domain: phys.phys.phys-optics spi sdv

text:
Jeffrey Bencteux

Pierre Pagnoux

Thomas Kostas

Sam Bayat

Michael Atlan

Holographic laser Doppler imaging of pulsatile blood flow

We report on wide-field imaging of pulsatile motion induced by blood flow using heterodyne holographic interferometry on the thumb of a healthy volunteer, in real-time. Optical Doppler images were measured with green laser light by a frequency-shifted Mach-Zehnder interferometer in off-axis configuration. The recorded optical signal was linked to local instantaneous out-of-plane motion of the skin at velocities of a few hundreds of microns per second, and compared to blood pulse monitored by plethysmoraphy during an occlusion-reperfusion experiment.

INTRODUCTION

One of the most commonly displayed clinical waveforms is the plethysmogram. In particular, finger plethysmography allows the non-invasive measurement of pulse wave amplitude, which reflects the changes in finger blood flow. It is usually recorded by a pressure sensor [START_REF] William | Review of medical physiology[END_REF] or a pulse oximeter which illuminates the skin and measures changes in optical absorption [START_REF] Ab Hertzman | Observations on the finger volume pulse recorded photoelectrically[END_REF][START_REF] Allen | Photoplethysmography and its application in clinical physiological measurement[END_REF][START_REF] Aymen | Photoplethysmography[END_REF]. Optical plethysmography is widely available, its feasibility has even been demonstrated using mobile phones [START_REF] Jonathan | Investigating a smartphone imaging unit for photoplethysmography[END_REF][START_REF] Scully | Physiological parameter monitoring from optical recordings with a mobile phone[END_REF]. However, it does not provide a spatially-resolved image. This limitation can be overcome by signal processing of regular video recordings in ambient light for time-dependent spatial and color fluctuations enhancement [START_REF] Wu | Eulerian video magnification for revealing subtle changes in the world[END_REF][START_REF] Balakrishnan | Detecting pulse from head motions in video[END_REF]. Other imaging approaches are designed to harness optical phase fluctuations of coherent laser light from the motion of body walls, either by single-point scanning detection [START_REF] Essex | A laser doppler scanner for imaging blood flow in skin[END_REF], direct image detection of temporal [START_REF] Draijer | Twente optical perfusion camera: system overview and performance for video rate laser doppler perfusion imaging[END_REF][START_REF] Leutenegger | Real-time full field laser doppler imaging[END_REF][START_REF] Kamshilin | Variability of microcirculation detected by blood pulsation imaging[END_REF] and spatial speckle contrast [START_REF] David | Laser speckle contrast imaging in biomedical optics[END_REF][START_REF] Humeau-Heurtier | Skin perfusion evaluation between laser speckle contrast imaging and laser doppler flowmetry[END_REF], or holographic interferometry [START_REF] Mathieu Leclercq | Evaluation of surface acoustic waves on the human skin using quasitime-averaged digital fresnel holograms[END_REF][START_REF] Dobrev | Optimization of a lensless digital holographic otoscope system for transient measurements of the human tympanic membrane[END_REF]. In this letter, we report on narrow band laser Doppler imaging of superficial pulsatile motion of the thumb by heterodyne holographic interferometry with a camera refreshed at 60 Hz. A transient blood flow interruption experiment in a healthy volunteer is performed and compared to blood pulse monitoring.

EXPERIMENTAL SETUP

The experimental laser Doppler imaging scheme designed for this study is sketched in Fig. 1. The apparatus consists of a fibered Mach-Zehnder optical interferometer for off-axis [START_REF] Leith | Reconstructed wavefronts and communication theory[END_REF] and frequency-shifting [START_REF] Aleksoff | Time average holography extended[END_REF][START_REF] Macovski | Efficient holography using temporal modulation[END_REF] holographic interferometry. The main optical radiation field is provided by a 150 mW, single-mode, fibered diode-pumped solid-state green laser (Cobolt Samba-TFB-150) at wavelength λ = 532 nm, and optical frequency ω L /(2π) = 5.6 × 10 14 Hz. The thumb is illuminated with about ∼ 10 mW of continuous optical power, over ∼ 40 mm × 40 mm. In the reference channel, an optical local oscillator (LO) is formed by optical frequency shifting. Two acousto-optic modulators (Bragg cells, AA Opto Electronic), driven with phase-locked signals at ω C and ω C + ∆ω, are used to shift the optical frequency of the laser beam from ω L to ω L + ∆ω. The carrier frequency ω C /(2π) is set to 200 MHz, at the peak response of the fibered acousto-optic modulators. The backscattered optical field E is mixed with the LO field E LO with a non-polarizing beam splitter cube. A Ximea MQ042MG-CM camera records interference patterns at a frame rate of τ -1 S = ω S /(2π) = 60 Hz. Each raw interferogram of 2048 × 2048 pixels, digitally acquired at time t is noted I(t) = |E(t) + E LO (t)| 2 . A small angular tilt ∼ 1 • between E and E LO ensures off-axis recording. The sensor is set ∼ 50 cm away from the object plane. A convergent lens of 50 mm focal length is present in the object channel, in order to widen the lateral field of view of the holographic detection to ∼ 50 mm. The backscattered laser optical field

E(t) = E exp [iω L t + iφ(t)
] undergoes a phase variation φ(t), as a consequence of out-of-plane motion of the illuminated tissue. It is mixed with the LO field from the reference channel

E LO (t) = E LO exp [iω L t + i∆ωt], that is tuned to a close-by intermediate frequency ω L + ∆ω.
The quantities E and E LO are complex constants and i is the imaginary unit. The magnitude of a given optical Doppler component is retrieved by frequency down conversion within the camera's temporal bandwidth, ensured by non-linear detection of the field E by the array of square-law sensors of the camera, that respond linearly with the optical irradiance

I(t) = |E(t) + E LO (t)| 2 .
The squared magnitude of the total field received, E + E LO , has cross-terms oscillating at the difference frequency of the fields E and E LO

I(t) = |E| 2 + |E LO | 2 + H(t) + H * (t) (1)
where

H(t) = EE * LO exp (iφ -i∆ωt)
is the heterodyne interferometric contribution, and * denotes the complex conjugate. This equation describes the temporal fluctuation of the recorded irradiance at a given pixel.

SIGNAL PROCESSING

Holographic image rendering, or spatial demodulation, is then performed onto each recorded interferogram I with a discrete Fresnel transform [START_REF] Goodman | Digital image formation from electronically detected holograms[END_REF]. In off-axis recording configuration, The Fresnel transform separates spatially the four interferometric terms of the right member of Eq. 1 [START_REF] Leith | Reconstructed wavefronts and communication theory[END_REF][START_REF] Cuche | Spatial filtering for zero-order and twin-image elimination in digital off-axis holography[END_REF]. After spatial demodulation of each interferogram, the heterodyne signal H(t) appears in the offaxis region of the hologram, processed as follows : First, the squared amplitude of the difference of two consecutive off-axis holograms is formed in order to cancel very low frequency noise contributions

S 2 = |H(t) -H(t -τ S )| 2 .
Then, to cancel laser intensity fluctuations, a normalization factor

S 2 0 = |H 0 (t) -H 0 (t -τ S )| 2 is formed.
The contribution H 0 is measured in a region of interest of the reconstructed hologram where the terms of Eq. 1 are not present [START_REF] Atlan | Laser doppler imaging, revisited[END_REF], and the brackets account for spatial averaging. In high heterodyne gain regime, i.e. when |E| 2 ≪ |E LO | 2 , the quantity S 2 0 is dominated by shot-noise of the LO [START_REF] Gross | Digital holography with ultimate sensitivity[END_REF][START_REF] Lesaffre | Noise and signal scaling factors in digital holography in weak illumination: relationship with shot noise[END_REF], which scales up linearly with |E LO | 2 . The averaged signal S 2 scales up linearly with

|E| 2 |E LO | 2 .
Hence the processed Doppler signal S 2 / S 2 0 is a heterodyne measurement of the optical power (or irradiance) |E| 2 , which does not depend on the LO power [START_REF] Magnain | Holographic laser doppler imaging of microvascular blood flow[END_REF].

