F Artemio-Schoulten

R Vauche

S Bourdel

E Muhr

J Gaubert

N Dehaese

F Hameau

H Barthelemy

Pulse Design Method for Complexity Reduction of IEEE IR-UWB Pulse Synthesizers

Keywords: IEEE 802.15.4 standard, IEEE 802.15.6 standard, Impulse-Radio Ultra-WideBand, pulse shaping, pulse synthesizer

This paper presents a sample & hold pulse envelope synthesis method and shows that only 5 (resp. 7) samples are necessary to respect the sidelobes rejection in adjacent channels of 18dB (resp. 20dB) required by the IEEE 802.15.4 (resp. 802.15.6) standard if a sample & hold Gaussian envelope is used. These required numbers of samples, which enable complexity reduction of IR-UWB emitters for IEEE standards, are validated using measurement results of a pulse synthesizer presented in this paper since a sidelobes rejection in adjacent channels of 21.8dB (resp. 21.4dB) is obtained when transmitting in the mandatory low band of the IEEE 802.15.4 (resp. 802.15.6) standard.

I. INTRODUCTION

Since the frequency band between 3.1GHz and 10.6GHz has been allocated to Ultra-WideBand (UWB) applications by the Federal Communication Commission (FCC) in 2002, several standards using this frequency band have been defined. Among them, two IEEE standards, the IEEE 802.15.4 standard introduced in 2011 and the IEEE 802.15.6 introduced in 2012, have chosen Impulse Radio (IR) to exploit this UWB frequency band instead of usual carrier modulations.

As for usual carrier modulations, a modulated IR-UWB signal must be compliant with a frequency mask. Considering the case of impulse modulations, it has been demonstrated that the emitted Power Spectrum Density (PSD) is directly proportional to the square modulus of the emitted pulse's spectrum [START_REF] Sparrow | High rate UWB CMOS transceiver chipset for WBAN and biomedical applications[END_REF]. Thus, one way to ensure this compliance is to use pulses with a spectrum which fits into the targeted frequency mask. For this reason, several particular pulses with no sidelobe have been defined for IR-UWB applications such as the ideal up-converted Gaussian pulse [START_REF] Vauche | A 100 MHz PRF IR-UWB CMOS Transceiver With Pulse Shaping Capabilities and Peak Voltage Detector[END_REF].

However, when pulses use the entire FCC frequency band, the antenna can mitigate constraints on sidelobes rejection since its frequency response generally fits with the FCC mask. Nevertheless, when considering IEEE standards, sidelobes rejection in adjacent channels is one of the more difficult issues to solve since the expected rejection is 18dB (resp. 20dB) for the IEEE 802.15.4 (resp. 802.15.6) and the adjacent channels are in the unfiltered part of the antenna frequency band.

Several pulse shaping methods have been introduced to limit sidelobe rejection issues. In the analog domain, pulse can be shaped by using modulated inverters or class C drivers as antenna driver [START_REF] Dokania | A 6μW, 100Kbps, 3-5GHz, UWB impulse radio transmitter[END_REF]. In digital domain, this can be done by enabling parallel antenna drivers [START_REF] Mercier | An Energy-Efficient All-Digital UWB Transmitter Employing Dual Capacitively-Coupled Pulse-Shaping Drivers[END_REF]. However, impairments such as Process, Voltage, and Temperature (PVT) variations can modify the shape of the emitted pulse, leading to the raise of the sidelobes in adjacent channels. To counterbalance for impairments, the pulse shape might be finely tuned as it can be easily done with digital shaping methods [START_REF] Zhang | The Design of an Energy-Efficient IR-UWB Transmitter With Wide-Output Swing and Sub-Microwatt Leakage Current[END_REF] [START_REF] Streel | SleepTalker: A ULV 802.15.4a IR-UWB Transmitter SoC in 28-nm FDSOI Achieving 14 pJ/b at 27 Mb/s With Channel Selection Based on Adaptive FBB and Digitally Programmable Pulse Shaping[END_REF]. However, the more the degrees of freedom an IR-UWB emitter has, the more complex it is, which unfortunately increases its area and power consumption.

In order to relax architecture complexity and design constraints, it is firstly shown in this paper that how the sidelobes rejection in adjacent channels required by the IEEE 802.15.4 (resp. 802.15.6) standard can be fulfilled with pulses shaped by a sample & hold Gaussian envelope with 5 (resp. 7) samples only. In a second time, a pulse synthesizer allowing the generation of pulses shaped by a sample & hold Gaussian envelope having 5 or 7 samples and their tuning is presented. Finally, measurement results of the pulse synthesizer are used to validate in practice the approach developed in this paper.

The paper is organized as follows. Section II details the synthesis of sample & hold Gaussian envelope. Next, the design of an UWB pulse synthesizer able to deal with this type of envelope is described in Section III. Finally, measurement results are shown and discussed in section IV.

II. SAMPLE & HOLD GAUSSIAN ENVELOPE SYNTHESIS

A way to ensure that a modulated IR-UWB signal is compliant with a frequency mask is to use pulses having a spectrum which can easily fit into this mask such as the upconverted Gaussian pulse. The up-converted Gaussian pulse pG(t) can be defined as follows:

( ) ( ) ( ) sin 2 G M G p t e t f t  =  ()
where the envelope eG(t) is the well-known Gaussian function which is here up-converted to the frequency fM (central frequency of the targeted frequency band). In this case, eG(t) is a function of the form:

( ) ( ) 2 exp G e t A t  = - ()
where A is the maximum of the Gaussian function and β a parameter which can be linked to the pG(t) bandwidth BW-XdB defined at X dB as follows [START_REF] Vauche | A 100 MHz PRF IR-UWB CMOS Transceiver With Pulse Shaping Capabilities and Peak Voltage Detector[END_REF]:

( ) ( ) 2 2 /10 2ln 10 XdB XdB BW  -   =    ()
Since the Fourier transform of a Gaussian is a Gaussian, the main advantage of this ideal pulse pG(t) is that its spectrum is a Gaussian centred on fM and also do not have sidelobes as shown in Fig. 1.

However, an up-converted Gaussian pulse has an infinite duration and also has to be truncated in practice according to a certain percentage of A. This truncated up-converted Gaussian pulse noted pTG(t) can be defined as follows:

( ) ( ) ( ) sin 2 TG M TG p t e t f t  =  ()
where the envelope eTG(t) is the truncated Gaussian which is a function of the form:

( ) ( ) ( ) TG G T e t e t t =  ()
with ΠT(t), the gate function equals to 1 for t between -T/2 and T/2 and 0 outside. Unfortunately, sidelobes occur due to this truncation as shown in Fig. 1 and the more truncated the Gaussian is, the higher the sidelobes are. The truncation percentage must also be moderated.

To allow pulses with finite time envelope, such as the truncated up-converted Gaussian pulse, to be generated using a digital approach, it is proposed to discretise this envelope using K samples having a duration τ. Thus, the sample duration must be equal to:

M Df  = ()
where D is an integer chosen to make τ a multiple of the sine period and also to be able to clock the pulse envelope generator with an oscillator working at fM. In this case, the number of samples K is given by:

KT  = ()
where K can be an integer if:

M T N f = ()
and if N is an integer equal to the product of K by D (T being the eTG(t) duration and also the pTG(t) duration). In the case of a truncated Gaussian envelope, this condition is fulfilled when particular percentages of A are used for truncation.

Considering these assumptions, the discretised truncated up-converted Gaussian pulses pDTG(t) can be defined as follows:

( ) ( ) ( )

sin 2 DTG M DTG p t e t f t  =  ()
where the discretised truncated Gaussian eDTG(t) is equal to:

( )

1 0 1 1 2 2 K DTG TG k K K e t e k t k      - = - -     = -   -+           ()
This approach can be extended to any envelope e(t) with a finite duration by replacing eTG(t) with e(t). However, this discretisation leads to sidelobes increase as shown in Fig. 2 and Fig. 3 and the lower K is, the higher sidelobes are.

Nevertheless, it can be demonstrated that it is possible to use a sample & hold envelope while limiting sidelobes magnitude if K and D are chosen according to the following inequality:

1 XdB q BW  -  ()
where q is a parameter which can be numerically computed and depends on the targeted rejection of sidelobes X in dB and the e(t) Fourier transform.

Finally, as shown in Fig. 2 (resp. Fig. 3), a pulse having a sample & hold Gaussian envelope with only 5 (resp. 7) samples can be compliant with the frequency mask of the mandatory low band channel C3 (resp. C1) defined in the IEEE 802.15.4 HRP UWB PHY (resp. 802.15.6 UWB PHY) regarding sidelobes rejection in adjacent channels between 3.1GHz and 10.6GHz. However, as shown in Fig. 2, other sidelobes, which must not be confused with sidelobes in adjacent channels circled in green, can be observed between 0.91GHz and 1.61GHz which is not compliant with FCC regulation. Nevertheless, AC coupling and filtering due to antenna will reduce enough these sidelobes since it is reasonable to consider that the antenna frequency response fits into the FCC mask.

III. DESIGN OF THE ASSOCIATED PULSE SYNTHESIZER

A. Pulse synthesizer architecture

The topology of the pulse synthesizer associated to the pulse design method is based on a H-bridge. Although a Hbridge has been already proposed for pulse shaping in UWB applications [START_REF] Vauche | A 100 MHz PRF IR-UWB CMOS Transceiver With Pulse Shaping Capabilities and Peak Voltage Detector[END_REF], the presented structure has been modified to allow a digitally control of the pulse envelope. In addition, the H-bridge is here driven by a differential Voltage Controlled ring Oscillator (VCO; detailed in [START_REF] Vauche | Ultra-wideband voltage controlled oscillator with commutable phases for BPSK implementation[END_REF]) as shown in Fig. 4.a (instead of a Voltage Controlled Delay Line such as in [START_REF] Vauche | A 100 MHz PRF IR-UWB CMOS Transceiver With Pulse Shaping Capabilities and Peak Voltage Detector[END_REF]) in order to allow the generation of high duration pulses as required by the IEEE standards. The VCO also controls alternatively the transmission gates couples (TGU1, TGD1) and (TGU2, TGD2) to alternate the current into the load which produces zero mean pulses. Thus, as represented in Fig. 4.a, the pulse envelope is controlled thanks to 4 transmission gates banks TGx (TG1 to TG4) which modify the current into the output load. Signals Sx (S1 to S4), provided by the shape control module, control the transmission gates banks TGx and their associated buffers. As shown in Fig. 4.a, this allows a pulse having a sample & hold envelope with a maximum of 7 samples to be generated. Finally, the magnitude of samples is controlled at the transmission gates bank (TGx) level by 4 control bits (Nxy). Indeed, when Sx activates a buffer, the control bits Nxy (Nx1 to Nx4) are applied to the TGxy (TGx1 to TGx4) gates that composed the TGx as presented in Fig. 4.a.

