and gB-IDVet ELISAs, respectively). Suboptimal specificity of gB ELISAs was emphasized by a comprehensive analysis of singleton reactors from certified BoHV1-free herds detected during two successive surveillance campaigns (from 2014 to 2016).

Characterization of 728 singleton reactors from 656 herds during the first campaign revealed that half of them corresponded to weak positive reactions. Analysis of new blood samples collected at a 6-week interval showed that these weak positive reactions fell into the negative range of gB ELISA, while those remaining stable could not be confirmed by either VNT or gE ELISA. These serological profiles were inconsistent with the humoral immune responses developed by infected animals. Indeed, antibodies directed against the gB protein appear early after infection (7-14 days post-infection) and raise to high and steady levels [START_REF] Kramps | A simple, specific, and highly sensitive blocking enzyme-linked immunosorbent assay for detection of antibodies to bovine herpesvirus 1[END_REF][START_REF] Babiuk | Immunology of bovine herpesvirus 1 infection[END_REF]König et al., 2003). Analysis of 1823 gB-positive sera from 1279 herds during the next campaign showed that 67% of singleton reactors corresponded to strong gB-positive reactions. However, most of them (93.6%) were not confirmed by gE ELISA despite the use of an upgraded cut-off, making this test as sensitive as gB ELISAs (Böttcher et al., 2012). Finally, epidemiological investigations following discovery of these singleton reactors as well as next annual surveillance controls failed to provide any sign of BoHV1 infection (data not shown).

Several factors may be responsible for the occurrence of nonspecific reactivity in BoHV1 ELISA, including batch variation of the ELISA used, bad sample quality, testing very fresh sera or samples collected soon after BoHV1-unrelated vaccination [START_REF] Beer | Diagnostic markers in the prevention of bovine herpesvirus type 1: possibilities and limitations[END_REF]OIE, 2018). Analytical schemes designed in this study for the characterization of singleton reactors (use of different validated batches, serum freezing, good concordance of results between laboratories) and subsequent epidemiological investigations (evaluation of the socalled "vaccination-phenomenon") allowed us to exclude all of these possible explanations.

Taken into account previous studies demonstrating the reliability of BoHV1 gB ELISAs [START_REF] Kramps | Evaluation of tests for antibodies against bovine herpesvirus 1 performed in national reference laboratories in Europe[END_REF], it was likely that singleton reactors with strong gB-positive reactivity might be attributed to serological cross-reactivity due to infection with other ruminant alphaherpesviruses. Bovine alphaherpesvirus 5 would probably induce severe clinical manifestations and has so far never been described in Europe. Infections with bubaline alphaherpesvirus 1 (BuHV1), caprine alphaherpesvirus 1 (CpHV1) and cervid alphaherpesvirus 1 (CvHV1) could also result in a gB-positive/gE-negative response. Both the number and the geographical localization of water buffalo herds are very limited in France, without any contact with cattle. Serological surveys revealed a very low prevalence of CvHV1 in wild ruminants [START_REF] Thiry | Serological survey of herpesvirus infections in wild ruminants of France and Belgium[END_REF][START_REF] Candela | Pathogens of zoonotic and biological importance in roe deer (Capreolus capreolus): seroprevalence in an agro-system population in France[END_REF], and a heterogeneous distribution of CpHV1 infection throughout the country [START_REF] Thiry | Serological evidence of caprine herpesvirus 1 infection in Mediterranean France[END_REF][START_REF] Suavet | First description of infection of caprine herpesvirus 1 (CpHV-1) in goats in mainland France[END_REF]. Therefore, it was unlikely that infections with these BoHV1-related viruses could accounted for the high number of epidemiological unfeasible singleton reactors reported from many regions of France. In contrast, Böttcher et al. (2012) showed that the seroprevalence for BoHV2 was higher in German dairy cattle herds with singleton reactors, compared to negative control herds. Moreover, the reactivity in indirect and gB ELISAs was significantly increased for cattle with BoHV2 antibodies. Our study demonstrated for the first time the presence of antibodies to BoHV2 in French cattle herds. Since BoHV2-positive reactions in ELISA was confirmed by VNT using homologous virus strain, it was unlikely that these serological results were either false-positive or induced by cross-reactions with a still unknown herpesvirus. BoHV2 infection appeared widely distributed throughout the country, with a calculated between-herd prevalence of 54% (CI95%: 40.4-67%). Remarkably, this between-herd prevalence was far smaller than the BoHV2 seropositivity among singleton reactors from BoHV1-free herds (89.8%). Furthermore, our results confirmed the crossreactivity of BoHV2 antibodies with BoHV1 ELISA tests, except gE ELISA. Altogether, these findings demonstrated that BoHV2 was responsible for the occurrence of singleton reactors in French BoHV1-free herds.