NARROWBAND APPARATUS RESPONSE

Two-phase demodulation of interferograms recorded with a sampling frequency approximately equal to the reciprocal of the exposure time (τ -1

S ≃ τ -1 E
) results in a narrowband detection with an apparatus response plotted in Fig. 2. For the backscattered field's irradiance |E| 2 , this response is [START_REF] Michael Atlan | Parallel heterodyne detection of dynamic light-scattering spectra from gold nanoparticles diffusing in viscous fluids[END_REF][START_REF] Verrier | Laser doppler holographic microscopy in transmission: application to fish embryo imaging[END_REF]

B(ω) = 1 ω 2 τ 2 E sin 2 ωτ S 2 sin 2 ωτ E 2 ( 2
)
where ω is the apparent frequency recorded by the camera, as a result of heterodyne beating. Let's assume that at the short time scale of the sampling process t < τ S = 2π/ω S , the local instant velocity is approximately constant. v(t) ≈ v(t -τ S ) ≈ v. In other words, its magnitude v is identified to the time-averaged value of the instant velocity during τ S , so we can simply write the local optical phase variation due to the transverse displacement of the skin as φ(t) = 2kvt = ωt, when the illumination and the scattered wave vectors are perpendicular to the skin's surface. The optical wave number is k = 2π/λ ≃ 1.2 × 10 7 rad.s -1 , and ω = 2kv. Now let's consider temporal variations of the instant velocity of norm v(t) at long time scales t > τ S . The light reflected by the skin undergoes a local time-dependent Doppler shift

ω(t) = 2kv(t) (3)
To assess a given instant velocity v 0 , the LO is detuned by ∆ω = 2kv 0 . According to Eq. 3 and Eq. 2, it results in the detection of Doppler-shifted light with a velocitydependent efficiency B (2k(v -v 0 )). The range of probed velocities v within the two first lobes of the apparatus response B is

[v -1 , v +1 ] = v 0 - ω S 2k , v 0 + ω S 2k (4)

EXPERIMENT

An experiment on the fingers of healthy volunteers undergoing arterial occlusion provoked with a pressure cuff was conducted. The pressure cuff was placed around the right arm. The cuff bladder pressure was measured using a transducer (MLT0670, ADInstruments, Oxford, UK). The signal was amplified, digitized at 200 Hz using an analog/digital converter (Powerlab; ADI Instruments), and recorded on a computer. The arterial pulse wave was monitored using a piezo-electric pulse transducer (TN1012/ST, ADInstruments) placed on the index of the imaged hand. The signal was recorded at 200 Hz. The thumb was illuminated by the green laser beam. A comfortable support for the forearm was used to avoid stray motion. A preliminary experiment was performed to monitor the Doppler response at three different detuning frequencies ∆ω/(2π) : 60 Hz, 600 Hz, and 1020 Hz; choosing a detuning frequency which is an integer number of times the sampling frequency is not mandatory, but is has the advantage of canceling efficiently staticallybackscattered light, according to the apparatus response (Fig. 2). These frequencies correspond to probed velocities of 16 µm.s -1 , 160 µm.s -1 , and 271 µm.s -1 , respectively. Fig. 3 shows plots over time of pulse waveforms (plethysmograms) and holographic Doppler signal 10 log 10 S 2 / S 2 0 (in dB) averaged in a region of interest, placed on the central part of the image of the thumb. At very low frequency detunings ( 100 Hz), motion artifacts prevented the measurement of pulsatile motion. When the frequency was set to ∆ω/(2π) = 1020 Hz, which corresponded to a measurement range of instant velocities [v -1 , v +1 ] = [255 µm.s -1 , 287 µm.s -1 ], centered at v 0 = ∆ω/(2k) = 271 µm.s -1 (Eq. 4), the Doppler signal appeared to be less prone to motion artifacts. The Doppler signal at 1020 Hz recorded over ∼ 8 s, and com-

CONCLUSION

In conclusion, we demonstrated wide-field imaging and monitoring of pulsatile motion of the thumb of a healthy volunteer. Narrowband optical Doppler video imaging in real-time was performed with a off-axis and frequencyshifting holographic interferometer. Numerical image rendering was performed by discrete Fresnel transformation and two-phase temporal demodulation at a 60 Hz framerate. The measured contrast was linked to the instant velocity of out-of-plane motion, of the order of a few hundreds of microns per second. Robust non-contact motion monitoring was achieved. This was illustrated by an occlusion-reperfusion experiment, in which the proposed detection scheme was compared to plethysmography.

FIG. 1 :

 1 FIG.1: Sketch of the fibered Mach-Zehnder heterodyne holographic interferometer. The main laser beam is split into two channels. In the object channel, the optical field E is backscattered by the skin. In the reference channel, the optical field ELO is frequency-shifted by two Bragg cells by ∆ω. A standard camera records interferograms of the diffracted fields E and ELO.

FIG. 2 :

 2 FIG.2: Apparatus response (Eq. 2), calculated for τS = τE, versus apparent (beating) frequency index n = ω/ωS. The ticks of the horizontal axis correspond to the zeros of the response at integer values of the index n of the beating frequency nωS and the probed velocity vn = (∆ω + nωS)/(2k).

FIG. 3 :

 3 FIG.3: Doppler signal (a,c,e), averaged in the central part of the image of the thumb (Fig.4), and concurrent blood volume signal (b,d,f), versus time. Detuning frequencies : ∆ω/(2π) = 60 Hz (a), ∆ω/(2π) = 600 Hz (c), ∆ω/(2π) = 1020 Hz (e). The synchronization accuracy of holographic and standard blood volume measurements is the order of ±0.5 s. a.u.: arbitrary units.

FIG. 4 :

 4 FIG. 4: Optical Doppler acquired for a detuning frequency ∆ω/(2π) = 1020 Hz, averaged in the central part of the picture, versus time. Composite images of the corresponding signal at low (i) and high (ii) signal level. Red color indicates signal presence. The whole sequence is available in this video

FIG. 5 :

 5 FIG. 5: Time traces of indicators monitored during a hypoperfusion experiment. Cuff pressure (a), plethysmogram [raw data (b), 5 s time-average of the magnitude (c)], holographic laser Doppler signal recorded at 1020 Hz and averaged spatially in the nail area [raw data (d), 5 s time-average (e)]. A time-lapse sequence of the composite Doppler image of the thumb is reported in this video.

Acknowledgments

We gratefully acknowledge the contributions of Anna Kieblesz, Renaud Boistel, Aurore Bonnin, and Claude Boccara, and financial support from Agence Nationale de la Recherche (ANR-09-JCJC-0113, ANR-11-EMMA-046), Fondation Pierre-Gilles de Gennes (FPGG014), région Ile-de-France (C'Nano, AIMA), the Investments for the Future program (LabEx WIFI: ANR-10-LABX-24, ANR-10-IDEX-0001-02 PSL*), and European Research Council (ERC Synergy HELMHOLTZ).


El Hadji

Malik Camara

Franck James

Philippe Breuil

Christophe Pijolat

Danick Briand

Nicolaas F De Rooij

MEMS-based Porous Silicon Preconcentrators Filled with Carbopack-B for Explosives Detection

Keywords: Micro gas preconcentrators, Preconcentration, Porous silicon, Explosives detection, DNT, Carbopack B

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

Introduction

The integration of micro-preconcentrators within handheld detectors has become standard for balancing the negative impact of the miniaturization on the sensitivity and selectivity of conventional analytical methods. Indeed, the preconcentrator acts as a "concentration amplifier" by trapping in relatively long time (up to min) and releasing in few seconds the target compound and thus enriching its concentration. The optimization of the design of the micro-device and of the experimental parameters is of importance for maximizing the "amplifying effect" of the preconcentrator. In summary, a high adsorption flow rate, a low desorption flow rate, a relatively long adsorption time and a very short desorption time are required [START_REF] Camara | Pijolat A micro gas preconcentrator with improved performance for pollution monitoring and explosives detection[END_REF][START_REF] Camara | Rooij Micro gas preconcentrator in porous silicon filled with a carbon absorbent[END_REF]. Besides the optimization of the adsorption-desorption parameters, the performance of the preconcentrator relies also on an optimum choice of the adsorbent material. This choice depends on the type of application and the nature of the gases to be detected [START_REF] Serrano | Hybrid preconcentrator/focuser module for determinations of explosive marker compounds with a micro-scale gas chromatograph[END_REF][START_REF] Martin | Stepnowski Microfabricated vapor preconcentrator for portable ion mobility spectroscopy[END_REF]. In the case of explosives detection, we are dealing with compounds having very low vapor pressures and concomitantly high adhesion energies which require high desorption temperatures and thus large power consumption. A severe drawback of high desorption temperatures is also the unavoidable decomposition of the accumulated target compounds and the ensuing difficulties in the compound identification in the final detection step. Therefore, adequate adsorbent materials must possess high internal surface area and low bonding energy toward explosive compounds. With these requirements in mind, the combination Carbopack-PS seems to be a very promising couple substrate-adsorbent. Indeed, as we have previously reported, PS allows reducing the desorption temperature of the couple adsorbent/adsorbed compound [START_REF] Camara | Pijolat A micro gas preconcentrator with improved performance for pollution monitoring and explosives detection[END_REF]. As a structural material, PS can be easily inserted into silicon micro-fluidic devices directly during their processing. In previous communications, we reported the development of a µGP for pollution monitoring [START_REF] Camara | Pijolat A micro gas preconcentrator with improved performance for pollution monitoring and explosives detection[END_REF][START_REF] Camara | Rooij Micro gas preconcentrator in porous silicon filled with a carbon absorbent[END_REF]. Here, we address the detection of 2, 4-dinitrotoluene with a Carbopack-PS µGP. The couple Carbopack-PS eases the desorption of DNT compounds and increases the "amplifying effect" of the µGP by offering supplementary adsorption sites through the PS pores.