B. Design of the pulse shaper

The pulse shaper is mainly composed of 4 banks of transmission gates (TGx) and each transmission gates bank is based on 5 parallel transmission gates (TGxy) as shown in Fig. 4.b. The voltage level of envelope samples is also controlled by changing the number of active TGxy which modifies the amount of current flowing through the output load. Since the current driven by one transmission gate depends on the width of the transmission gate transistors, the transmission gates have been sized to achieve levels which are distributed around the ones required by the sample & hold Gaussian envelope compliant with IEEE standards, considering that when the x th TGx is activated, all other previous ones (i.e. all TGx from 0 to x-1) are also activated. It is worth to mention that the smallest transmission gate TGx0 of each TGx is directly controlled by Sx to tune the samples level around its nominal value. However, TG10 is not implemented in order to completely disable TG1 which allows the generation of pulses having an envelope with K=5 or 7 samples.

C. Design of the shape control module

To synchronize the pulse shaper with the H-Bridge, the same VCO is used to clock the H-bridge and the shape control module. However, to maintain a constant bandwidth over the different channels, the time duration of the envelope must be constant whatever the VCO oscillation frequency (fM) is. To this end, the shape control module shown in Fig. 4.c is in charge of tuning the samples duration which is determined by two consecutive edges of the control signals Sx and Sx+1 as represented in Fig. 5. Thus, a programmable frequency divider driven by the VCO has been designed to generate a clock (SAMPLE_CLK in Fig. 4.c) having a period equal to D/fM with D [START_REF] Vauche | A 100 MHz PRF IR-UWB CMOS Transceiver With Pulse Shaping Capabilities and Peak Voltage Detector[END_REF][START_REF] Dokania | A 6μW, 100Kbps, 3-5GHz, UWB impulse radio transmitter[END_REF][START_REF] Mercier | An Energy-Efficient All-Digital UWB Transmitter Employing Dual Capacitively-Coupled Pulse-Shaping Drivers[END_REF][START_REF] Zhang | The Design of an Energy-Efficient IR-UWB Transmitter With Wide-Output Swing and Sub-Microwatt Leakage Current[END_REF]. The value of D is then set by the couple of bits MzS. Considering a given channel mask, D can be set to 2 for the lowest channels which leads to a sample duration of 2/fM whereas for the highest channels, D is set to 5 which leads to a sample duration of 5/fM. To address larger bandwidth, it is also possible to set D to a low value while using a high fM. Finally, the outputs (Ei in Fig. 4.c of the counter (CML Counter in Fig. 4.c) clocked by the SAMPLE_CLK signal are used to generate the control signals Sx with conventional logic gates. However, only Current Mode Logic (CML) gates are used in the shape control module since the CML gates can operate at higher frequencies than the CMOS ones.

IV. MEASUREMENTS RESULTS

The proposed programmable digital pulse synthesizer has been implemented using a 65nm technology from ST Microelectronics and a supply voltage of 1.2V. As shown in Fig. 6.a, the manufactured chip, which is pad limited, has a die area of 1.56mm² but a core area of 0.05mm² since it uses only MOS transistors and since others functions not discussed in this paper have been implemented such as a Phase-Locked Loop (PLL) to control the built-in VCO. Moreover, a Serial Peripheral Interface (SPI) slave has been added to make easier the set of Nxy (resp. MzS) bits which control the envelope samples magnitude (resp. duration). Finally, as shown in Fig. 6.b, the test chip has been soldered on a dedicated Printed Circuit Board (PCB) for measurement purposes. have been obtained by varying Nxy and MzS around the initial values obtained using the ideal pulse introduced in section II. This has led to 802.15.4 (resp. 802.15.6) compliant pulses regarding the sidelobes rejection in adjacent channels with a peak-to-peak voltage of 0.71V (resp. 0.76V) and a sidelobes rejection of 21.8dB (resp. 21.4dB), which leads to an emitted energy of 1.22pJ (resp. 1.72pJ) on a 2x50Ω load whereas the measured power consumption is 1.9nJ/pulse. Performances of the proposed pulse synthesizer are summarized in Tab. I and compared with state-of-the-art works. From the authors knowledge, it appears that it is the first time than pulses shaped by a sample & hold envelope with only 5 samples have been used to respect sidelobes rejection in adjacent channels required by the 802.15.4 standard, which will pave the way to less complex UWB emitters. Finally, to highlight the ability of the presented pulse synthesizer to shape the pulse envelope and also its spectrum, the shorter (resp. longer) envelope measured for K=5 and D=2 (resp. K=7 and D=5) is shown in Fig. 9.a (resp. Fig. 9.b) and shows a sidelobes rejection in adjacent channels higher or equal to 20dB.

V. CONCLUSION

In this paper, a sample & hold pulse envelope synthesis method has been introduced and shows that only 5 (resp. 7) samples are necessary to respect sidelobes rejection in adjacent channels required by the IEEE 802.15.4 (resp. 802.15.6) standard. Next, this has been validated using measurement results of a pulse synthesizer implemented with a 65nm CMOS technology from ST-Microelectronics. Authors hope that this work will pave the way to less complex UWB emitters and also reduce their area and power consumption. Pcons@<PRF> a (W@Hz) 600/700µ @1M 5.7µ@ 50k 4.36m@ 7,8M 379µ@ 10M 380µ@ 13,62M

a Mean Pulse Repetition Frequency (<PRF>)

Fig. 1 .Fig. 2 .

 12 Fig. 1. Up-converted Gaussian pulse truncated at 22% and compliant with the channel 3 frequency mask of the IEEE 802.15.4 standard.

Fig. 3 .

 3 Fig. 3. Example of a pulse with sample & hold Gaussian envelope compliant with the channel 1 of the IEEE 802.15.6 standard (K = 7, D = 2 ; the highest sidelobes in adjacent channels are circled in green).

Fig. 5 .

 5 Fig. 5. Chronograms of the shape control module.

Fig. 4 .

 4 Fig. 4. Architecture of the pulse synthsizer (a), the transmission gates bank (b), the shape control module (c).

Fig. 6 .

 6 Test chip photo (a) and test PCB photo (b).

Fig. 7 .

 7 Fig. 7. Pulse for the channel 3 of the IEEE 802.15.4 standard (mandatory).

Fig. 8 .

 8 Fig. 8. Pulse for the channel 1 of the IEEE 802.15.6 standard (mandatory).

  Fig. 9. Example of pulses with the shorter (a) and the longer (b) envelope having a sidelobes rejection higher or equal to 20dB.

TABLE I

 I

		.	STATE OF THE ART COMPARISON
		This Work	[3]	[4]	[5]	[6]
	Techno.	65nm		130nm	90nm	130nm	28nm FDSOI
	IEEE Standards	15.4 / 15.6	No	15.4	No	15.4
	Vpp (V)	0.71 / 0.76	0.5	0.71	0.94	0.35
	Sidelobe reject. (dB) 21.8 / 21.4	20	20.7	24	25
	PRF (pulse/s)	15.6M	5M	15,6M	10M	27.24M
	Ep (pJ)	1.22 / 1.72	1.3	2.5	3.6	0.64
	Fine tuning	yes		no	yes	yes	yes
	Number of envelope samples	5 / 7		NA	7	7	9 or 18


F A Schoulten

A A Mariano

R Vauche

S Bourdel

J Gaubert

N Dehaese

H Barthelemy

Low Power Ultra-Wide Band Pulse Generator based on a Duty-Cycled 2-ASK Emitter

Keywords: BPSK, IEEE standards, Impulse Radio, Low Power, OOK, Power Gating, UWB, VCO

This paper presents an ultra-wideband pulse generator based on a duty-cycled 2-ASK emitter able to generate OOK and BPSK modulated carriers. This emitter uses a differential architecture which has been designed in FD-SOI 28 nm CMOS technology with a supply voltage of 1.0 V. It presents a fast enough transient response to entirely cover the 3.1-10.6 GHz FCC band. Moreover, it can be quickly turnedon and turned-off which allow 3 ns pulses to be generated and also, low power impulse communications to be implemented. The emitter is built around a wideband Voltage Controlled Oscillator (VCO) which is able to generate at its outputs large oscillations from 1.0 GHz to 13.9 GHz. When the emitter is used as a 3 ns pulse generator centred on 7987 MHz, its energy consumption is 152.7 pJ/pulse, and the static power consumption is 3.83 μW. This leads to a mean power consumption of 5.36 µW (resp. 19.1 µW and 156 µW) for a pulse repetition frequency of 10 kHz (resp. 100 kHz and 1 MHz), which highlights the interest of impulse modulations for low power radio-frequency communications.

I. INTRODUCTION

The eminent rise of the Internet of Things (IoT) along with the growing number of devices connected to Wireless Personal Area Network (WPAN) and Wireless Body Area Network (WBAN) will require wireless systems that must offer high-data quality transfer within short and medium range [START_REF] Wang | Body Area Communications: Channel Modeling, Communication Systems, and EMC[END_REF]. A promising technology that is well suited for WPAN and WBAN applications is the Ultra-wide Band Impulse Radio (IR-UWB), which is a communication technique based on short duration pulses transmission. The UWB communications have attracted interest of researchers since the Federal Communication Commission (FCC) has authorized the use of impulse modulations inside the UWB frequency band between 3.1 GHz and 10.6 GHz in 2002 [START_REF][END_REF][START_REF] Hamalainen | UWB Theory and Applications[END_REF]. The duration of the associated pulses is typically the order of nanosecond and its energy can be spread on a frequency band of the order of GHz.

The IR-UWB communications lead to a high energy efficiency on the transmitter side thanks to their duty-cycled nature. Indeed, the transmitter chain must be operational only during the pulses emission, which make it suitable for low power applications. Thus, the power consumption of UWB transmitters is proportional to data rate. Moreover, it is possible to produce short time domain pulses with simple and low power architecture. For this reason, the IR-UWB emitters can intrinsically present a low-power consumption which is reinforced by the low authorized power spectral densities that are orders of magnitude lower than mobile communications signals [START_REF] Goavec | An efficient method of power spectral density estimation for on-chip IR-UWB transmitter self-calibration[END_REF], in addition to high bit rate due to the use of short duration pulses.

Among the IR-UWB pulse generator reported architectures, one uses voltage-controlled oscillators (VCO) combined with a shaper circuit [START_REF] Artemio-Schoulten | Pulse Design Method for Complexity Reduction of IEEE IR-UWB Pulse Synthesizers[END_REF], meanwhile, other uses baseband pulses combiner, using delay cells [6]. Both works target an IR-UWB pulse generator whose generated pulse shape fits in spectral masks defined by the IEEE 802.15.4 standard, that defines 15 channels divided in 3 groups and whose bandwidth is between 499 MHz and 1355 MHz (Fig. 1). To be compliant with this standard, an UWB device must cover at least 1 channel from these 3 groups, where the mandatories channels are 0, 3 and 9. In this paper, a 2-ASK emitter with a sufficient range to cover the FCC band (3.1-10.6 GHz) and which can be quickly turned-on and turned-off to be used as a pulse generator is reported. It consists in a VCO followed by a buffer stage and an antenna buffer. The VCO can be switched on and off through a logic signal and its quick response to start or stop dispenses the use of a mixing stage. Moreover, this VCO has a differential output voltage whose phase can be quickly commuted, which enables bipolar modulations such as BPSK. Therefore, it can be used for IR-UWB communications as well as for the traditional 2-ASK communications based on a modulated carrier. Finally, although communications based on carrier modulation require an oscillator operating all the time, an interesting feature of this circuit is that it can be powered down between two consecutives emitted pulses and then consumes only during a pulse transmission. This paper is organized as follows. Section II discusses IR-UWB pulse generation and presents the pulse generator architecture. Section III focuses on the differential VCO architecture designed with a FD-SOI 28nm design kit. In section IV, simulation results demonstrate that the proposed emitter covers all the 3.1-10.6 GHz band and can be used for IR-UWB pulse generation.