In France, the surveillance of dairy cattle farms is performed by serological bulk-milk testing. Farms with positive results are then retested using blood samples from animals older than 24 months, like beef cattle herds. Testing of blood samples is performed in a sequential manner. Firstly, pools of 10 sera are analyzed by indirect ELISA. Then, all sera from a positive pool are tested individually by gB ELISA. The panel of negative sera used in this study to evaluate the performances of gB ELISAs originated from 35 certified herds, including 15 beef cattle herds. Interestingly, while these herds were controlled as negative by indirect ELISA based on pooled sera, nine of them contained animals which tested both positive for BoHV2 and weak positive by gB ELISA. Therefore, testing of blood samples using pooled sera appeared to be a suitable strategy for limiting the serological drawback induced by BoHV2.

Conclusion

Obviously, serological tests should be enough sensitive to protect BoHV1-free herds from introduction of a latently infected animal. Due to cross-reactions with other bovine alphaherpesviruses, these tests should be also highly specific to avoid inappropriate withdrawal of the certified-free status. Consequently, ongoing eradication programs of BoHV1 in Europe generally rely on complex analytical schemes from routine to confirmatory diagnosis, including different tests (indirect ELISA, gB and gE blocking ELISAs, VNT), some of them being applied to different matrix (milk and serum). These monitoring systems are costly and time-consuming, preventing farmers to any trade for a long period. Our results highlighted that testing of pooled sera and re-evaluation of the cut-offs of gB ELISAs could be alternative diagnostic strategies for limiting nonspecific reactivity.

  All ELISA tests commercially available in France and registered at the time of the study for the detection of BoHV1 antibodies in serum were used, including two whole-virus indirect ELISAs (Idexx IBR individual [Idexx] and ID Screen IBR Mixte Indirect [IDVet]), three gB blocking ELISAs (Idexx IBR gB X3 [Idexx], ID Screen IBR gB competition [IDVet] and BioLisa kit IBR gB Ab [Biosellal]), and two gE blocking ELISAs (Idexx IBR gE [Idexx] and ID Screen IBR gE competition [IDVet]). The assays were performed according to manufacturer's instructions. BoHV2 antibodies were detected by using the ID Screen BHV2 indirect ELISA from IDVet.

Figure 1 .

 1 Figure 1. gB ELISA reactivity of sera from BoHV1-free herds with negative or

Figure 2 .

 2 Figure 2. Analytical schemes of surveillance program for BoHV1 in France during the

Figure 3 .

 3 Figure 3. ELISA reactivity of singleton positive sera detected during the surveillance

  Fig.1

Table 1 .

 1 Diagnostic specificity (Sp) and sensitivity (Se) of gB ELISAs. Analyses were performed on 281 positive sera from 15 herds with BoHV1 outbreaks and 716 negative sera from 35 BoHV1-free herds. The performances of gB ELISAs were estimated on the basis of the manufacturer's cut-off values and by a ROC analysis.