Implementation of PS µGPs

2.1. Design and fabrication Our µGPs are microchannels on a silicon chip with a square shape of 11 x 11 mm 2 . They consist in one or two interconnected channels with a depth of 150µm and a width of 500µm. The "mono serpentine" design (made with one channel) is suitable for adsorbents having low adsorption capacity for which a longer residence time is preferable, whereas the "biserpentine" is more adequate for adsorbents with high adsorption capacity for which a long residence time is not really necessary (Figure 1a). Microchannels are then sealed with a glass cap on which are fixed the metallic capillaries used as fluidic connection. Compared to our previous designs [START_REF] Camara | Pijolat A micro gas preconcentrator with improved performance for pollution monitoring and explosives detection[END_REF][START_REF] Camara | Rooij Micro gas preconcentrator in porous silicon filled with a carbon absorbent[END_REF], the interest of having the fluidic connections on the glass cover is that there is no limitation on the capillary size since inlet/outlet are not dipped into the silicon wafer (Figure 1b). Our micro-devices include also a platinum micro-heater patterned on the backside by clean-room process (Lift-off). a) b) Figure 1

: a) Picture of two silicon µGPs in mono and bi-serpentine designs b) 3D Image of our new design of µGPs in bi-serpentine design with connected capillaried onto the cover

Prior the sealing of the glass cover, the silicon pores were grown within the microchannels by anodic etching in a bath of HF (50%) and EtOH in a ratio of 1:1. The size, shape and depth of pores depend on several parameters such as the doping and the resistivity of the wafer, the applied current density and the etching time [START_REF] Camara | Pijolat A micro gas preconcentrator with improved performance for pollution monitoring and explosives detection[END_REF]. In this study, 525um thick n-type silicon wafers with resistivity of 5Ω.cm were used to generate macroporous silicon. To obtain a pore depth of about 100µm, the current density and the etching time were fixed at 80mA/cm 2 and 20min, respectively. Since the growth of the pores is conical, a few micrometers of silicon was etched by reactive ion etching (RIE) in order to open these pores (Figure 2).

Implementation

The implementation of the PS µGP consists in setting up the electric and fluidic connections and inserting the adsorbent material into microchannels. The electric connection of the patterned heater was made with gold wires and the heating element obtained with this process allows a fast heating rate of 60°C/s (Figure 3a). Two different methods were used for filling µGPs with Carbopack B: either by fluidic solution after the sealing of the glass cover or in a granular form before its sealing (Figure 3b).

a) b) c) Figure 3: Pictures of a) a Pt heater on the backside of our µGP b) a µGP in bi-serpentine design filled with Carbopack B and bonded with a glass cover and ) a µGP in bi-serpentine design with sealed capillaries at the glass cover.

Once the devices filled with Carbopack, the fluidic connections were made with metallic capillaries (ID = 500um) and fixed with a ceramic cement using a special procedure so as not to block connectors during the sealing (Fig. 3c). The combination of large capillaries and deep etched microchannels allows reaching flow rates up to 330mL/min.

Results and discussion

Our test bench is composed of a gas generator (Owlstone Nanotech) using permeation tubes, mass flow-meters and a climatic chamber containing the preconcentrator device and the detection system. The climatic chamber is maintained at 40°C to avoid any condensation between the µGP and the detector. The preconcentration experiments consist first in injecting through the µGP 200ppb of DNT during 5 minutes at a flow rate of 167mL/min. This step is followed by its desorption at 250°C with a heating rate of 60°C/s. The DNT compound is desorbed and brought into the detector at constant flow rate of 167 mL/min. The results obtained with our newly designed PS µGPs filled with Carbopack B have demonstrated a better desorption of the DNT compound in contrast to our previous µGPs tested in the same operating conditions (Figure 4). Furthermore, experiments realized with our µGPs have also shown a higher desorption peak with the combination Carbopack-PS in opposition to non-porous µGPs filled with the same amount of Carbopack and to PS µGPs free of adsorbent too (Figure 5).

In fine, PS substrate presents the main advantage of increasing the adsorption capacity of the µGP by offering supplementary adsorption sites through its pores and by acting as a binding surface for the Carbopack particles. It also eases the desorption of DNT from the Carbopack adsorbent by reducing its desorption temperature.

Conclusions and perspectives

A porous silicon micro gas preconcentrator filled with Carbopack B has been implemented and successfully tested for the detection of explosives. The significant impact of the couple Carbopack-Porous silicon on the preconcentration of explosives has also been demonstrated. We are currently focusing of the improvement of this µGP by using different PS substrates from micro to macro-PS and also by optimizing experimental parameters for shortening the detection duty cycle.

Figure 2 :

 2 SEM Image of a PS µGP: a) top view of the outlet channels and b) magnified view of the pores within channels.

Figure 4 :

 4 Figure 4: DNT desorption peak after an adsorption of 200ppb@5min in our old (left) and new (right) designs of PS µ-GP filled with Carbopack B with adsorption and desorption flow rates of 167mL/min.

Figure 5 :

 5 Figure 5: Comparison of different adsorbents (PS and Carbopack B separately and in combination) towards DNT: Desorption peaks obtained after an adsorption of 200ppb@5min with adsorption and desorption flow rates of 167mL/min.


F James

P Breuil

C Pijolat

M Camara

D Briand

A Bart

R Cozic

Development of a MEMS preconcentrator for micro-chromatography analyses

Keywords: Gas Preconcentration, Adsorption, Desorption, Detection, Micro Gas Chromatograph

This paper presents the development of MEMS gas preconcentrator (GP) for the detection of trace level Volatile Organic Compounds (VOCs). Besides the development of the MEMS gas preconcentrator, we have also worked on the coupling of this preconcentration device with a micro gas chromatograph (µ-GC) for achieving an accurate detection system for gas trace monitoring. Preliminary results have highlighted a significant contribution of the MEMS GP on the µ-GC efficiency and a detection limit below 35ppbv for different vapors like toluene, vinyl acetate monomer, chloroform and methylisobutylketone was reported.

Introduction

Process control presents a major challenge for the chemical industry to ensure product quality, cost control, maintaining productivity and risk control. The direct analysis within the heart of the process is the most effective way, but most of the gas analysis processes are hampered by the limit of detection. So in the field of analytical techniques, the preprocessing of the gases is generally a very important step, especially to get a preconcentration effect in order to increase the limit of detection. Moreover, one major trend is to miniaturize analytical techniques for in situ analysis. The development of µ-GC system has driven an important research effort. Portable gas chromatographs or micro-gas chromatographs (µ-GCs) provide an analytical tool that permits a quick analysis of volatile organic compounds (VOC) for online monitoring. Adding a preconcentration step should allow increasing the sensitivity of miniaturized analyzer [START_REF] Lewis | Frye-Manson Recent Advancements in the Gas-Phase MicroChemLab[END_REF]. Therefore we have worked on the integration of our MEMS gas preconcentrator into a µ-GC module already developed and commercialized by SRA Instruments Company. The design of the MEMS gas preconcentrator and the experimental parameters were optimize in regards to the future application. Experiments conducted with preconcentrator filled carbon nanopowder and associated to a micro chromatograph have shown an accurate separation and identification of various VOCs in trace within a gas mixture.

Preconcentrator's design and adsorbent material

The main part of the device used in this study is a silicon microchannel (20x85 mm 2 , thickness 500µm) obtained by deep reactive ion etching (DRIE) and designed with fluidic micro-structures at the inlet/outlet leading to a preconcentration chamber with a volume of 14 µl. The microchannels were produced on a 5 Ω•cm, n-type, (100)-oriented 1mm µm-thick silicon wafers. The first step of the fabrication was the DRIE with a thick photoresist used as an etch mask. Then the micro-channels were bonded with a Pyrex ® glass wafer by anodic bonding. Finally capillaries were sealed at the inlet/outlet with ceramic cement and a glass paste. The inlet/outlet has a depth of 500 µm, corresponding to the diameter of the capillaries used as fluidics interconnections [START_REF] Pijolat | Rooij Application of carbon nano-powders for a gas micro-preconcentrator[END_REF]. The preconcentrator allows desorbing at relatively high temperature (230°C) all the molecules collected after an adsorption step made at ambient temperature thanks to an adsorbent which is described below. On the backside of the silicon device, a platinum resistance was screen printed to act as a heater with a heating rate of 40°C/s (Figure 1). In this study, the device was designed [START_REF] Camara | Pijolat A micro gas preconcentrator with improved performance for pollution monitoring and explosives detection[END_REF] to ease the introduction of a carbon nanopowder (activated charcoal from Aldrich 95m 2 /g) used as adsorbent (Figure 2).

Tests on laboratory bench

First of all, the preconcentrator was tested with a laboratory setup. A micro-PID was used as a gas analyzer. Toluene has been chosen as a representative of Volatiles Organic Compound. The carrier gas was air. Calibrated concentrations of this vapor were generated by using a permeation tube and a gas generator. Results reported (Figure 3) show the response of the PID analyzer after an adsorption of 1 ppm toluene during 5 min at 10L/h. When the temperature is increased up to 230°C a desorption peak of toluene is clearly observable. The results have shown a preconcentration factor of 35 (ratio of outlet to inlet concentration). The preconditioning effect of the device filled with carbon powder is therefore demonstrated since no desorption peak has been observed when this device has no carbon powder inside under the same conditions.