II. IR-UWB PULSE GENERATION

A. Pulse generator architecture

For an IR-UWB transmission, the data can be transmitted using different values of the emitted pulses parameters like amplitude, position, or polarity. In the case of a pulse amplitude modulation (PAM), the signal emitted by the UWB antenna can be expressed as a combination of single pulses, such that:

( ) 0 k k s k s t d p t D  =   =  -      ()
where dk is the k th emitted symbol taken from a M-ary alphabet D, Ds is the symbol rate, and p(t) is the generated pulse waveform. For an OOK modulation, the alphabet D is:

•     : 0,1 k Dd=
and for an BPSK modulation, the alphabet D is:

•     : 1,1 k Dd=- 
The signal from (1) can also be generated by the UWB-IR emitter based on gated oscillator as proposed here, whose architecture is displayed in Fig. 2. The main parameters of the pulse generation by an oscillator are the oscillation duration τp and the oscillation frequency fm, that respectively determines the generated pulse bandwidth and its central frequency. To charge a 50 Ω UWB antenna, a buffer stage is required and consists here of push-pull circuit and differential buffers. The buffer stage and antenna, placed right after the differential VCO, are not detailed in this paper.

In order to achieve a pulse with a central frequency inside the UWB FCC band, the emitter proposed here is built around a differential VCO having a frequency control, and start-up and stop command. The pulse duration is controlled here by the time that the oscillator is operational (signal "Enable" with high logic level) and the VCO frequency can be largely using "Voltage Control". To enable BPSK modulations, the output phases can be exchanged using the logic signal "Phase". Finally, the emitter can be turned-on or turned-off through the logic signal "ON/OFF".

B. Pulse waveform generated by gated oscillators

For a gated oscillator generating sinusoidal waveform signals, the pulse shape p(t) can be written such that:

( ) ( ) ( ) sin 2 m p t A f t t  =    () ( ) 1 2 2 0 p p if t t if otherwise   -    =   ()
where A is the maximum pulse magnitude and (t), the rectangular function having a width of τp. Notice that the pulse shape is here a sinusoidal waveform with a rectangular windowing, with a duration controlled. Shifting to frequency domain, the mono-lateral Fourier transform of the emitted pulse can be expressed as:

( ) ( ) ( ) ( ) sinc exp 2 p mp A P f f f j     + = - - ()
where φ is the sinewave phase. For a BPSK impulse modulation, the power spectral density is proportional to the squared magnitude P + (f).

The duration τp of p(t) can be expressed as a function of the oscillation frequency fm as follows:

2 p M N f  = ()
where N is the integer number of sinusoidal half-periods of the generated pulse. However, the minimal number of halfperiods of the sinewave, in relation of the bandwidth BW-XdB, can be written:

min M XdB f N BW  - = ()
where α is coefficient that has a value of 1.8, 3 and 10.8 when the attenuation XdB is 3 dB, 10 dB and 18 dB respectively. From ( 5) and ( 6), the pulse minimum width can be expressed as:

min 2 p XdB BW   - - = ()
Finally, it can be noted that an attenuation of 10 dB is enough to be compliant with FCC requirements. However, an attenuation of 18 dB is for example necessary to be compliant with the IEEE 802.15.4 standard.

III. PROPOSED VCO DESIGN

The gated voltage-controlled oscillator is the main circuit of the 2-ASK emitter which allows this last to be used as an IR-UWB pulse generator. It is based on a ring oscillator architecture whose principle is to propagate a signal on this close loop chain. Thus, it consists here of an odd number of cascaded delay cells, connected in a close loop chain, as indicated in the Figs. 3 and4. This architecture is interesting given that it does not require inductor neither capacitor and it is able to provide high frequency oscillations, and multiphase outputs to the detriment of the phase noise. The instability condition, required for oscillations start, comes from the number of delay cells, where the minimum necessary is two for differential architecture, and three for non-differential architecture. Indeed, a ring with one delay cell is not feasible because the transistor will be self-biased, presenting only a DC component current. Also, the delay time of cells defines the oscillation frequency. The proposed VCO design includes buffer cells, in order to make the oscillation frequency independent of the VCO load. The delay cell and buffer cell schematic are presented in Figs. 5 and6. "Vctrl" is a voltage which control the oscillation frequency. The signals "ON" and "OFF" control the pulse duration, while the signal "PDB" turns-on or turnsoff the IR-UWB pulse generator. Meanwhile, the signals "INV" and "INV\" control the VCO output signal phase. The truth table of the delay cell and buffer cell are displayed in Table I. The push-pull, placed between the gated VCO and the buffer stage, has been added to amplify the VCO output current, thus providing a better signal dynamic and improving the driving capability especially for high frequency.

0 0 0 0 0 0 0 1 0 0 1 1 1 0 0 1 0 1 1 1 1 1 1 0
IV. SIMULATION RESULTS This section presents simulation results of the 2-ASK emitter when it is used as a pulse generator. It has been designed using the FD-SOI 28 nm CMOS design kit from STMicroeletronics, with a supply voltage of 1.0 V. In the design process, the possible parasitic capacitances at each node have been considered. The values of the parasitic capacitances were extracted from a parasitic extraction of another previous design. Fig. 7 shows the VCO frequency and its always-on power consumption as a function of the oscillation frequency control voltage ("Vctrl")., in addition to the always-on power consumption of the IR-UWB emitter. From Fig. 7, it can be noticed that the proposed design is able to cover all the 3.1-10.6 GHz, where the maximum frequency reached is 13.2 GHz. The average (resp. minimum and maximum) VCO gain is about -18.1 GHz/V (resp. -20.8 GHz/V and -10.6 GHz/V). When "Enable" and "PDB" have a high logic level, the always-on power consumption of the proposed IR-UWB emitter and the gated oscillator have a value of 45.7 mW and 11.1 mW respectively, for a VCO frequency of 7987 MHz. When "Enable" and "PDB" has a low logic level, the pulse generator is turned-off and has a total power consumption of 3.83 μW. Thanks to the oscillation frequency range, the proposed pulse generator is able to address the central frequencies of all the IEEE 802.15.4 channels. In Fig. 8 and Fig. 9, pulses obtained through transient simulations and targeting a central frequency similar to the ones of the mandatory channels 3 (4482 MHz) and 9 (7987 MHz) of IEEE 802.15.4 standard are shown. They have a bandwidth BWX=-10dB (α=3) equals to 500 MHz and also can be used outside the context of IEEE standards as allowed by FCC requirements since pulses spectrum side lobes present a power spectrum density lower than 10 dBc. However, this is not enough to be fully compliant with IEEE standards. For this reason, an additional pulse shaper circuit should be added to the proposed pulse generator to synthesize a pulse with a particular envelop which fulfils in the same time IEEE standards requirements and the FCC ones.

To obtain a 10 dB bandwidth of 500 MHz, α has been set to 3 which leads using [START_REF] Haapala | A fully integrated digitally programmable pulse shaping 6.0-8.5 GHz UWB IR transmitter frontend for energy harvesting applications[END_REF] to τp = 3 ns. Thus, the "Enable" input must have a high logic level for 3 ns, given that the VCO has a fast response time. Regarding the simulations, signal "ON" from Fig. 4 (resp. signal "PDB" from Fig. 4) have high logic level for 3 ns (resp. 3.6 ns). It can be noted that the PDB high level duration must be higher than the ON high level duration in order to let biasing currents and voltages to be initialized. Finally, when DATA=1, the signal "INV" has low logic level, otherwise, the signal "INV" has high logic level which allows DATA to change the generated pulse phase to 180° (BPSK modulation). As shown in Fig. 8 and Fig. 9, it can be noticed, for the same central frequency, that the two pulses with different value of DATA are exactly in phase opposition and therefore have the same frequency response. Given that the IR-UWB signals presents a gated nature, the average power consumption can be expressed in terms of the pulse repetition frequency (PRF), such that:

( ) c AC DC P PRF E PRF P =  + ()
where EAC is the energy consumed per generated pulse, and PDC the static power that is consumed when no pulse is generated. It can be noted that this last is here equal to the leakage power consumption since the pulse generator circuit can be fully turned-off. Both parameters EAC and PDC can be extracted from at least two measured average power consumption at different values of PRF. Moreover, [START_REF] Park | An all-digital 12 pJ/pulse IR-UWB transmitter synthesized from a standard cell library[END_REF] highlights the particularity of impulse communications which is that the power consumption increases with the data rate. For the proposed pulse generator, when

V. CONCLUSION

In this paper, a low power IR-UWB pulse generator based on a duty-cycled 2-ASK emitter has been presented. It is composed of a gated and differential voltage-controlled oscillator and a buffering stage. This circuit has been designed in FD-SOI 28 nm CMOS technology from STMicroelectronics. The simulations results show that the proposed gated VCO is fully capable to cover the 3.1-10,6 GHz band, regulated by the FCC, and thus to potentially address every channel of the IEEE 802.15.4 standard. Nevertheless, to be fully compliant with the IEEE standard spectrum requirements, an additional pulse shaper circuit is required and should be designed. In terms of power consumption, the VCO achieves an always-on power consumption of 11.1 mW whereas the full pulse generator has an always-on power consumption of 45.7 mW. Finally, the pulse generator has an energy consumed per pulse of 152.7 pJ when it generates 3 ns pulses centred on 7 987 MHz and a static power consumption of 3.83 µW (leakage consumption). Therefore, the circuit total power consumption can be reduced until this limit by reducing the data rate.

Fig. 1 .

 1 Fig. 1. IEEE 802.15.4a standard channels frequency plan.

Fig. 2 .

 2 Fig. 2. Proposed IR-UWB emitter based on gated oscillator architecture.

Fig. 3 .

 3 Fig. 3. Proposed VCO topology.

Fig. 4 .

 4 Fig. 4. Proposed gated differential VCO architecture.

Fig. 5 .

 5 Fig. 5. VCO delay cell schematics.

Fig. 6 .

 6 Fig. 6. VCO buffer cell schematics.

Fig. 7 .

 7 Fig. 7. VCO frequency and IR-UWB emiter always-on power consumtpion in terms of VCO voltage control.

Fig. 8 .

 8 Fig. 8. IR-UWB pulse generator simulated response for a BW-10dB of 500 MHz (τp-min = 3 ns) centered on 4482 MHz (channel 3).

Fig. 9 .