	Tables
	gB ELISA	Manufacturer's cut-off	Sp (%) Se (%)	Cut-off	ROC analysis Sp (CI 95%)	Se (CI 95%)
	Idexx	45 ≤ (1-S/P)% < 55	97.5	100.0	75%	100.0 (99.5-100.0)	99.6 (98.0-99.9)
	IDVet	45 < (S/P)% < 55	93.2	100.0	20%	100.0 (99.5-100.0)	99.3 (97.4-99.8)
	Biosellal	45 ≤ (1-S/P)% < 55	99.2	100.0	60%	99.9 (99.2-100.0) 100.0 (98.7-100.0)
	Sp, specificity; Se, sensitivity; CI, confidence interval.

Table 2 .

 2 gB ELISA reactivity of singleton reactors detected during the surveillance campaign 2014-15. Sera were classified as weak or strong positive samples in gB ELISA on the basis of new cut-off values defined by a ROC analysis.

	Day of blood sampling	gB-ELISA/OL Reactivity Nb. of sera	gB-ELISA/NRL weak strong neg. pos. pos.
	Day 0	weak pos.	459	76	292	91
		strong pos.	269	6	29	234
	Day ≥ 6 weeks neg.	242	NT	NT	NT
		weak pos.	129	21	93	15
		strong pos.	13	0	10	3
	OL, official laboratories; NRL, national reference laboratory for IBR.
	NT, not tested.

Acknowledgements

We thank all farmers and veterinarians, the local farmers' organizations for animal health protection (GDS), the French association for health and environment (Afse) and the French national network of local veterinary laboratories who sent biological samples, analytical and/or epidemiological data for BoHV1 monitoring and subsequent studies. Written consent was obtained from all farmers whose provided biological samples for reference and

Funding

This work was supported by grants from Anses, GDS France and Région Nouvelle-Aquitaine.

between laboratories, kits and successive blood samples, the discrimination of sera on the basis of their reactivity in gB ELISA was only performed at OL level using a single blood sample. The status of herds was determined according to the number of strong positive reactors, but independently of the number of weak positive reactors. Herds showing only weak positive reactors kept their BoHV1-free status, whereas those with more than three strong positive reactors were stated as infected herds and all seropositive animals were vaccinated and/or removed. In cases of herds having no more than three strong positive reactors, all positive sera (including weak and strong samples) were submitted to analysis at the NRL. The gE ELISA (from Idexx) was chosen in place of VNT as confirmatory test, allowing analysis of a great number of samples. The cut-off of the gE ELISA was set at 0.9, making this test as sensitive as the gB-Idexx (Böttcher et al., 2012). A total of 1823 gBpositive sera from 1279 herds were analyzed by gE ELISAs (Fig. 3). The gB-Idexx and gB-IDVet tests were applied on 68% and 32% of sera, respectively. Strong positive reactors (n=1221) accounted for 67% of seropositive animals. Seventy-eight (6.4%) of these animals also tested positive in gE ELISA. Six (1%) out of the 602 weak positive reactors were found positive in gE ELISA. Overall, gE-positive reactions were found in 5.4% of herds. A fraction of the gE-negative samples was analyzed by VNT, including 64 weak and 324 strong positive reactors. All weak positive reactors tested negative, and 9 (2.8%) strong positive reactors were found positive.

Prevalence of BoHV2 among singleton reactors

In view of the performances of blocking ELISAs used and the absence of epidemiological link between singleton reactors and IBR outbreaks, animal movement or accidental vaccination, it was likely that these gB-positive/gE-negative reactions mainly resulted from antigenic cross-reactivity with a BoHV1-related herpesvirus. Since BoHV2

Conflict of interest statement

All authors deny any financial and personal relationships with other people or organizations that could inappropriately influence this work.

Authors' contributions

SV, DN-M and SM designed the study. DN-M and SM facilitated the collection and the use of specimens. IB, SS and BC performed the laboratory analyses. SV performed the data analyses and wrote the manuscript. MT and DN-M revised the manuscript critically. All co-authors read and approved the final manuscript.