Coupling between a preconcentrator and a micro-chromatograph

Experiments were conducted with a preconcentrator associated to a micro gas chromatograph (µGC) in collaboration with SRA Instruments Company (Figure 5). The capillary column used was an OV1 type, 12 m long and has allowed the mixture separation of compounds in less than 5 minutes. Considering the removal and purging steps, the complete analysis cycle does not exceed 20 minutes for this application. First, we have studied a mixture of toluene, vinyl acetate monomer, chloroform and méthylisobutylketone (MIBK) with initial concentrations range of 10 ppmv, and have obtained preconcentration factor (ratio of peak areas with and without preconcentration step at the same experimental conditions) of 600 to 800 (Figure 4). This concept of miniaturized preconcentrator has allowed the detection of VOCs in concentration range of 35 ppbv while maintaining the speed of the µ-GC analysis. This experiment has also demonstrated significant preconcentration factors (up to 200) with a maximum about 800) (Table 1).

Conclusion

These results have shown that with the micro-preconcentration device we can increase the detection limits of the coupling micro gas chromatograph for different vapors. The capacity of the carbon nanopowder to adsorb a mixture of different VOCs has been proved. This study has allowed developing a preconcentration step for better VOC analysis with these experimental conditions. However studies of the adsorption temperature influence and of the humidity should be performed to determine the effectiveness of this process for outdoor air or a specific atmosphere. Moreover, another challenge is to realize the same approach for explosive analysis which required more technology. Indeed explosive compounds are mainly characterized by a low vapor pressure and a strong bond on solid matters which makes their detection very difficult.

Figure 1 :

 1 Figure 1: Design and schematic device composition; a) platinum heater, b) MEMS, c) glass cover, d) capillaries

Figure 2 :

 2 Figure 2: a) SEM Image of carbon nanopowder b) Filled preconcentration device in a box

Figure 3 :

 3 Figure 3: Sensor response for a preconcentrator filled with 1 mg of activated carbon nanopowder when exposed to 1 ppm of toluene during 5 minutes and desorbed at 230°C at 10L/h

Figure 5 :

 5 Figure 5: ppb range analysis of VOCs mixture with interference by micro-chromatography

Table 1 .

 1 Preconcentration factors for different VOCs.

	Target Gas	Concentration (ppb)	Preconcentration factors
	Toluene	37	200
	Vinyl acetate monomer	39	800
	Chloroform	46	600
	Méthylisobutylketone	35	200


Riad Mazloum

Jordan Augé

Dario Rossi

Timur Friedman

Errors Announcing 32-bit ASNs in BGP Routes

teaching and research institutions in France or abroad, or from public or private research centers.

I. INTRODUCTION

BGP (Border Gateway Protocol) is the globally implemented inter-domain routing protocol. It glues together the thousands of networks (autonomous systems or ASes) that form the Internet. An AS learns how to route its traffic toward destinations in other ASes through BGP. Some BGP routers publicly share their routing tables and route updates. Projects such as Route Views [1] and RIPE-RIS [START_REF]RIPE-RIS[END_REF] collect this information and make it available to the community. BGP feeds are the main source of data for most scientific research on inter-domain routing and AS relationships. One of the attributes carried by BGP routes is the AS Path, which describes the sequence of ASes to be traversed toward the destination prefix.

There are some filtering and sanitizing steps on BGP feeds that the scientific community performs before using the datasets, such as considering only routes that seem to be stable ( [START_REF] Dimitropoulos | AS relationships: inference and validation[END_REF], [START_REF] Luckie | AS relationships, customer cones, and validation[END_REF] for instance), or by removing routes that contain loops [START_REF] Luckie | AS relationships, customer cones, and validation[END_REF]. Recent research on spurious BGP routes states that one of the public BGP routers has announced hundreds of thousands of BGP routes that do not exist in reality [START_REF] Luckie | Spurious routes in public bgp data[END_REF].

We point in this work at another source of bias that to our knowledge has not been reported previously in the scientific literature, where we identify BGP routes that carry malformed AS Paths because of the employment of AS23456 (also called AS_TRANS) which is used solve the transition from 16-bit ASNs to 32-bits ASNs. We show how this phenomenon highly impacts our observations of what we call multi-exit routing. Multi-exit routing happens when an AS uses multiple nexthop ASes toward a given destination prefix. Figure 1 illustrates the general case when there are two next-hops: routers in X announce two different routes ρ and ρ ′ toward a destination prefix p, each route having a different next-hop AS, Y and Y ′ respectively. We define and then quantify multi-exit routing in [START_REF] Mazloum | Violation of interdomain routing assumptions[END_REF], where we also show that ASes that do such routing seem to violate some commonly used routing assumptions. We applied in this work our methodology to snapshots over ten years to Collaboration through the LINCS laboratory. Full institutional affiliation of UPMC Sorbonne Universités authors: Sorbonne Universités, UPMC Univ Paris 06, UMR 7606, LIP6, F-75005, Paris, France. quantify ASes that are doing multi-exit routing, which allowed us to discover yet another source of artifacts in the BGP feeds that is caused by the employment of AS_TRANS. We show how it highly impacts quantification of multi-exit routing where the number reaches its peak in 2010 with a more than doubled number of multi-exits caused by spurious instances. We also explain how this phenomenon affects research making use of BGP feeds.

II. TRANSITION TO 32-BIT ASNS

Each AS is identified by a unique AS Number, or ASN. ASNs used to be 16-bits numbers. Then, as the number of ASes has increased, this numbering is no longer enough to identify all of the ASes. In 2007, the move was made to 32-bit ASNs (RFC 4893 [START_REF] Vohra | BGP support for four-octet Autonomous System (AS) number space[END_REF]). In this representation, all ASNs are encoded on 32-bits, even those that are 16-bit numbers. However, not all BGP routers can handle AS Paths when they contain 32-bit ASNs. RFC 4893 introduces AS23456 (also called AS_TRANS) to provide a solution that assures compatibility between old BGP routers and AS Paths containing 32-bit ASNs. Whenever a new BGP router forwards routes to an old one it rewrites the AS Path using only 16-bit numbers. ASNs that can be represented on 16-bits are directly written as a 16-bit number, but all those that cannot are replaced by the number 23456 at each occurrence, which is a 16-bit number. In order to preserve the original AS Path that contains the real ASNs, the router writes it down into a new BGP attribute, which is the AS4-PATH. AS4-PATH is defined as an optional, transit BGP attribute, which means that when a router does not recognize it, it forwards it to its neighbors as it is, guarantying that it will not be lost. The old BGP router treats the AS Path as any normal one and may forward the route to its neighbors. Whenever this route reaches a new BGP router, this router will extract the original AS Path from the AS4-PATH attribute, rewrites the AS Path in 32-bit encoding and replaces each occurrence of 23456 with the corresponding 32-bit ASN.

III. AS_TRANS ARTIFACTS

If the method described earlier is implemented correctly then any new BGP router should be reporting AS Paths that do not contain AS234561 . What we have found is that some of the routers that provide public BGP feeds clearly understand 32-bit ASNs as they report paths that contain such ASNs, however some of those same routes carry also AS234562 . Then we looked at AS Paths of different routes toward the same destination. In some cases, one of the AS Paths has an extra AS hop compared to the other, which is AS23456, located between the AS that originated the route (which has a 32-bit ASN) and the AS after it. In other cases, we saw two AS Paths, one having AS23456 as the origin AS followed by an AS (which also has a 32-bit ASN), and one having this AS as the origin AS followed by AS23456. Both of the previous cases make us think that there is a misconfiguration somewhere in the AS that originated the route. In consequence, we may observe false multi-exits where there are two next-hops, one is the real AS, as seen from a BGP route, and the other is AS23456, as seen from another BGP route.

We considered BGP routes from Route Views and RIPE-RIS, the two commonly used public BGP data sources. A snapshot at a given instant is obtained by extracting all of the BGP routes that were used at that instant. We took snapshots on the first of August of each year from 2005 to 2014.

The total height of the bars in Figure 2 represent the number of ASes doing multi-exit routing that are observed after applying the method in [START_REF] Mazloum | Violation of interdomain routing assumptions[END_REF] on the ten snapshots. The first remark on this figure is the spike in number of ASes in 2010. After investigation, we found first that almost all ASes are observed only in the 2010 snapshot. Then we remarked that most of them seem to be small ASes that have a couple of prefixes. Those ASes are distributed all around the world (we looked at the countries where they exist) and they are the origin of the routes that form the multi-exits (those routes concern their own prefixes). At last, we found that those cases are formed by two next-hop ASes, one of them is always AS23456 and the other one is the same AS that comes right after 23456 in the first AS Path, which means that they are false multi-exits.