 9 Fig. 9. IR-UWB pulse generator simulated response for a BW-10dB of 500 MHz (τp-min = 3 ns) centered on 7987 MHz (channel 9).

TABLE I

 I

		.	VCO CELLS TRUTH TABLES
		Delay Cell			Buffer Cell
	IN	ON	OUT	IN	INV	OUT

  [START_REF] Hamalainen | UWB Theory and Applications[END_REF] ns pulses centred at 7987 MHz are generated, Pc is equal to 1.53 mW (resp. 15.27 mW) for a PRF of 10 MHz (resp. 100 MHz), which leads to an a EAC of 152.7 pJ/pulse and a PDC of 3.33 µW. PDC is also very close to the stand-by power consumption (3.83 µW) of the pulse generator, the small difference coming from the precision with which Pc was obtained in simulation. Thus, the power consumption of the proposed pulse generator for this type of pulses is 5.36 µW (resp. 19.1 µW and 156 µW) for a pulse repletion frequency of 10 kHz (resp. 100 kHz and 1 MHz). Finally, a comparison with the state of the art is done in table II. It can be noted that the proposed pulse generator is the only one which is able to cover all the FCC band (3.1-10.6 GHz) in addition to have the greatest output voltage amplitude level.

TABLE II

 II

		.	STATE OF THE ART COMPARISON
		This Work a	[5]	[6]	[7]	[8]
	Techno.	FD-SOI 65 nm	130 nm	65 nm	90 nm
	Power Supply (V)	1	1.2	1.2	1.2	0.9
	Fc (GHz)	3-12	3-5	3-5	6.5-8	3.1-5
	BW (X=3dB) 0.5-1.3 0.5-1.3	0.5-1.3	0.5	0.5-1.4
	Amplitude (V)	0.53	0.4	0.45	0.45	0.05-0.06
	Eac (pJ/pulse)	152.7	1.9	146	2.1	8-12
	Static power (μW)	3.83	NA	100	0.48	170
	Architecture	LO based	LO based	Edge Combiner	Edge Combiner	LO based
			a Simulation results with parasitic capacitances


Darya Urusova

Lenore Carias

Yining Huang

Vanessa C Nicolete

Jean Popovici

Camille Roesch

Nichole D Salinas

Sebastien Dechavanne

Benoit Witkowski

Marcelo U Ferreira

John H Adams

Michael L Gross

Christopher L King

Niraj H Tolia
email: niraj.tolia@nih.gov.

Structural basis for neutralization of P. vivax by naturally acquired

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

suggesting a DBP-based vaccine will reduce infection [START_REF] Grimberg | Plasmodium vivax invasion of human erythrocytes inhibited by antibodies directed against the Duffy binding protein[END_REF] . However, the successful design of a DBP-II-based vaccine may be limited by strain-specific immune responses due to the polymorphic nature of DBP [START_REF]Strain-specific duffy binding protein antibodies correlate with protection against infection with homologous compared to heterologous plasmodium vivax strains in Papua New Guinean children[END_REF][START_REF] Tsuboi | Natural variation within the principal adhesion domain of the Plasmodium vivax duffy binding protein[END_REF] , and the presence of immunodominant but non-protective epitopes within DBP [START_REF] Ntumngia | Design and immunogenicity of a novel synthetic antigen based on the ligand domain of the Plasmodium vivax duffy binding protein[END_REF][START_REF] Chen | Structural analysis of the synthetic Duffy Binding Protein (DBP) antigen DEKnull relevant for Plasmodium vivax malaria vaccine design[END_REF] . Despite the polymorphic nature of DBP, broadly conserved epitopes of three inhibitory murine monoclonal antibodies (mAbs) have been identified in subdomain 3 of DBP-II [START_REF] Chen | Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein[END_REF] . These epitopes are distant from the dimer interface or DARC binding site [START_REF] Chen | Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein[END_REF] . Furthermore, human vaccination with DBP-II elicits antibodies that block the in vitro binding of four alleles of DBP to DARC suggesting broadly-neutralizing epitopes of human antibodies may exist within DBP-II [START_REF] Payne | Human vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies[END_REF][START_REF] Singh | Malaria vaccine candidate based on Duffy-binding protein elicits strain transcending functional antibodies in a Phase I trial[END_REF] .

The identification of broadly-conserved human neutralizing-antibody epitopes that contribute to naturally acquired immunity is essential for the improved rational design of potent strain-transcending DBP-based vaccines. Here, we present the study of DBP-II with two human neutralizing monoclonal antibodies 053054 and 092096. These human mAbs were produced by sorting individual DBP-II-specific B cells from a Cambodian patient with naturally acquired DBP-II-blocking antibodies and then isolating, sequencing, and cloning the variable regions from human IgG heavy and light chains. Structures of DBP-II antibody complexes were determined by X-ray crystallography, and epitopes further mapped by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and mutational studies. Both antibodies inhibit binding of DBP to red blood cells, and 092096 neutralizes P. vivax in ex vivo experiments. Polysera from patient populations competes with binding of 092096 to DBP. We show that these naturally-acquired human antibodies neutralize P. vivax by targeting the DARC-binding site and dimer interface of P. vivax DBP. This work forms a strong foundation for the rational design of potent strain-transcending DBP-based vaccines against P. vivax.

RESULTS

Isolation of human monoclonal antibodies 053054 and 092096

Igh and Igl PCR products from 98 individual B cells were sequenced from one Cambodian donor, and 16 B cell clonal groups as defined by Ab V heavy chain sequences with the same inferred VH and JH germline sequences, identical CDR3 length, and the same or very similar CDR3 sequences. One or two clones were selected from each group and expressed as full-length IgG1, thereby creating monoclonal antibodies (mAbs). mAbs from eleven clones recognized DBP-II. We selected one mAb from two different clonal groups corresponding to two of the larger clonal groups in terms in the number of DBP-II-specific B cells isolated by single cell sorting. These two mAbs were designated as 092096 and 053054.

Structures of human antibodies 053054 and 092096 in complex with DBP-II

We solved two crystal structures of DBP-II in complex with a single-chain variable fragment (scFv) generated from the human mAbs 053054 and 092096 that were isolated from a Cambodian patient (Fig. 1a,c and Supplementary Table 1). The quality of the electron density maps clearly defined the contact sites of the DBP-II/antibody interface in both structures (Supplementary Fig. 1a,b). Both mAbs bind to the same face of DBP, although the orientation of the heavy and light chains with respect to DBP differs (Supplementary Fig. 2). The interacting residues showed a significant overlap between the two epitopes (Supplementary Table 2).

The discontinuous conformational epitope for 053054 is composed of amino acids D264-A281 and Q356-N372 belonging to two outer helices of subdomain 2, and E249 of the Nterminal helix in subdomain 1 (Fig. 1a,b and Supplementary Table 2). mAb 092096 binds to the discontinuous conformational epitope comprised of residues L270-K289, A355-W375, E249 and Y219 (Fig. 1c,d and Supplementary Table 2). All complementarity determining regions (CDRs) of both antibodies contact DBP (Supplementary Table 2), and the buried surface area and shape complementarity of the interactions are within standard parameters (Supplementary Fig. 1c,d). Strikingly, these epitopes differ from previously reported epitopes for broadly conserved inhibitory murine mAbs located in SD3 of DBP-II (Fig. 1e). The overlap of human epitopes in DBP coupled with distinct modes of binding indicate multiple avenues for antibody engagement of the surface comprised of residues D264-K289 and Q356-W375 in DBP.

Mechanism of neutralization

Binding of 053054 or 092096 does not cause conformational changes within DBP-II as there are negligible structure differences between antibody-bound, unbound and DARC-bound DBP (Supplementary Table 3). However, structural comparison of DBP-II/053054 and DBP-II/092096 with DBP-II/DARC complex revealed that mAbs 053054 and 092096 engage residues in the DARC-binding cleft and dimer-interface of DBP-II (Fig. 2). DBP uses residues F261-T266, L270-K289 and Q356-K367 to engage DARC ectodomain and residue range F261-F267, L270-Y278 and E352-Q356 to form a dimer interface [START_REF] Batchelor | Red Blood Cell Invasion by Plasmodium vivax: Structural Basis for DBP Engagement of DARC[END_REF] . These segments overlap considerably with the 053054 and 092096 epitopes suggesting that antibody association prevents DBP dimerization and DARC binding (Fig. 2c and2f).

Human antibodies 053054 and 092096 block DBP binding to RBCs and neutralize P. vivax invasion ex vivo

The structural studies suggest that the mAbs 053054 and 092096 directly block DBP binding to DARC on red blood cells and would, therefore, neutralize P. vivax merozoites. To test this hypothesis, we examined inhibition of DBP-II binding to RBCs (Fig. 3a). Both human mAbs completely blocked the binding with IC 50 values for 053054 and 092096 as 4.88 ± 1.06 nM and 1.63 ± 1.07 nM, respectively while an isotype control, 043038, could not inhibit binding. 092096 was chosen for its higher potency to test the human mAb's ability to neutralize P. vivax invasion of human reticulocytes ex vivo. Using P. vivax-infected erythrocytes obtained from Brazilian patients we showed that 092096 neutralizes P. vivax invasion with 43% invasion inhibition at a human mAb concentration of 100 μg/mL compared to no antibody (p = 0.0001) (Fig. 3b). We further performed the P. vivax ex vivo assay with Cambodian clinical isolates to assess the ability of this mAb to neutralize parasites from distinct geographical locations. Compared to no antibody 092096 neutralizes P. vivax invasion with 47.4% neutralization at a human mAb concentration of 100 μg/mL (p =0.002) and 86.6% neutralization at a human mAb concentration of 500 μg/mL (p = 0.0007) (Fig. 3c). Note that the degree of invasion inhibition is fairly consistent for the different parasite isolates. This result indicates a single human mAb that targets the receptor-binding residues and the dimer interface of DBP can neutralize invasion of multiple P. vivax isolates, from distinct geographical locations.

Neutralizing epitopes are widely recognized in patient populations

We examined whether the epitope recognized by 092096 is widespread in patient populations exposed to P. vivax (Fig. 3d,e). Functional mAbs found in individuals with high levels (≥80%) of blocking antibody activity to DBPII who resided in P. vivax-endemic regions of Cambodia recognize similar epitopes as 092096 (Fig. 3d). For example, the serum sample at the top right in Fig. 3d (in small box) shows 100% blocking activity of DBP-II binding to DARC N-terminus at a dilution of 1:20, and mAb 092096 (dilution of 1:50) competed for 90% of the blocking activity. The ability of mAbs 092096 and 087086 (a nonblocking mAb to DBPII) to compete with polysera stratified as to whether participants had 80-100% blocking activity versus lower blocking activity of 40-79% (Fig. 3e). mAb 092096 demonstrated clear competition with high-blocking activity polysera. These data demonstrate the epitopes identified here are widely recognized by sera in patient populations.