We remove all routes that contain AS23456 in their AS Path from the dataset, then we do again the quantification of multi-exit ASes (which is represented by hashed bars in Figure 2). We can see, looking at white bars, that there were no observations related to AS23456 before 2007, then increase reaching a peak in 2010, and fall after that. We recall ASN assignment policy to understand what happened. If an network wants to become an autonomous system it has to do a request to obtain an ASN. The ASN assignment policies3 state that 32bit ASNs were available since 1st January 2007 upon request, which explains that no observations were before this year. Then from 1st January 2009, 32-bit ASNs were assigned by default (except for networks that ask for a 16-bit ASN). After that, since 1st January 2010, there were no distinction and ASNs were assigned from 32-bit pool. The peak in 2010 can be explained that those ASes were new ASes that were assigned 32-bit ASNs, and perhaps, since the AS is a new AS, some misconfiguration can appear, but do not last long time as they are solved. The number of ASes that are observed to do multi-exit routing in 2010 decreases to less that the half after removing routes containing AS23456. Interestingly, there are still false multi-exits in 2014 indicating that there are still misconfigured BGP routers. For the first look, such artifact seem to affect only multi-exit quantifications, but a second look shows that these routes can affect research where the inexistent link between an AS and AS23456 can be seen as an alternative route. Such data is employed by research on identifying the origin of routing events for instance, or on inferring alternative routes between two ASes in cases of network changes (we omit citing examples of such research due to lake of space), which means that indeed research making use of BGP feeds should be aware of those routes.

IV. CONCLUSION

Fig. 1 .

 1 Fig. 1. Multi-exit routing general case

Fig. 2 .

 2 Fig. 2. Fraction of ASes falsely observed to do multi-exit routing

Unless the route has been aggregated.

To see how frequent this ASN is observed, we refer the reader to Hurricane Electric website (http://bgp.he.net/AS23456). We can see in this web page a list of hundreds of prefixes that seem to be originated by this AS and a list of ASes that seem to be interconnected with it.

We refer the reader to http://www.ripe.net/ripe/docs/ripe-525 and http:// www.apnic.net/policy/asn-policy#6.3 for more details.

We described in this paper one of the artifacts of 32-bit representation of AS Numbers that can be observed in public BGP feeds. Through observations of multi-exit routing, we show how greatly it biases quantification results (53% of the observations in 2010 are false). Finally, we explain how those routes can bias also research making use of BGP feeds.


Jin Hong Kim

Philippe Gripon

Mun Fidaa Bouezzedine

Sik Jeong

Seung-Wook Chi

Seong-Eon Ryu

Hyo Jeong Hong
email: hjhong@kangwon.ac.kr

Mun Sik Jeong

Enhanced humanization and affinity maturation of neutralizing anti-hepatitis B virus preS1 antibody based on antigen-antibody complex structure

Keywords: humanized antibody, specificity-determining residue grafting, affinity maturation, HBV, preS1, virus neutralization

come   L'archive ouverte pluridisciplinaire

Introduction

Over the last three decades, monoclonal antibodies (mAbs) have emerged as powerful human therapeutics due to advances in antibody engineering technologies [START_REF] Buss | Monoclonal antibody therapeutics: history and future[END_REF]. Nonhuman antibodies induce an immune response in humans, which limits their therapeutic use [START_REF] Shawler | Human immune response to multiple injections of murine monoclonal IgG[END_REF]. To overcome this problem, humanized antibodies have been constructed by grafting the complementarity-determining regions (CDRs) of murine mAbs onto human framework regions (FRs), which is commonly referred to as CDR grafting [START_REF] Jones | Replacing the complementarity-determining regions in a human antibody with those from a mouse[END_REF]. Simple grafting of CDRs, however, often decreases affinity, because some FR residues directly contact the antigen or support conformation of the CDR loops [START_REF] Queen | A humanized antibody that binds to the interleukin 2 receptor[END_REF][START_REF] Al-Lazikani | Standard conformations for the canonical structures of immunoglobulins[END_REF]. Therefore, humanized antibodies are usually constructed by CDR grafting while retaining those mouse FR residues that influence antigenbinding activity [START_REF] Winter | Humanized antibodies[END_REF][START_REF] Safdari | Antibody humanization methods -a review and update[END_REF]. These humanized antibodies are generally less immunogenic in patients than chimeric antibodies that are composed of mouse variable domains and human constant domains; however, they may still be immunogenic since the CDRs are not human [START_REF] Hwang | Immunogenicity of engineered antibodies[END_REF][START_REF] Harding | The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions[END_REF]. Therefore, to minimize the immunogenicity of humanized antibodies, only the mouse CDR residues that influence antigen binding (i.e., specificity-determining residues, or SDRs) are retained, while those that are not involved in antigen binding are replaced by human counterparts [START_REF] Padlan | Anatomy of the antibody molecule[END_REF][START_REF] Gonzales | SDR grafting of a murine antibody using multiple human germline templates to minimize its immunogenicity[END_REF][START_REF] Yoon | Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody[END_REF][START_REF] Kim | Construction of a humanized antibody to hepatitis B surface antigen by specificity-determining residues (SDR)-grafting and de-immunization[END_REF]. SDR-grafted humanized antibodies exhibit less immunogenic potential than CDR-grafted humanized antibodies.

HBV is a worldwide public health problem that affects 240 million chronic carriers, who have a high risk of developing hepatocellular carcinoma [START_REF] Ganem | Hepatitis B virus infection--natural history and clinical consequences[END_REF]. The HBV envelope contains three related surface glycoproteins called the large (L), middle (M), and small (S) proteins.

These proteins are the product of a single open reading frame that is divided into preS1, preS2, and S regions [START_REF] Heermann | Large surface proteins of hepatitis B virus containing the pres sequence[END_REF]. The S protein is encoded by the S region; the M protein contains the preS2 and S antigens; and the L protein contains the preS1, preS2, and S antigens. The three antigens have been shown to elicit virus-neutralizing antibodies [START_REF] Dreesman | Antibody to hepatitis B surface antigen after a single inoculation of uncoupled synthetic HBsAg peptides[END_REF][START_REF] Heermann | Immunogenicity of the gene S and Pre-S domains in hepatitis B virions and HBsAg filaments[END_REF][START_REF] Itoh | A synthetic peptide vaccine involving the product of the pre-S(2) region of hepatitis B virus DNA: protective efficacy in chimpanzees[END_REF][START_REF] Klinkert | Pre-S1 antigens and antibodies early in the course of acute hepatitis B virus infection[END_REF][START_REF] Neurath | Antibodies to synthetic peptides from the preS1 region of the hepatitis B virus (HBV) envelope (env) protein are virus-neutralizing and protective[END_REF]. In particular, the preS1(amino acids 2-48, ayw subtype) of the L protein and the common "a" determinant of the S antigen have been shown to play an essential role in viral infectivity [START_REF] Neurath | Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus[END_REF][START_REF] Pontisso | Identification of an attachment site for human liver plasma membranes on hepatitis B virus particles[END_REF][START_REF] Seyec | Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain[END_REF][START_REF] Gripon | Infection of a human hepatoma cell line by hepatitis B virus[END_REF][START_REF] Glebe | Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus[END_REF][START_REF] Gripon | Efficient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein[END_REF][START_REF] Salisse | A function essential to viral entry underlies the hepatitis B virus "a" determinant[END_REF].

Recently a cellular receptor for the pre-S1 sequence was identified as sodium taurocholate transporter (NTCP) [START_REF] Yan | Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus[END_REF], while heparin sulfate proteoglycan (HSPG) was recognized as a binding partner for the "a" determinant [START_REF] Sureau | A conformational heparan sulfate binding site essential to infectivity overlaps with the conserved hepatitis B virus a-determinant[END_REF]. For immunoprophylaxis of HBV infection, hepatitis B immune globulin (HBIG) prepared from pooled human anti-HBsAg plasma is administered to infants born of HBsAg-HBeAg-positive mothers, to susceptible individuals with acute exposure to infectious HBV-containing material, and to orthotopic liver transplant patients with chronic HBV-related liver disease [START_REF] Beasley | Prevention of perinatally transmitted hepatitis B virus infections with hepatitis B immune globulin and hepatitis B vaccine[END_REF][START_REF] Mcgory | Improved outcome of orthotopic liver transplantation for chronic hepatitis B cirrhosis with aggressive passive immunization[END_REF][START_REF] Terrault | Prophylaxis in liver transplant recipients using a fixed dosing schedule of hepatitis B immunoglobulin[END_REF]. However, the currently available HBIG is not an ideal source of antibody due to its limited availability and low specific activity. Virus-neutralizing mAbs against the preS1 and S antigens would be an effective alternative for immunoprophylaxis of HBV infection.

We previously generated a murine mAb (KR127) that recognizes amino acids 37-45 (NSNNPDWDF) of the preS1 of adr subtype, corresponding to aa 26-34 of ayw subtype, and subsequently constructed a humanized antibody (HzKR127) by CDR grafting. HzKR127 retained the same epitope specificity and affinity as KR127 and exhibited HBV-neutralizing activity in chimpanzees [START_REF] Ryu | Mouse monoclonal antibodies to hepatitis B virus preS1 produced after immunization with recombinant preS1 peptide[END_REF][START_REF] Maeng | Fine mapping of virus-neutralizing epitopes on hepatitis B virus PreS1[END_REF][START_REF] Hong | In vivo neutralization of hepatitis B virus infection by an anti-preS1 humanized antibody in chimpanzees[END_REF]. In addition, we determined the crystal structure of the HzKR127 antigen-binding fragment (Fab) and its complex with the epitope to understand the structural mechanisms of the antigen-antibody interaction [START_REF] Chi | Broadly neutralizing anti-hepatitis B virus antibody reveals a complementarity determining region H3 lid-opening mechanism[END_REF].

In the present study, based on the structural information, we further humanized HzKR127 by SDR grafting and also improved its affinity by mutating two heavy-chain CDR residues.

The resulting humanized antibody (HzKR127-3.2) exhibited higher HBV-neutralizing activity than KR127 and lower immunogenic potential than HzKR127. The enhanced virusneutralizing activity was achieved by an increase in the association rate. This information will be useful for the design and development of potent antibody therapeutics against HBV, and this advanced humanized antibody may be a good candidate for immunoprophylaxis of HBV infection.

Materials and methods

Cell culture

HEK293T, ACHN, B16F1, and SCK-L1 [START_REF] Min | L1 cell adhesion molecule is a novel therapeutic target in intrahepatic cholangiocarcinoma[END_REF] cells were grown in DMEM (Invitrogen) supplemented with 10% FBS. CHO-DG44 cells were grown in DMEM/F12 (Invitrogen) with supplements as described previously [START_REF] Hong | In vivo neutralization of hepatitis B virus infection by an anti-preS1 humanized antibody in chimpanzees[END_REF]. HepaRG cells were cultured and differentiated as previously described [START_REF] Gripon | Infection of a human hepatoma cell line by hepatitis B virus[END_REF] and maintained after seeding into 96-well plates in William's E medium (Gibco Life technologies) supplemented with 5% fetal calf serum, 2% DMSO, 5 mg/L insulin, 5 × 10 -6 M hydrocortisone, 5 µg/L sodium selenite, 20,000 UI/L penicillin, and 20 mg/L streptomycin. All cells were cultured in 5% CO 2 in a 37°C humidified incubator.

Construction, expression, and purification of humanized antibodies

Humanized VH and VL genes were synthesized by GeneArt (Germany) and sequentially subcloned into the EcoRI-ApaI and HindIII-BsiWI sites, respectively, of pdCMV-dhfrC-cA10A3 containing human C 1 and C . The resulting expression plasmids were introduced into HEK293T cells using Lipofectamine (Invitrogen). The culture supernatant was subjected to affinity chromatography on a protein A-sepharose column (Millipore), and the protein concentration was determined with a NanoDrop (Thermo Scientific) based on the molar extinction coefficient. The integrity of the purified protein was analyzed by SDS-PAGE.

Affinity determination

Competition ELISA was performed as described previously [START_REF] Oh | A new epitope tag from hepatitis B virus preS1 for immunodetection, localization and affinity purification of recombinant proteins[END_REF]. Briefly, a solution containing 5-10 ng of each antibody and various concentrations (10 -10 -10 -6 M) of GST-preS1 (amino acids 1-56) as a competing antigen were preincubated at 37°C for 3 h. The mixtures were then added to each well, which had been coated with 100 ng of GST-preS1.

Bound antibody was detected by indirect ELISA.

For affinity determination by Octet Red, anti-human Fc-coated biosensor tips (ForteBio, 18-0015) were activated in PBS with 0.1% w/v bovine serum albumin (0.1% PBA) for 20 min by agitating 96-well microtiter plates (Greiner bio-one, 655209) at 1000 rpm and then saturated with 2 g/mL antibody for 10 min. This typically resulted in capture levels of 1 nm.

GST-preS1 was prepared as a 2-fold serial dilution (6.25, 12.5, 25, 50, and 100 nM) in 0.1% PBA and separately incubated with the antibody bound on the tips. Association and dissociation rates were measured for 15 and 30 min, respectively. All measurements were corrected for baseline drift by subtracting a control sensor exposed to running buffer only.

Operating temperature was maintained at 30°C. Data were analyzed using a 1:1 interaction model (fitting global, Rmax unlinked by sensor) with ForteBio data analysis software 7.0.

Western blot analysis

GST-preS1 (amino acids 1-56) and each of the alanine replacement mutants of the preS1 (amino acids 37-47) were expressed in E. coli DH5 cells as described previously [START_REF] Oh | A new epitope tag from hepatitis B virus preS1 for immunodetection, localization and affinity purification of recombinant proteins[END_REF].

Protein extracts were subjected to 12% SDS-PAGE and western blot analysis with KR127 or humanized KR127 antibody (1 g/ml), followed by anti-mouse or human IgG (Fc-specific)-HRP conjugate (1:5000 v/v, Thermo Scientific).

Flow cytometric analysis

Cells were incubated with 1 g of antibody in 100 l of PBA for 60 min at 4°C. After washing three times with PBA, the cells were incubated with a fluorescein isothiocyanate-conjugated anti-hFc antibody (BD Pharmingen) for 30 min at 4°C. Propidium iodidenegative cells were analyzed for antibody binding using FACSCalibur (Becton Dickinson).

In vitro HBV infection and neutralization assays

In vitro HBV infection and neutralization assays were carried out using differentiated HepaRG cells and adr or ayw subtype of HBV particles. The ayw viral particles were produced by transient transfection of HepG2 cells [START_REF] Sells | Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA[END_REF] with plasmid (pHBVEcoR1-) containing an overlength HBV genome originally described in Gripon et al. [START_REF] Gripon | Myristylation of the hepatitis B virus large surface protein is essential for viral infectivity[END_REF], and supernatants from the transfected cells were concentrated fifty-fold by PEG precipitation as previously described [START_REF] Seyec | Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain[END_REF]. The genomic sequence of the ayw subtype corresponds to the sequence (genebank accession number V01460.1) published by Galibert et al. [START_REF] Galibert | Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli[END_REF], corresponding to genotype D by online genotyping analysis [START_REF] Hayer | HBVdb: a knowledge database for Hepatitis B Virus[END_REF]. The adr particles were produced and concentrated by the same procedures using the pHBV 5.2 [START_REF] Ryu | In vitro neutralization of hepatitis B virus by monoclonal antibodies against the viral surface antigen[END_REF] containing the preS1 sequence [START_REF] Kim | Efficient expression, purification and characterization of hepatitis B virus preS1 from Escherichia coli[END_REF] that was identified as genotype C by the online genotyping analysis.

For in vitro HBV infection, HepaRG cells were seeded at a density of 6 × 10 4 cells per well (containing 100 µl of culture medium) and were infected 6 days later with the adr or ayw viral particles (about 6 x 10 6 viral genomic equivalent). HBV infection was also performed in the presence of 4% PEG 8000 and a five-fold diluted inoculum. PEG was added during the infection process, which allows a great enhancement of viral infectivity without compromising the specificity of the infection process as previously described [START_REF] Gripon | Reproducible high level infection of cultured adult human hepatocytes by hepatitis B virus: effect of polyethylene glycol on adsorption and penetration[END_REF]. To evaluate binding of antibody to the viral particles present in the HBV inocula, ELISA plates were coated with either KR127 or HzKR127-3.2 (1 µg/ml) in a carbonate buffer overnight at room temperature. After saturation with 5% fetal calf serum for 2 h at room temperature, serially diluted HBV inocula were added and incubated for 4 h at room temperature. The bound viral particles were then detected with an anti-HBs antibody conjugated with peroxidase from a HBs detection kit (Bio-Rad).

For the neutralization assay, the adr or ayw viral particles (5 µl) were preincubated with 5 µl of antibody at the indicated concentrations at room temperature for 30 min and then incubated with cultured HepaRG cells covered with 100 µl of the culture medium for 24 hours in the absence or presence of 4% PEG 8000. The infected cells were washed with the medium and further incubated for 10 days, with the medium changed every 2 days. On day range of the assay and the HBsAg concentration was determined with an ELISA kit (Bio-Rad).

Results

Construction of humanized antibody HzKR127-3 by SDR grafting

We previously constructed an anti-preS1 humanized antibody (HzKR127) from the murine mAb KR127 by CDR grafting. In HzKR127, the CDRs of mouse VH and VL were grafted into human germline segments DP7-JH4 and DPK12-JK4, respectively, while eleven FR residues in the mouse VH and five FR residues in the mouse VL that were thought to influence the CDR loops or to stabilize the structure of the variable regions were retained [START_REF] Hong | In vivo neutralization of hepatitis B virus infection by an anti-preS1 humanized antibody in chimpanzees[END_REF]. The amino acid sequences of the VH and VL of KR127 and HzKR127 are shown in Figure 1. In this study, we further humanized HzKR127 by SDR grafting and increased its affinity by site-directed mutagenesis.