Mutations and ELISA reveal overlapping binding sites with different binding requirements

The epitopes were evaluated using surface mutant libraries of DBP-II and ELISA. Both mAbs 053054 and 092096 recognized wild type Sal-1 DBP-II and mutants 17 and 19, with substituted residues outside either epitope, equally well (Fig. 4a, and Supplementary Fig. 3). In contrast, mAb 053054 did not bind to mutants 18 and 20 as these mutants contain mutations in the epitope for 053054. Consistent with overlapping epitopes between the two mAbs, mAb 092096 also lost binding to mutant 20. Mutant 18, however, had no effect on binding to 092096. This divergence in binding to mutant 18 is caused by interaction differences due to the altered orientation of heavy and light chains of 053054 and 092096 with respect to DBP (Fig. 4b,c and Supplementary Fig. 2). Although residues substituted in mutant 18, F267, Y271, K274 and Y278, are bound to the core of 053054 and interact with CDR3 on both the heavy and light chains (Fig. 4b), they only bind to the periphery of 092096 and predominantly interact with its light chain (Fig. 4c). On the contrary, residues Y363, K367 and K370 substituted in mutant 20 interact with the CDR3 on the heavy chain in 092096 and contact the core of 053054 through CDRs 1 and 2 of its light chain. Therefore, this region of DBP is crucial for binding to both mAbs.

HDX-MS determination of the epitope for 053054 and 092096

Hydrogen deuterium exchange mass spectrometry (HDX-MS) was used to independently assess the epitopes for mAbs 053054 and 092096 in solution (Fig. 4d). In excellent agreement with the structures, peptide 268-281 is significantly attenuated from exchange upon binding to 053054, whereas no difference is detected for this peptide with 092096. Regions 288-298 and 364-379 show substantial HDX attenuation against 092096 predominantly on peptide 364-373. Peptide 364-373 also responds for 053054 but with less attenuation and converging kinetics indicate weaker binding for 053054. In contrast, peptides 288-298 and 374-379 are silent with 053054 (Fig. 4d). The remaining peptides either show minor differences in deuterium exchange upon antibody binding or no difference including the broadly conserved epitope for murine antibodies 2D10, 2H2 [START_REF] Chen | Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein[END_REF] (Fig. 4d, last panel, and Supplementary Fig. 4 and5).

Polymorphisms within or in the vicinity of the epitopes for 053054 and 092096 do not affect antibody binding to DBP

Having determined the human neutralizing epitopes and the mechanism of antibody neutralization, we examined the DBP variation within epitopes. We first determined antibody affinities for 053054 and 092096 to the reference strain Sal-1 DBP by using biolayer interferometry (BLI) (Supplementary Table 4 and Supplementary Fig. 6 and7). The steady state equilibrium dissociation constants were found to be 7.44 ± 0.36 nM for 053054 and 7.63 ± 0.29 nM for 092096, respectively. BLI also provides the association and dissociation rates that can inform the half-life of antibody binding. In both cases, the dissociation rates were remarkably slow, indicating antibody binding leads to a stable, longlived complex. The slow off rate is consistent with the HDX data that show no convergence of HDX kinetics at long exchange times. These results indicate that the two antibodies have comparable binding affinity.

A comparative alignment of 599 DBP sequences from diverse isolates of P. vivax revealed a number of polymorphisms within the epitopes for 053054, 092096, and adjacent residues (Supplementary Table 5). The most common polymorphisms individually and in combination are R263S and N372K. Sal-1, the strain that 053054 and 092096 were isolated and crystalized with, contains R263 and N372 at these positions. R263 is located in a disordered segment at the periphery of the 053054 epitope and therefore is not visible in the crystal structure of the complex (Fig. 5a). R263 is also distant from the epitope of 092096. Nevertheless, to determine the effect of polymorphism within or adjacent to the epitopes on the binding of 053054 and 092096 to DBP, the single mutants and the double mutant were generated and analyzed for the antibody binding by ELISA. All variants retained robust binding to both antibodies (Fig. 5b). We further quantified binding of antibodies to the DBP variants by BLI. Binding affinities showed no difference in equilibrium dissociation constants for the natural DBP variants compared to Sal-I DBP-II (Supplementary Table 4 and Supplementary Fig. 6 and7). In addition to the most frequent polymorphism, we tested whether the lower frequency polymorphisms L288F, I374M and T359R affected binding by BLI (Supplementary Table 4 and Supplementary Fig. 6 and7). I374M occurs most frequently in combination with N372K, and T359R occurs most frequently with R263S. Introducing these mutations into DBP also had no effect on antibody affinity or binding parameters. These results demonstrate that the polymorphisms within the epitopes do not affect antibody binding.

and 092096 heavy and light chain sequences show limited development from the germline genes

The lack of sequence similarity between mAbs 053054 and 092096 (Fig. 6a,b) indicates they do not evolve from a common progenitor. Nevertheless, both mAbs recognize overlapping epitopes in DBP. We analyzed the CDRs of the neutralizing antibodies to DBP to determine how drastic a deviation from germline the paratopes are and how dramatically the antibodies would need to develop. Nucleotide and amino acid sequences were analyzed by NCBI/ IgBlast and IMGT/V-Quest to identify the germline V, D and J genes (Fig. 6c-f). The analysis showed that 053054 and 092096 heavy and light chain sequences are very close to the germline genes with a minimal number of junction insertions (Supplementary Table 6). A limited number of somatic hypermutations create direct contacts between the antibodies and DBP: two (T62 and S110) of fourteen contact residues in the 053054 heavy chain; four (E173, H196, T237 and E240) of fifteen contact residues in the 053054 light chain; two (Y108 and F109) of twenty residues in the 092096 heavy chain; and three (R175, P177 and D197) of fourteen residues in the 092096 light chain (Supplementary Table 2). The low number of somatic hypermutations reflects that the mAbs underwent limited affinity maturation to achieve broadly neutralizing activity (Supplementary Table 6).

DISCUSSION

We found that naturally acquired human antibodies target the DARC-binding site and dimer interface in PvDBP-II. The epitope mapping data are consistent and demonstrate the epitope for mAb 053054 spans residues E249, D264-A281, Q356-N372 of helices belonging to SD2. This epitope overlaps with the epitope for 092096 that comprises residues of Y219, E249, L270-K289 and Q356-W375. These findings result in a clear mechanism for inhibition and neutralization. These human antibodies function by preventing DBP-II binding to the N-terminus of DARC and are neutralizing. Strikingly, we show that a single human mAb 092096 that targets the receptor-binding residues and dimer interface of DBP can neutralize invasion of reticulocytes by multiple P. vivax isolates from distinct geographical locations.

The human mAb epitopes are distinct from broadly neutralizing epitopes of murine mAbs [START_REF] Chen | Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein[END_REF] and this diversity of epitopes residing in different subdomains of DBP-II is reminiscent of receptor-binding site [START_REF] Schmidt | Viral receptor-binding site antibodies with diverse germline origins[END_REF] and stem [START_REF] Ekiert | Antibody recognition of a highly conserved influenza virus epitope[END_REF] epitopes in influenza hemagglutinin. Although 053054 and 092096 mAbs do not share a germ line lineage, they target overlapping epitopes of DBP involved in DARC engagement. Our results suggest the development of neutralizing mAbs can arise from multiple progenitor B cells with straightforward CDR affinity maturation to result in high neutralization and breadth. The epitopes for broadly neutralizing human antibodies presented in this study are, therefore, an excellent platform to develop DBP-II based vaccines.

DBP is highly polymorphic, and this variation may confound vaccine development [START_REF] Ceravolo | Naturally acquired inhibitory antibodies to Plasmodium vivax Duffy binding protein are short-lived and allele-specific following a single malaria infection[END_REF][START_REF] Vanbuskirk | Antigenic drift in the ligand domain of Plasmodium vivax duffy binding protein confers resistance to inhibitory antibodies[END_REF] . Identification of highly conserved epitopes of neutralizing antibodies will have a profound positive effect on the development of a DBP-based vaccine. One caveat of the invasion assays presented here is that the isolates tested were not sequenced to identify the diversity of DBP variants used in the invasion assays. Therefore, we examined the breadth of binding of the antibodies to DBP variants that encompassed polymorphisms within the epitopes. We demonstrate that the most prevalent polymorphisms within or adjacent to the epitopes, as determined by comparative analysis of 599 DBP sequences, were residues N372K, R263S, I374M, L288F, and T359R. As we showed, these polymorphisms individually and in combination do not affect antibody binding to DBP-II. This suggests that human neutralizing antibodies have evolved to compensate for polymorphisms and variation in DBP likely due to repeated exposure to P. vivax, thereby becoming broadly neutralizing antibodies.

A significant challenge for successful vaccination is the need for complex maturation of broadly neutralizing antibodies from their precursors requiring long developmental pathways. For example, broadly neutralizing antibodies that target HIV must undergo dramatic development including multiple rounds of affinity maturation to result in CDRs capable of neutralization breadth and involved extensive lengthening of the CDRs [START_REF] Verkoczy | Role of immune mechanisms in induction of HIV-1 broadly neutralizing antibodies[END_REF] . This has had a fundamental impact on the immunization scheme necessary to generate suitable broadly neutralizing antibodies for HIV. In contrast, the number of somatic hypermutations seen here reflects that 053054 and 092096 underwent limited affinity maturation for broadly neutralizing activity. This limited development for P. vivax-neutralizing antibodies differs from the extensive development needed for HIV-neutralizing antibodies, and suggests immunization with DBP-based vaccines will likely lead to the ready production of neutralizing mAbs.