For SDR grafting, mouse residues that are buried and/or not involved in antigen binding are replaced by human counterparts, based on the previously determined three-dimensional structure of the antigen-antibody complex [START_REF] Chi | Broadly neutralizing anti-hepatitis B virus antibody reveals a complementarity determining region H3 lid-opening mechanism[END_REF].For the VH, nine mouse FR residues (Val12, Ala28, Ser30, Ile48, Lys66, Ala67, Leu69, Ala78, and Phe91) were replaced by human residues, while two mouse FR3 residues (Ala71 and Lys73) that are involved in the interaction with HCDR2 loop were retained. Ala71 is in hydrophobic interaction with Ile51 and Pro53, and Lys 73 is in hydrogen bonding with Gly54 (Fig. 2A). For the VL, three mouse FR residues (Leu3, Ser43, and Lys45) were replaced by human residues, while two mouse FR2 residues (Leu36 and Arg46) were retained. Leu36 forms a tight interaction with Trp103 of VH in the bottom of the antigen-binding pocket and is close to the Glu98 side chain of the HCDR3 loop (Fig. 2B). Thus, mutation of Leu36 to Tyr is likely to disturb the conformation of the antigen-binding pocket and prohibit optimal binding to the antigen. Residue Arg46 forms a salt bridge with Asp97 in HCDR3, which stabilizes the conformation of HCDR3 in the free structure (Fig. 3A). In addition to the FR residues, two LCDR2 residues (Lys53 and Leu54) were replaced by human residues because they are not involved in antigen binding (Fig. 2C).

The designed humanized VH and VL sequences were synthesized and combined with human C 1 and C , respectively, to construct expression plasmid pdCMV-dhfrC-HzKR127-3. This plasmid DNA was introduced into HEK293T cells, and the resulting humanized antibody (HzKR127-3) was purified from the culture supernatant. Affinity determination by competition ELISA indicated that the affinity of HzKR127-3 for GST-preS1 was slightly lower than that of the original KR127 antibody (Supplemental Fig. S1). Affinity determination by Octet Red indicated that HzKR127-3 had approximately 3-fold lower affinity (K D ) compared to KR127; this decrease was due mostly to an increase in the dissociation rate (Supplemental Fig. S2A andS2B and Table 1).

Affinity maturation of HzKR127-3

In our previous structural study, a comparison of the structures of free HzKR127 Fab and its complex with the preS1 epitope revealed that the HCDR3 loop undergoes the most noticeable conformational change between the free and complex structures, illustrating a lidopening mechanism for antigen binding [START_REF] Chi | Broadly neutralizing anti-hepatitis B virus antibody reveals a complementarity determining region H3 lid-opening mechanism[END_REF]. As shown in Figure 3A, the Asp97 residue is not involved in a direct interaction with the preS1 antigen; rather, it forms a salt bridge with Arg46 in the light chain FR2, which stabilizes closing of the HCDR3 lid in the free structure.

However, upon antigen binding, the HCDR3 loop lid is opened and an antigen-binding pocket is thereby created, leading to tight locking in of the peptide (Fig. 3B). Residue Asp97 is located at the apex of the HCDR3 lid and undergoes the largest movement, with a 9.6 Å shift in the main chain upon lid opening (Supplemental Fig. S3). Alanine mutation of the Asp97 would disrupt the salt bridge, making the HCDR3 loop more flexible. This could facilitate the structural transition from the closed to the open state, thus leading to an increase in affinity. We therefore substituted an alanine for Asp97 of HzKR127-3. The resulting antibody (HzKR127-3.1) was transiently expressed in HEK293T cells, purified, and subjected to affinity determination by competition ELISA followed by Octet Red. The affinity of HzKR127-3.1 (8.13 × 10 -10 M) was 4.9-fold higher than that of HzKR127-3 (3.97 × 10 -9 M), and the increase in affinity was achieved mostly by a decrease in the dissociation rate (Supplemental Fig. S1 andS2C and Table 1).

In an attempt to further increase the affinity of HzKR127-3.1, Asn58 in HCDR2 of HzKR127-3.1 was mutated to alanine and subjected to competition ELISA, which showed an increase in affinity (data not shown). This indicates that Asn58 is not optimal for antigen binding. We speculated that the replacement of Asn58 by alanine, which has a smaller side chain, would give the preS1 epitope easier access to the antigen-binding pocket, which could result in a faster association rate (Fig. 2D). Therefore, Asn58 was replaced by the human counterpart (Ser58) as a first choice. The resulting humanized antibody (HzKR127-3.2) was transiently expressed, purified, and subjected to affinity determination. The affinity of HzKR127-3.2 (4.93 × 10 -10 M) was 1.6-fold higher than that of HzKR127-3.1, and the increased affinity was achieved mostly by an increase in the association rate (Supplemental Fig. S1 and S2D and Table 1).

Analysis of potential immunogenicity of HzKR127-3.2

The VH and VL sequences of HzKR127 and HzKR127-3.2 were analyzed with ProPred [START_REF] Singh | ProPred: prediction of HLA-DR binding sites[END_REF] to identify potential T cell epitopes that bind to MHC II molecules (HLA-DR). This is based on the fact that the peptide-MHC II complexes are recognized by helper T cells and trigger the activation and differentiation of helper T cells to stimulate the immune response.

As summarized in Table 2, potential T cell epitopes detected at the junction between HCDR2 and FR3 (HCDR2/FR3) or in the light chain FR2 of HzKR127 were eliminated in HzKR127-3.2, while those detected at the junction between light chain FR2 and LCDR2 (FR2/LCDR2) were greatly reduced.

Specificity of HzKR127-3.2

To confirm the antigen-binding specificity of HzKR127-3.2, a series of GST-preS1 (amino acids 1-56) proteins that individually carry an alanine replacement mutation at each position in the preS1 epitope were expressed in E. coli and subjected to western blot analysis with KR127 or HzKR127-3.2. As shown in Figure 4A, HzKR127-3.2 exhibited the same epitope specificity as that of KR127.

In addition, to determine whether HzKR127-3.2 does not exhibit off-target activity, flow cytometric analysis was performed. To prepare preS1-expressing cells (293T-S1-L1) as a positive control, preS1 (amino acids 37-47) was fused to the N-terminus of the integral membrane protein L1 cell adhesion molecule (L1CAM). The fusion protein was then transiently expressed in HEK293T cells. HzKR127-3.2 bound to the surface of the 293T-S1-L1 cells but did not bind to preS1-negative cells such as HEK293T, L1CAM-overexpressing human cholangiocarcinoma (SCK-L1), human renal carcinoma (ACHN), mouse melanoma (B16F1), and Chinese hamster ovary (CHO) cells (Fig. 4B).

Evaluation of HBV-neutralizing activity of HzKR127-3.2

Because HzKR127-3.2 exhibited higher affinity for the preS1 antigen than KR127, we compared their virus-neutralizing activities by in vitro HBV-neutralization assay. To begin with, we confirmed binding of the antibodies to the viral particles. As shown in Figure 5A, the antibodies bound to the ayw and adr particles in a dose-dependent manner, and HzKR127-3.2 bound more efficiently compared to KR127. Next, HepaRG cells were infected with either the adr or ayw subtype of HBV particles, which had been preincubated with different concentrations (0.2-200 g/ml) of each antibody. The infected cells were then cultured for 10 days, and the growth medium was changed every 2 days. On day 10 postinfection, HBsAg secretion by the infected cells was measured by ELISA. As shown in Figure 5B and 5C, the HBsAg secretion rate gradually decreased with increasing antibody concentration, demonstrating the specificity of the neutralization assay, and HzKR127-3.2 exhibited enhanced virus-neutralizing activity against both adr and ayw subtypes compared to KR127.

We also performed the HBV infection and neutralization assay in the presence of 4% PEG 8000. The presence of PEG increases the infectivity and thus allowed to use five-fold diluted inoculum without compromising infectivity [START_REF] Gripon | Reproducible high level infection of cultured adult human hepatocytes by hepatitis B virus: effect of polyethylene glycol on adsorption and penetration[END_REF]. Because this assay has a higher ratio of antibody molecules to viral particles compared to the assay in the absence of PEG, it was expected to improve the neutralization activity of the antibodies, although it is not under physiological conditions. Indeed the infection level was higher since HBsAg production reached 100 ng/ml, and an almost complete neutralization was observed with the highest antibody dose, while HzKR127-3.2 neutralized the viral particles more efficiently than KR127 (Supplemental Fig. S4).

Discussion

We had previously constructed the CDR-grafted humanized antibody HzKR127; the epitope specificity and affinity of HzKR127 are the same as those of the original murine mAb, KR127. HzKR127 virus-neutralizing activity was confirmed in chimpanzees. Subsequently, the crystal structures of HzKR127 Fab and its complex with the epitope revealed that HzKR127 exerts a lid-opening mechanism for antigen binding, one of the largest conformational changes in antigen-antibody recognition. In the present study, we successfully optimized HzKR127 through SDR grafting and affinity maturation by rational design based on the antigen-antibody complex structure. A total of 15 (12 in FR, one in HCDR2, and two in LCDR2) mouse residues in HzKR127 were replaced by human residues, and Asp97 in HCDR3 was replaced by alanine. The resulting humanized antibody (HzKR127-3.2) exhibited 2.5-fold higher affinity for the preS1 antigen and enhanced virusneutralizing activity against both adr and ayw subtypes of HBV compared to KR127.