The findings from this study of DBP will inform neutralization of and vaccine development for an important family of parasite invasion ligands. DBP is a member of the Erythrocyte Binding like (EBL) family of Plasmodium red cell invasion proteins that includes EBA-175 [START_REF] Orlandi | Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum[END_REF][START_REF] Klotz | Binding of Plasmodium falciparum 175-kilodalton erythrocyte binding antigen and invasion of murine erythrocytes requires N-acetylneuraminic acid but not its O-acetylated form[END_REF][START_REF] Sim | Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum[END_REF][START_REF] Tolia | Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum[END_REF][START_REF] Chen | Structural and Functional Basis for Inhibition of Erythrocyte Invasion by Antibodies that Target Plasmodium falciparum EBA-175[END_REF][START_REF] Salinas | Critical Glycosylated Residues in Exon Three of Erythrocyte Glycophorin A Engage Plasmodium falciparum EBA-175 and Define Receptor Specificity[END_REF][START_REF] Salinas | A quantitative assay for binding and inhibition of Plasmodium falciparum Erythrocyte Binding Antigen 175 reveals high affinity binding depends on both DBL domains[END_REF] , EBA-140 [START_REF] Mayer | Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175[END_REF][START_REF] Narum | A novel Plasmodium falciparum erythrocyte binding protein-2 (EBP2/BAEBL) involved in erythrocyte receptor binding[END_REF][START_REF] Lobo | Glycophorin C is the receptor for the Plasmodium falciparum erythrocyte binding ligand PfEBP-2 (baebl)[END_REF][START_REF] Lin | Crystal and solution structures of Plasmodium falciparum erythrocyte-binding antigen 140 reveal determinants of receptor specificity during erythrocyte invasion[END_REF][START_REF] Malpede | Molecular basis for sialic acid-dependent receptor recognition by the Plasmodium falciparum invasion protein erythrocyte-binding antigen-140/BAEBL[END_REF][START_REF] Lopaticki | Reticulocyte and erythrocyte binding-like proteins function cooperatively in invasion of human erythrocytes by malaria parasites[END_REF][START_REF] Persson | Erythrocyte-binding antigens of Plasmodium falciparum are targets of human inhibitory antibodies and function to evade naturally acquired immunity[END_REF] , EBA-181 [START_REF] Lopaticki | Reticulocyte and erythrocyte binding-like proteins function cooperatively in invasion of human erythrocytes by malaria parasites[END_REF][START_REF] Gilberger | A novel erythrocyte binding antigen-175 paralogue from Plasmodium falciparum defines a new trypsin-resistant receptor on human erythrocytes[END_REF][START_REF] Mayer | Polymorphism in the Plasmodium falciparum erythrocyte-binding ligand JESEBL/ EBA-181 alters its receptor specificity[END_REF][START_REF] Maier | Polymorphisms in erythrocyte binding antigens 140 and 181 affect function and binding but not receptor specificity in Plasmodium falciparum[END_REF] , and EBL-1 [START_REF] Mayer | Glycophorin B is the erythrocyte receptor of Plasmodium falciparum erythrocytebinding ligand, EBL-1[END_REF] . EBL family members have either a single or double tandemly arranged Duffy Binding Like (DBL) domains that Plasmodium parasites use for receptor-binding and attachment to the host erythrocytes. This study has established that a single monoclonal antibody that targets the receptor binding residues of DBP can neutralize P. vivax parasites, and underscores the importance of targeting the functional receptor-binding residues of other EBL ligands. In addition, DBP and EBA-175 undergo receptor-induced dimerization required for invasion [START_REF] Batchelor | Dimerization of Plasmodium vivax DBP is induced upon receptor binding and drives recognition of DARC[END_REF][START_REF] Batchelor | Red Blood Cell Invasion by Plasmodium vivax: Structural Basis for DBP Engagement of DARC[END_REF][START_REF] Tolia | Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum[END_REF][START_REF] Chen | Structural and Functional Basis for Inhibition of Erythrocyte Invasion by Antibodies that Target Plasmodium falciparum EBA-175[END_REF] . mAbs 053054 and 092096 also block the dimer interface of DBP suggesting that antibody disruption of oligomeric interfaces in antigens could be a general method for neutralization. Defining the full complement of mechanisms for neutralizing DBL domains using antibodies will aid in the direct design of highly efficient immunogens against malaria. Although additional receptor-ligand interactions have been identified with proposed roles in P. vivax malaria [START_REF] Ntumngia | A Novel Erythrocyte Binding Protein of Plasmodium vivax Suggests an Alternate Invasion Pathway into Duffy-Positive Reticulocytes[END_REF][START_REF] Gruszczyk | Transferrin receptor 1 is a reticulocyte-specific receptor for[END_REF] , a P. vivax vaccine will likely include DBP given the central role of the DBP:DARC interaction in infection.

In conclusion, we studied two human neutralizing monoclonal antibodies 053054 and 092096 that target P. vivax DBP isolated from an individual with naturally acquired immunity to P. vivax and with high levels of blocking antibodies to DBP. These two monoclonal antibodies arose from different clonal groups, bind overlapping epitopes in DBP, and compete with mAbs found extensively in multiple patients with blocking antibody activity to DBP. We characterized their interaction with DBP, confirmed their high neutralizing potential, structurally defined their epitopes and proposed a mechanism for neutralization. This work extends our knowledge of the function of parasitic ligands and methods to leverage the immune response for neutralization. These naturally acquired human antibodies appear to have a short pathway for development with broadly neutralizing capacity. Monoclonal Abs to DBP-II following immunization of mice recognized a distinct set of epitopes that do not target the receptor binding domain. Therefore, current vaccination regiments may not favor targeting the receptor-binding domain and dimer interface of DBP to generate antibodies capable of blocking parasite invasion. Furthermore, while naturally acquired immunity can result in the desired neutralizing antibodies described here, naturally acquired immunity requires repeated exposure to the parasite and develops over the lifetime of a patient. The structure definition of conformational epitopes provides a solid foundation for focusing the immune response to the neutralizing human antibody epitopes identified through immunogen design and structural vaccinology. This work enables the design of immunogens that primarily elicit antibodies to the neutralizing epitopes identified here and to avoid or reduce targeting immunodominant but non-protective epitopes within DBP. Improved immunogens will aid in the design of successful and efficient DBP-based vaccines for malaria.

ONLINE METHODS

Cell staining and sorting of antigen-specific memory B cells

Blood samples were obtained from Cambodians, Brazilians and Papua New Guineans residing Pv endemic areas 2,51-53 . Samples were screened for blocking Abs to PvDBPII (see below). A subset of Cambodian and Brazilian adults with high levels of blocking Abs to PvDBPII (>80% binding inhibitory activity at a titer 1:10 or higher) donated up to 200 ml of peripheral venous blood. Peripheral blood mononuclear cells (PBMC) were from prepared from the venous blood and cryopreserved. Institutional Single cells were identified and sorted according to previously described techniques [START_REF] Wardemann | Expression cloning of human B cell immunoglobulins[END_REF] from cryopreserved PBMC without activation. B cells from cryopreserved PBMC were enriched using immunomagnetic positive selection with anti-CD19 magnetic MACS beads (MiltenyiBiotec). Cells were washed at least twice with 5 ml FACS buffer with 3 mM EDTA and adjusted to a cell density of 1-2×10 6 cells/ml. Cells were stained with mouse antihuman CD20 (PE-Cy5.5, Invitrogen) and anti-human IgG Abs (PE-Cy7 clone G18-145; Becton, Dickinson and Co., BD) along with DBPII-or TTCF-prepared tetramers using streptavidin coupled with allophycocyanin (BD) and SYTOX Green Dead Cell Stain (Invitrogen) to gate out dead cells. Stained CD19+ cells were sorted on a BD FACSAria II based on size and complexity and individual DBPII-or TTCF-specific CD20+, IgG+ memory B cells were single cell sorted directly into 4μl mRNA extraction buffer on a cooled 96-well metal block. After cells were collected, plates were frozen immediately on dry ice and stored at -80°C until further processing.

cDNA synthesis

The 96-well plates with single cells were thawed on ice; a cold volume of 7 μl containing 300ng random hexamers (Qiagen Operon), 12U Rnasin (Promega) and 0.9% NP-40 (Thermo Scientific Pierce) was added to each well. After thorough pipetting and rinsing, wells were capped, centrifuged at 4°C, heated to 68°C in a thermal cycler for 5 min and placed on ice for at least 1 min. Reverse transcription was performed with the addition of 7 μl containing 3.6 μl 5X reverse transcriptase buffer, 10 U RNAsin (Promega), 62 U Superscript III RT (Invitrogen), 0.62 μl dNTPs 25 mM each (Omega Bio-Tek) and 1.25 μl 0.1 M DTT (Sigma). All wells were capped, the plate placed in a cold rack and vortexed for 10 sec before centrifugation at 300xg. Thermal cycler conditions for reverse transcription were as follows: 42°C 5 min, 25°C 10 min, 50°C 60 min, 94°C 5 min and 4°C hold. When completed, 10 μl of nuclease-free PCR water was added to each well.

Ig gene amplification

Immediately following cDNA synthesis, IgG genes (Igg) were amplified in a total of 20 μl per well for the first round of nested PCR for IgG heavy chain (Iggh), IgG kappa (Iggκ) and IgG lambda (Iggλ), utilizing primers (Supplemental Table 1) as previously described [START_REF] Tiller | Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning[END_REF] . In brief, a master mix was prepared consisting of 15.58 μl water, 2 μl 10X HotStar PCR buffer (Qiagen), 0.065 μl 5' primer mix, 0.065 μl 3' primer, 0.2 μl dNTP solution and 0.09 μl HotStarTaq per well, to which 2 μl cDNA from individual sorted B cells were added and Igg amplified under the following conditions: thermal cycle PCR at 94°C for 15 min; 50 cycles at 94°C for 30 sec, then 58°C (Iggh and Iggκ) or 60°C (Iggλ) for 30 sec, then 72°C for 55 sec; then one cycle at 72°C for 10 min. Second round of nested PCR for Iggh, Iggκ and Iggλ utilized 2 μl of first-round PCR product with second-round primers [START_REF] Tiller | Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning[END_REF] and the same master mix protocol, with the following conditions: thermal cycle PCR at 94°C for 15 min; 50 cycles at 94°C for 30 sec, then 58°C (Iggh and Iggκ) or 60°C (Iggλ) for 30 sec, then 72°C for 45 sec; then one cycle at 72°C for 10 min. The PCR product generated was purified and sequenced, with V(D)J genes determined using IMGT/V-Quest [START_REF] Giudicelli | IMGT/V-QUEST: IMGT standardized analysis of the immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences[END_REF] .

Specific V(D)J region amplification and cloning

Primers specific with restriction enzyme sites for V and J regions were used to amplify the first-round PCR product to generate a fragment for cloning based on previously described primers 55 . PCR product was purified, restriction enzyme digested, cloned into Iggh, Iggκ or Iggλ expression vectors and chemically transformed into 5 μl aliquots of TOP10 E. coli cells (Thermo Fisher Scientific). Successful transformants were screened by PCR amplification utilizing a vector-specific primer paired with an insert-specific primer, sequenced and compared to the second-round PCR product sequence.

Definition of clonal groups

Clonal groups were based on heavy chain nucleotide sequences. Any PCR product with >0.8% nucleotide sequences with a Phred score <20 was excluded. From PCR-amplified sequences, we determined heavy chain alleles using IMGT/V-QUEST (http://www.imgt.org, international ImMunoGeneTic information system). A clonal group was defined by Ab V heavy chain sequences with the same inferred VH and JH germline sequences, identical CDR3 length, and the same or very similar CDR3 sequences (CDR, complementaritydetermining region), i.e. >72% similarity of each CDR3 amino acid sequence. Clonal grouping was determined using Sequence Manipulation Suite: Ident and Sim [START_REF] Stothard | The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences[END_REF] using Abspecific clusters as previously defined [START_REF] Mirsky | Antibody-specific model of amino acid substitution for immunological inferences from alignments of antibody sequences[END_REF] .

MAb expression and purification

Two plasmids that included coding sequences for full-length IgG1 heavy and light chains were transfected into HEK293-H cells using polyethyleneimine (PEI). Five hundred micrograms PEI were incubated for 25 min at room temperature with 250μg of each plasmid and then added to the HEK293-H at a density of 1×10 6 cells/ml in total volume of 500ml [START_REF] Wardemann | Expression cloning of human B cell immunoglobulins[END_REF][START_REF] Tiller | Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning[END_REF] . Transfected cells were adapted for growth in Freestyle 293 serum-free expression medium (Gibco, Thermo Fisher Scientific) under suspension conditions of 37°C and 8.5% CO2. Cells were centrifuged 96 h post transfection and culture medium was harvested, filtered through a 0.22 μm filter and supernatants concentrated 20 times using a 50 kDa cut-off Vivaflow 50 System (Vivasciences). One volume of IgG binding buffer (Thermo Scientific Pierce) was added and IgG was purified on a Protein A HP HiTrap column (GE Healthcare) eluted with IgG elution buffer (Thermo Scientific Pierce) and neutralized with 1M Tris pH 9.0. Proteins were concentrated and buffer exchanged with PBS using Amicon Ultra4 10kDa. Protein concentrations were determined on Nanodrop (Thermo Fisher Scientific) and sample purity was analyzed by SDS-PAGE.