Moreover, it retained the same epitope specificity as that of KR127 and did not show any offtarget activity, as assessed by flow cytometric analysis. In addition, T epitope analysis suggested that HzKR127-3.2 may be less immunogenic in humans than HzKR127.

Considering that HzKR127 exhibited virus-neutralizing activity in chimpanzees, HzKR127-3.2 might exhibit higher in vivo virus-neutralizing activity than HzKR127 and thus may be more effective in the immunoprophylaxis of HBV infection. HzKR127-3.2 may be particularly useful for repeated administration to humans because of its minimized immunogenic potential.

KR127 and HzKR127-3.2 target amino acids 37-45 from the L protein of adr subtype, corresponding to amino acids 26-34 of ayw subtype. This epitope lies in preS1 region of the L protein which is involved in HBV infectivity, since the target of the peptide(amino acids 2-47, ayw subtype) was identified as NTCP and shown to be a functional receptor for HBV entry [START_REF] Yan | Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus[END_REF]. Therefore, our antibodies target a region crucial for HBV infectivity and thus were expected to block entry. However, Bremer et al. reported that antibodies against preS1(1-21)

of ayw subtypes reacted well with non-myristoylated preS1 peptides but only weakly with myristoylated preS1 peptides and did not neutralize the infectivity of HBV, whereas antibodies to neighboring sequences neutralized very well [START_REF] Bremer | N-terminal myristoylation-dependent masking of neutralizing epitopes in the preS1 attachment site of hepatitis B virus[END_REF]. The data suggest that antibodies to preS1 of ayw subtypes are likely to bind well to HBV and neutralize viral infectivity. Indeed, we observed that HzKR127-3.2 recognizing preS1(37-45) of adr subtypes bound to the viral particles of both ayw and adr subtypes and neutralized them efficiently. Analysis of antigen-antibody interaction of HzKR127-3.2 indicated that seven residues in the epitope sequence, except Ser38 and Asn39, are essential for antibody binding (Figure 4). Among the essential residues, five residues (Asn37, Pro41, Asp42, Trp43, and Asp44) are highly conserved among genotypes [START_REF] Yan | Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus[END_REF], while the other two residues (Asn40 and Phe45) are infrequently changed to serine and leucine or histidine, respectively in some genotypes. Neutralization efficiency of HzKR127-3.2 against these genotypes remains to be determined.

Regarding the correlation between kinetic parameters and virus-neutralizing potency, studies with mAbs against respiratory syncytial virus (RSV) F protein have shown that a direct relationship exists between the association rate and RSV-neutralizing activity [START_REF] Wu | Ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization[END_REF][START_REF] Wu | Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract[END_REF][START_REF] Bates | Reversion of somatic mutations of the respiratory syncytial virusspecific human monoclonal antibody Fab19 reveal a direct relationship between association rate and neutralizing potency[END_REF],

while studies with other viral pathogens, such as human immunodeficiency virus or rotavirus, have shown that virus-neutralizing activity can be determined predominantly by the dissociation rate [START_REF] Vancott | Dissociation rate of antibody-gp120 binding interactions is predictive of V3-mediated neutralization of HIV-1[END_REF][START_REF] Kallewaard | Functional maturation of the human antibody response to rotavirus[END_REF]. In case of HBV-neutralizing antibody, a recent study showed no correlation between the affinity of anti-S monoclonal antibodies and their ability to neutralize HBV infection [START_REF] Shirazi | Monoclonal antibodies to various epitopes of hepatitis B surface antigen inhibit hepatitis B virus infection[END_REF]. In this study, HzKR127-3.2 exhibited 2.5-fold higher affinity for the preS1 and enhanced in vitro virus-neutralizing activity against both adr and ayw subtypes of HBV compared to KR127, while the association rate was 2.3-fold faster and the dissociation rate was slightly slower. These data indicate that the enhanced virus-neutralizing activity was achieved by an increased association rate, which suggests that the association rate of anti-preS1 antibodies may play a predominant role in HBV neutralization. To the best of our knowledge, this is the first study to show a direct relationship between the association rate and neutralizing potency in antibody-mediated HBV neutralization. Our findings may provide insights that will help to facilitate the engineering of potent antibody therapeutics for HBV immunoprophylaxis. GST-preS1 (amino acids 1-56) protein (WT) and a series of alanine replacement mutants were expressed in E. coli and subjected to 12% SDS-PAGE (bottom) followed by western blot analysis with KR127, HzKR127-3.1, or HzKR127-3.2 (Top). The protein band corresponding to the GST-preS1 is marked. (B) HzKR127-3.2 was subjected to flow cytometric analysis using preS1-expressing HEK293T cells (293T-S1-L1) and preS1negative cells (HEK293T, SCK-L1, ACHN, B16F1, and CHO).

-------A-VK-------V-------T-T -YY-H --R-A-------M- I-N-SG-S-S-AQ--Q- HzKR127 --------A-V--------V---------- ----- --R-A--------- -----------AQ--Q- HzKR127-3 --------A-VK-------V-------T-T ----- --R-A-------M- -----------AQ--Q- HzKR127-3.1 --------A-VK-------V-------T-T ----- --R-A-------M- -----------AQ--Q- HzKR127-3.2 --------A-VK-------V-------T-T ----- --R-A-------M- ---------S-AQ--Q- FR3 HCDR3

Highlights

• Anti-hepatitis B virus preS1 humanized antibody was constructed by SDR grafting.

• SDR-grafted humanized antibody was affinity-matured by mutating two HCDR residues.

• Affinity matured humanized antibody exhibits enhanced HBV-neutralizing activity.

• Enhanced HBV-neutralizing activity was achieved by an increased association rate.

Fig. 1 .

 1 Fig. 1. Amino acid sequences of VH (A) and VL (B) of murine antibody KR127 and humanized antibodies (HzKR127, HzKR127-3, HzKR127-3.1 and HzKR127-3.2). DP7 and DPK12 are the human Ig VH and V germ-line segments, respectively. The dashes indicate identical amino acid residues.

Fig. 2 .

 2 Fig. 2. Crystal structure of the HzKR127 Fab-preS1 complex. (A) Interaction between the VH FR3 residue (Ala71H or Lys73H) and HCDR2 (H2) loop. (B) Interaction between the VL FR2 residue (Leu36L) and Trp103H in FR4. (C) Location of the LCDR2 residues (Leu54L and Lys53L). (D) Location of Asn58H in HCDR2. The VH and VL are shown in blue and in pink, respectively. The CDR loops and preS1 peptide are colored yellow and green, respectively.

Fig. 3 .

 3 Fig. 3. Hydrogen bonds between the HCDR3 lid (yellow) and neighboring residues in the free (A) and preS1-bound (B) HzKR127 Fab. The residues involved in the hydrogen bonds are labeled, and the hydrogen bonds are indicated as dotted lines. The preS1 peptide is shown in green. The light and heavy chains are colored pink and blue, respectively.

Fig. 4 .

 4 Fig. 4. Validation of epitope specificity (A) and off-target activity (B) of HzKR127-3.2. (A)

Fig. 5 .

 5 Fig. 5. In vitro HBV binding (A) and neutralization (B and C) assays of HzKR127-3.2 against ayw or adr subtypes of HBV.

  ------S----P---------------H-D-----Y -Y--K---P-QL----V-NRF-HzKR127 ---------S----P------------------------------S----P----------------------------K---P-Q----S------------S------V--------------------V----A B

Table 2 .

 2 Assessment of potential immunogenicity of humanized antibodies by T-cell epitope analysis. Prediction of HLA-DR binding sites by ProPred analysis (www.imtech.res.in/raghave/proped/index.html)

	Antibody	HzKR127	HzKR127-3.2	HzKR127	HzKR127-3.2	HzKR127	HzKR127-3.2
	Location	HCDR2/FR3	FR2 in VL	FR2/LCDR2
	Peptide sequence	YNGKFKGKA	YAQKFQGRV	WLLQKPGQS	WLLQKPGQP	IYLVSKLDS	IYLVSNRDS
	MHC
	Class II
	molecules
	that
	Bind to
	peptide

Acknowledgments

This work was supported by Ministry of Health and Welfare grant A050260 and by a 2014 research grant from Kangwon National University (No. 120140400). This work was also financially supported by ANRS (Agence nationale de recherche contre le sida et les hépatites virales). Fidaa Bouezzedine was recipient of a fellowship from the Ligue Nationale Contre le Cancer. Manipulations of pathogens were performed in the biosafety level 3 containment laboratory core facility of the Biology and Health Federative Research Structure of Rennes (Biosit).