Protein expression and purification

Sal-1 DBP-II WT and DBP-II mutants were prepared as described [START_REF] Batchelor | Dimerization of Plasmodium vivax DBP is induced upon receptor binding and drives recognition of DARC[END_REF][START_REF] Batchelor | Red Blood Cell Invasion by Plasmodium vivax: Structural Basis for DBP Engagement of DARC[END_REF][START_REF] Chen | Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein[END_REF] . scFvs were produced by expression and refolding from E. coli. The light chain variable region was linked to the heavy chain variable region by using a (GGGGS) 3 linker, cloned into the pET28(a+) expression vector using restriction sites NcoI/XhoI and expressed in E. coli.

Inclusion bodies were solubilized in 6 M guanidinium hydrochloride overnight at 4 °C and refolded with 400 mM L-Arginine, 2 mM reduced glutathione and 0.2 mM oxidized glutathione in 50 mM Tris, pH 8.0, 10 mM EDTA and 0.1 mM PMSF at 4 °C for 24 h. Refolded scFv were concentrated in Amicon®Stirred Cells using Biomax® 10 kDa Ultracentrifugation Discs and purified by size exclusion chromatography on GF200 (GE Healthcare) into 10 mM HEPES pH 7.4, 100 mM NaCl.

Protein crystallization and data collection

Purified DBP-II and 053054 -scFv or 092096 -scFv were mixed in a 1.2:1 (antigen:antibody) molar ratio and incubated at room temperature for 30 minutes. Complexes was purified by size exclusion chromatography (GF200 -GE Healthcare) in 10 mM HEPES pH 7.4, 100 mM NaCl. Crystals were grown by hanging-drop vapor diffusion by mixing 1 μL of complex at 7 mg/mL for DBP-II/053054 -scFv or 10 mg/mL for DBP-II/092096 -scFv with 2 μL of reservoir (0.2M ammonium sulfate, 0.1 M sodium citrate pH 5.6, 25% w/v PEG 4,000 for DBP-II/053054 -scFv or 12% w/v PEG 6,000, 0.1 M MES, pH 6.0 for DBP-II/ 092096 -scFv ). Crystals were flash frozen with 30% ethylene glycol as cryoprotectant in liquid nitrogen. Data for DBP-II/053054 -scFv were collected at the beamline 4.2.2 of the Advanced Light Source. Data for DBP-II/092096 -scFv were collected at the beamline 23-ID-D (GM/CA) of Advanced Photon Source. Both data sets were processed with XDS 59 .

Structure solution and analysis

The DBP-II/053054 -scFv and DBP-II/092096 -scFv structures were solved by molecular replacement in PHASER [START_REF] Mccoy | Phaser crystallographic software[END_REF] by using PDB 4NUV and scFv domains modeled by the PIGS server [START_REF] Marcatili | automatic prediction of antibody structures[END_REF] as search models. Refinement was performed using PHENIX [START_REF] Adams | PHENIX: building new software for automated crystallographic structure determination[END_REF] and COOT [START_REF] Emsley | model-building tools for molecular graphics[END_REF] . Summary of diffraction data and refinement statistics are given in Supplementary Table 1. The quality of the models was assessed using the MolProbity server [START_REF] Davis | MolProbity: all-atom contacts and structure validation for proteins and nucleic acids[END_REF] . All structures have good Ramachandran statistics with no outliers. 94.89 % and 5.11% of residues for DBP-II/ 053054 -scFv or 93.80 % and 6.20% of residues for DBP-II/092096 -scFv in favored and allowed regions of Ramachandran plot, respectively. Interaction interfaces were determined using PDBePISA [START_REF] Krissinel | Inference of macromolecular assemblies from crystalline state[END_REF] . Software used in the project was installed and configured by SBGrid [START_REF] Morin | Collaboration gets the most out of software[END_REF] .

Hydrogen-Deuterium Exchange Mass Spectrometry

Before HDX footprinting experiments, 10 μM PvDBP-II was incubated with 12 μM of mAb in PBS for 30 min at 25 °C. The apo-state sample was 10 μM PvDBP in PBS. Continuous HDX on apo-and holo-state samples were performed after diluting into D 2 O buffer at the ratio of 1:5 and incubate for 10, 30, 60, 120, 900, 3,600, and 14,400 s, as described previously [START_REF] Chen | Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein[END_REF] followed by on-line pepsin digestion and reversed-phase HPLC separation. Peptides were analyzed by a Thermo LTQ-FT mass spectrometer (Thermo Scientific). Deuterium uptakes of various time points were analyzed using HDX Workbench (Scripps, FL).

ELISA

The ELISAs were performed as previously described [START_REF] Salinas | Critical Glycosylated Residues in Exon Three of Erythrocyte Glycophorin A Engage Plasmodium falciparum EBA-175 and Define Receptor Specificity[END_REF] . Briefly, BSA, Sal1 DBP-II, and DBP-II mutants were coated on plates overnight at 4 °C, washed with PBS/Tween-20, and blocked with 2% BSA in PBS/Tween-20 for 1 h at room temperature. The plates were washed with PBS/Tween-20 and then incubated with each mAb (053054, 092096) in a concentration 250ng/ml for 1 h at room temperature. The plates were washed with PBS/ Tween-20 and incubated with an anti-human antibody conjugated to HRP for 30 min at room temperature. After a final wash step with PBS/Tween-20 ELISA, substrate TMB was added to the plates. The reaction was quenched by addition of 0.2 M sulfuric acid, and the absorbance at 450 nm was measured using a POLARstar Omega (BMG Labtech) plate reader.

Biotinylation of BirA-Tagged Sal-1 DBP-II

BirA-tagged Sal-1 DBP-II was buffer exchanged into Phosphate buffered saline. Then 50 μL of BiomixA (Avidity), 50 μL of BiomixB (Avidity), and 10 μL of 5 mM D-biotin (Avidity) were added to the protein along with BirA ligase, followed by overnight incubation at 4 °C. The biotinylation was confirmed by Western Blot using Streptavidin HRP-conjugate (Thermo Scientific). Before use, the reaction mix was buffer-exchanged into PBS.

Biolayer Interferometry Binding Assays

The binding affinity of purified Sal1 DBP-II protein with human mAbs was monitored by BLI conducting on an Octet-Red96 device (Pall ForteBio) by using Streptavidin (SA) biosensors (ForteBio). The biotinylated DBP-II was loaded onto biosensors until saturation, typically 1 μM for 15 min, in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% P20 surfactant with 3% BSA. The mAb scFvs (analyte) were applied at two-fold serial dilution concentrations (100 nM to 1.675 nM). Association and dissociation were measured at 25 °C for all mAbs. The real-time data were analyzed using Biaevaluation 4.1 (GE Healthcare). Steady-state equilibrium concentration curves were fitted using a 1:1 binding model.

Fluorescence Activated Cell Sorting Assay

Biotinylated purified DBP-II protein in concentration of 10 nM was incubated with ten different concentrations of 053054 or 092096 (from 0.03 nM to 1 μM interval of concentrations) for 1 h at RT. Red blood cells were added and incubated for an additional 1 h at RT. To detect bound DBP Alexa Fluor 488 Streptavidin conjugate (Fisher) was added to the mix and incubated at RT for 1 h followed by washing twice with PBS. FITC labeling was measured by flow cytometry. An IC 50 value was calculated in GraphPad Prism from three independent biological replicates. IC 50 curve for the isotype control mAbs 043038 was plotted as a negative control.

P. vivax invasion assay

P. vivax ex vivo assays were independently performed with clinical isolates from Brazil and Cambodia. Cryopreserved P. vivax infected erythrocytes (iRBC) obtained from Brazilian donors with acute vivax malaria were thawed, enriched using Percoll density-gradients, immediately re-suspended in 100 μl IMDM plus 10% human AB serum with GlutaMax (1:100) to a hematocrit of 6% and cultured with a gas mix of 5% O 2 , 5% CO 2 , 90% N 2 at 37 °C until established. The cultures were performed in quadruplicate from a single isolate of P. vivax and incubated with 092096 or isotype control comprising anti-TT monoclonal antibody 043038 at a concentration of 100 μg ml-1 and examined for parasite viability and maturation three times: after 20 h of initial incubation (initial growth from ring stages into early trophozoites, since trophozoites and schizont stages of the parasites do not survive cryopreservation), after additional incubation for 38 h, at which time most of the parasite have matured to schizont stages prior to merozoite release, parasite cultures were supplemented with fresh blood cells enriched for reticulocytes to ~2.5-3.0% at a ratio of 1:1 and additional incubation for 24 h to allow for new erythrocyte invasion. For examination, two smear slides were prepared for each culture well and stained with Giemsa. Rings and early trophozoites per 20,000 RBC were counted blindly three times. Cytochalasin D (5 μg/ mL), a cell-permeable fungal toxin that inhibits merozoite invasion of erythrocytes, was used as a positive invasion inhibitor control.

For the assay with Cambodian isolates, cryopreserved iRBCs obtained from Cambodian patients with acute P.vivax malaria were thawed and cultured in IMDM medium (Gibco) supplemented with 0.5% Albumax II (Gibco), 2.5% heat-inactivated human serum, 25 mM HEPES (Gibco), 20 μg/ml gentamicin (Sigma) and 0.2 mM hypoxanthine (C-C Pro) for ~24 or ~48h until a majority of schizont stage parasites were noted. The schizont-infected erythrocytes were enriched using Percoll-KCl as described [START_REF] Rangel | Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays[END_REF] , then mixed at a ratio of 1 erythrocyte to 1 with reticulocytes enriched from cord blood and labeled with CellTrace Far Red dye. The cultures were incubated for ~8h in a final volume of 100 μl in 96 well plates or 20 μl in 384 well plates, in presence of the human monoclonal antibodies 092096 or anti-TT MoAb 043038 while a positive invasion inhibitor control used the mouse anti-DARC monoclonal antibody 2C3 [START_REF] Russell | A reliable ex vivo invasion assay of human reticulocytes by Plasmodium vivax[END_REF] at 100 μg/ml. Cells were stained with DNA stain Hoechst 33342 post invasion and examined by flow cytometry. Reticulocytes which were Hoechst 33342 and Far Red positive were scored as new invasion events. Invasion of reticulocytes ranged from 0.19% to 6.52% (media alone) for the six experiments, each of which corresponded to one of six P. vivax isolates.

DBP-II patient population ELISAs and binding inhibition of DBPII to DARC fusion protein

To assess Ab blocking activity, plasma from Pv-exposed individuals was incubated with DBP-II at specified concentrations. We then measured levels of binding inhibition of DBP-II to a fusion protein containing amino acids 1-60 from the DARC N-terminal region fused to the Fc region of human IgG (nDARCIg) [START_REF] Grimberg | Plasmodium vivax invasion of human erythrocytes inhibited by antibodies directed against the Duffy binding protein[END_REF][START_REF] Choe | Sulphated tyrosines mediate association of chemokines and Plasmodium vivax Duffy binding protein with the Duffy antigen/receptor for chemokines (DARC)[END_REF] . Pooled plasma samples with high blocking activity from Papua New Guinea or Brazil served as positive controls. Diluent alone or pooled samples from North Americans not exposed to malaria were used for negative controls. Percent inhibition was calculated by (1-(optical density [OD] of test sample/OD of negative control) x100).

Competition experiments

For assays measuring competition between mAbs and naturally-acquired Abs, plates were coated with mAbs at 0.5 μg/ml per well and remained overnight at 4 °C, then washed with PBST and blocked with 3% BSA in PBST for 2 h at room temperature. Serum from naturally immune subjects, at a dilution of 1/50, was pre-incubated with biotinylated DBP-II Sal I (20 ng/ml) for 20 min at room temperature. The serum mixture was added to plates and incubated 1 h at room temperature. Detection of DBP-II was achieved using High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific) as described for competition assays between individual mAbs.

DBP epitope conservation analysis

A total of 599 sequences representing global variation in DBP-II [START_REF] De Sousa | Worldwide genetic variability of the Duffy binding protein: insights into Plasmodium vivax vaccine development[END_REF] was obtained from GenBank (accession codes XM_001608337.1, DQ156519, AF289480-AF289483, AF289635-AF289653, AF291096, AY970837-AY970925, AF469515-AF469602, U50575-U50590, DQ156513, DQ156515, DQ156522-DQ156523, AF215737-AF215738, AF220657, AF220659-AF220667, FJ491142-FJ491241, EF219451, EF368159-EF368180, EF379127-EF379132, EF379134, GU143914-GU144013, DQ156520, EU812839-EU812960, and EU860428-EU860438), aligned using ClustalW [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF] , and inspected in JalView [START_REF] Waterhouse | Jalview Version 2--a multiple sequence alignment editor and analysis workbench[END_REF] . All 599 sequences contained complete coverage for the 053054 and 092096 epitopes.

Antibody evolution analysis

To compare heavy and light chains of 053054 and 092096 with germlines we used IMGT/V-Quest, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis [START_REF] Brochet | IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis[END_REF] .

Statistical analyses

Analysis of ELISA data was performed using GraphPad Prism version 6.03 (GraphPad Software). because of limited number of parasites available for different culture conditions. The isotype matched mAb 043038 serves as an additional negative control. Statistical comparison between non-inhibitory mAb 043038 (n=2) and neutralizling mAb 092096 was performed with one-way ANOVA and Dunnett's test (p =0.002 for 100 μg/mL mAbs concentration, n=5, p =0.0007 for 500 μg/mL mAbs concentration, n=3). d, Human mAbs compete with epitopes recognized by serum from individuals with high levels of DBPII-specific blocking activity (≥80%) residing in Cambodia. Y-axis indicates the level of blocking activity using a DBPII:DARC binding assay, which measures the overall blocking activity in a serum sample. The x-axis is the percentage of blocking activity in the sample for which a given mAb competed. e, The box and whisker plots (median blocking activity, 25% and 75% interquartile ranges, and 95% confidence intervals) examine the degree to which mAbs 092096 and 087085 compete with serum blocking activity stratified as to whether participants had 80-100% blocking activity (n=33 for both mAbs, p<0.0001) versus lower activity of 40-79% (n=31 for both mAbs, p=0.34). The sample size for the two strata is shown in the scatter plots. Difference in blocking activity was examined using two-tailed Mann-Whitney U test.

  review boards from the United States National Institutes of Health (NIAID protocol #08-N094, Clinicaltrials.gov NCT00663546), Cambodian Ministry of Health, University Hospital of the University of Sao Paulo (1025/10), National Human Research Ethics Committee of the Ministry of Health of Brazil (551/2010), Medical Research Advisory Counsel of Papua New Guinea (PNGIMR No 1409, PNG MRAC No. 1400, and UH IRB No. 04-14-19) and University Hospitals of Cleveland Medical Center approved the protocols. Written informed consent was obtained from all study participants or their parents/guardians.

Figure 1| .

 1| Figure 1|. Structural definition of human antibody epitopes in DBP. a, Overall structure and epitope for the 053054 complex. Gray -DBP-II. Dark blue -053054 heavy chain. Orange -053054 light chain. Red -epitope. b, orthogonal detailed view of the epitope for 053054 in DBP. Gray -DBP-II. Dark blue -DBP residues contacted by the 053054 heavy chain. Orange -DBP residues contacted by the 053054 light chain. Beige -DBP residues contacted by both heavy and light chains. c, Overall structure and epitope for the 092096 complex. Gray -DBP-II. Light blue -092096 heavy chain. Pink -092096 light chain. Red -epitope. d, orthogonal detailed view of the epitope for 092096 in DBP. Gray -DBP-II. Light blue -DBP residues contacted by the 092096 heavy chain. Pink -DBP residues contacted by the 092096 light chain. Beige -DBP residues contacted by both heavy

Figure 2| .

 2| Figure 2|. Neutralizing mAbs 053054 and 092096 block DARC-binding site and dimer interface. a, Overlay of the 053054 epitope with the DARC-binding residues in DBP. Orange -overlap between the 053054 epitope and the DARC binding site. Yellow/Orange -DARC binding site. Red/Orange -the 053054 epitope. b, Overlay of 053054 epitope with the dimer interface of DBP. Purple -overlap between the 053054 epitope and the dimer interface. Blue/Purple -dimer interface. Red/Purple -the 053054 epitope. c, Mapping the 053054 epitope on the tetramer structure of DARC-bound DBP reveals the epitope overlaps with the DARC binding residues and dimer interface. Grey -DBP. Green -DARC monomer 1. Purple -DARC monomer 2. Red -epitope of 053054. d, Overlay of the 092096 epitope with the DARC-binding residues in DBP. Orange -overlap between the 092096 epitope and

Figure 3| .

 3| Figure 3|. Human antibodies block DBP binding to RBCs and P. vivax invasion. a, Inhibition of DBP-II binding to RBCs. The isotype matched mAb 043038 was used as a negative control. Data shown are mean ± SD of three biological replicates. b, Neutralization data for a Brazilian isolate of P.vivax invasion of human reticulocytes by human mAb 092096. Individual data points represent the total number of P. vivax infected cells per 20,000 RBCs and are shown as mean ± 1 SEM, for one P. vivax isolate performed in four biological replicate cultures in one experiment. Statistical differences between 092096 and no invasion inhibitor control (No Abs = medium alone) and non-inhibitory mAb 043038 were analyzed by one-way ANOVA and Dunnett's test (p =0.0001 and 0.0006, respectively). Cytochalasin D, an actin inhibitor, served as a positive control for the inhibition of invasion. c, Neutralization data for Cambodian isolates of P.vivax invasion of human reticulocytes by human mAb 092096. Each data point represent results from one of five P. vivax isolates and the results presented as mean ± 1 SEM. These data represent results from five separate experiments, each with a different P. vivax isolate. All experiments had no Ab control and 4 out of 5 experiments used mouse anti-DARC monoclonal antibody 2C3 as a positive control and one experiment used heparin as positive control (11% invasion relative to no antibody, not shown in figure). The other controls were performed with only some experiments

Figure 5| .

 5| Figure 5|. Strain-transcending human mAbs overcome polymorphisms located within the vicinity of their epitopes. a, Location of polymorphisms R263S and N372K on DBP-II surface shown in DBP-II/ 053054 and DBP-II/092096 structures. Polymorphic residues 263 and 372 shown in ball representation in yellow and green, respectively. The location of R263 was modeled as this residue is located in a disordered segment that is not visible in either structure. b, ELISA for 053054 and 092096 against Sal-I DBP-II wild type and three DBP-II natural variants, R263S, N372K and double mutant R263S/N372K. Data are shown as mean ± SD, calculated from three independent experiments. Statistical differences between wild type Sal-1 DBP and the mutants were analyzed by one-way ANOVA and Dunnett's test (****p < 0.0001).

Figure 6| .

 6| Figure 6|. Monoclonal antibody maturation. Sequence identity analysis for 053054 and 092096 mAbs by a, heavy and b, light chain alignment. CDRs 1-3 are marked at the top of the alignment. Similar residues are highlighted gray, identical black. c-f, Amino acid sequence alignment of the variable heavy chain and light chain of the mAbs (c and d) 053054 and (e and f) 092096 against the germline V, D and J gene members with the highest sequence similarity defined by NCBI/ IgBlast and IMGT/V-Quest indicated in each alignment. Complementarity determining regions (CDR 1-3) are shown at the top of the alignment. Amino acid mutations are highlighted green. Non-silent somatic hypermutations in CDRs are indicated by black stars. Red triangles mark insertions in the junction region. The secondary structure elements are

Nat Microbiol. Author manuscript; available in PMC 2019 November 27.

ACKNOWLEDGMENTS

Funding: This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, the Extramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (R56 AI080792 to NHT; contract HHSN272201400018C to NHT, JHA, CLK and MLG; R01 AI064478 to JHA, NHT and CLK; and P41 GM103422 to MLG), the Veterans Affairs Research Service (BX001350 to CLK) and the Burroughs Wellcome Fund (to NHT). Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP, 2009/52729-9 to MUF). VCN is supported by a scholarship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) of Brazil, which also provides a senior researcher scholarship to MUF. We thank Y. Colin for the gift of the murine 2C3 anti-DARC. We thank J. Nix and ALS Beamline 4.2.2 supported by contract DE-AC02-05CH11231, and the Facility of the Rheumatic Diseases Core Center under award number P30AR048335. We also thank GM/CA-CAT beamlines 23-ID-D at the Advanced Photon Source, Argonne National Laboratory. Our special thanks to Dr. Christopher Nelson for assistance in the analysis of BLI data. We also thank Dr. Audrey Odom-John for the use of a plate reader, and J. Patrick Gorres for his assistance in preparing this manuscript for publication.

DATA AND SOFTWARE AVAILABILITY

Atomic coordinates and structural factors have been deposited in the Protein Data Bank with accession codes 6OAN and 6OAO (https://www.rcsb.org/structure/6OAN and https:// www.rcsb.org/structure/6OAO).

COMPETING INTERESTS

The authors declare no competing interests.

SUPPLEMENTARY INFORMATION Supplementary Information is available online.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material. 4 for full peptide coverage of DBP). The HDX data were determined in duplicate, and data are presented as mean +/-SD. For determinations where the deviation is not seen, it is smaller than the size of the data point. The statistical validity of the curves was also assured by making the measurement over seven time points. Given that the purpose of the HDX kinetic plots is to assign binding or no-binding, those assignments were made visually without further statistical analysis.