Camille Hespel
email: camille.hespel@univ-orleans.fr

Jean-Bernard B Blaisot
email: jean-bernard.blaisot@coria.fr

Xandra Margot
email: xmargot@mot.upv.es

S Patouna

Armelle Cessou

B Lecordier

Influence of Nozzle geometry on

Introduction

Injection is one of the key parameters in engine control. Indeed, combustion processes in Diesel engines greatly depend on the characteristics of the injection. Atomization and mixing are the first two processes of a long and complex chain which ends in the formation of pollutants. Thus, in the context of pollutant emission reduction, advanced knowledge of injection features is still needed. It is wellknown that the atomization of the jet strongly depends on the nozzle geometry. The shape of the nozzle affects the cavitation phenomenon in the nozzle and therefore the spray characteristics. To visualise the internal flow and the structure of cavitation in multi-holes and real-size nozzles is not obvious. This has been done on large-scale transparent nozzles by [START_REF] Soteriou | Diesel Injector Laser Light Sheet Illumination of the Development of Cavitation in Orifices[END_REF] or on singlehole real-size nozzle by [START_REF] Badock | Investigation of cavitation in real size diesel injection nozzles[END_REF]. Here the internal flow is investigated through numerical simulations while the jet emanating from the nozzle exit is characterized experimentally. This work is a part of a program which aims at improving our capacity to predict in a fine way all the processes of atomization. Numerical simulations of the flow in the nozzle as well as experiments on the spray emanating from the nozzle have been done to characterize the injection process.

Complementary results obtained through experiments and CFD simulations give a better understanding of the injection.

The effect of the nozzle geometry on the atomization processes is straightforward. This study is focused on the comparison of two nozzle geometries. Two 6-hole nozzles are used, one with cylindershaped holes (Kf0) and the other with cone-shaped holes (Ks1.5). The main difference between these two nozzles concerns the cavitation that is expected to occur more frequently for Kf0 nozzle.

Experimental conditions and measurement techniques used in this work are presented in the next section. A comparison of the results for the two nozzle geometries follows, based on spray features measured during the stationary phase of injection. The particular behavior of the injectors during transient phases is then presented for experiments done in multi-injection conditions. A conclusion ends this paper.

Methodology

For the analysis presented in this work, an experimental set-up described below was used that included the injection system and the optical techniques for the measurement of the macro-and microscale characteristics of the spray. In addition, some CFD calculations of the internal nozzle flow were performed to help explain the behavior of the sprays, which is known to be influenced by the presence of cavitation.

Injection system and test bomb

Injection Nozzles

Two different six holes Common Rail Diesel solenoid injectors working under injection pressure of up to 1500 bar have been considered in this investigation, one with cylinder-shaped holes (type Kf0) with an orifice diameter of 147 µm, the other with cone-shaped holes of type Ks1.5 with nozzle exit diameter of 138 µm. The geometry of the holes has been measured with a particular technique described in [START_REF] Macián | New technique for the determination of the internal geometry of Diesel nozzle with the use of silicone methodology[END_REF], whereby a special type of silicone is used to obtain an internal mould of the nozzles ( Fig. 1). Photographs of the moulds are taken with a microscope, and then loaded on a computer aided design (CAD) software with the appropriate scale factor. The dimensional characterization of the nozzle can thus be obtained. This experimental technique is useful for the study of the Diesel nozzle internal flow, as it allows establishing relationships between the internal geometry, the flow characteristics and the subsequent spray behavior. The nozzle geometries and operation conditions have been chosen in order to obtain different cavitation levels. The cylindrical holes promote the cavitation, while the cone-shaped holes limit significantly this phenomenon.

Only one of the six jets is investigated in each case. In order to avoid the interaction between jets, a small deflector that does not change the behavior of the injector is placed over the nozzle (Fig. 2).

Measurement conditions

The experiments were performed in a high pressure bomb with different optical accesses. The operating conditions are summarized in table 1. The test oil was dodecane, a non-fluorescent fuel with physical properties similar to those of the Diesel fuel (ρ f =750 kg/m3). The reference time was given by the start of activation (SOA) of the main injection. Therefore, for the double injection the beginning of pilot injection was -416.16 µs or -832.3 µs for 5°CA and 10°CA respectively. The hydraulic delay was 306 µs for the Kf0 nozzle and 340 µs for the Ks1.5 nozzle for a back-pressure of 1 MPa. The difference between hydraulic delays disappeared for a back-pressure of 5 MPa

Internal nozzle flow CFD approach

Some calculations were computed with the commercial CFD code STAR-CD, v 4.06 for the different measurement conditions, with the objective of better understanding the influence of the inner flow characteristics on the spray evolution.

Considering the 60 o periodic symmetry of the six-hole injector, the computational domain meshed was a 60 o sector, based on the exact geometry of one of the six nozzles chosen as being representative of all. The calculations were performed for the full lift configuration (250 μm). Symmetry boundaries were defined to model the geometric periodicity, and constant pressure was prescribed at both the inlet and outlet boundaries. A grid independence study allowed determining the mesh fineness, which resulted in a total of about 600.000 cells, with a cell size of 10 µm approximately.

All calculations presented here were performed using the conventional k-ε turbulence model with hybrid near wall treatment. The cavitation model implemented in this code is based on the Rayleigh equation [START_REF] Rayleigh | On the pressure developed in a liquid during the collapse of a spherical cavity[END_REF]], which links the rate of change of the bubble radius with the local pressure. In this model both the liquid and vapor densities are constant and there is no slip between bubbles and liquid. Details of the complete numerical approach set-up are given in [START_REF] García | Analysis of the cavitating flow in real size diesel injectors with fixed and moving needle lift simulations[END_REF] It should be noted that the working liquid was Diesel fuel with the corresponding properties.

Imagery

Large field

The experimental setup for shadowgraphy consists of a CCD-camera with a lens of 50 mm focal length illuminated by a pulsed source in an on-axis configuration. A light panel connected to a stroboscope provides a uniform background illumination.

The field of view is 69x53mm² with a resolution of 11pix/mm. At least 30 pictures per operating point were analyzed to provide the mean penetration length and cone angle of the spray. Penetration length up to 80 mm was measurable thanks to the inclined position of the jet in the field of view.

Small field

Granulometry measurements were performed by imagery. The setup includes the same camera, a long working distance microscope and nanolite source (flash duration about 11ns) ( 2005; Fdida and Blaisot 2010).

).

Fig. 3 Experimental setup of the granulometry by imagery

A lens arrangement provides a uniform illumination over the field of view which is 650x496 µm² (resolution =1170 pix/mm). A specific image processing analyzes the gray levels images in order to determine the diameter and the shape of droplets in a control volume limited by the field of view and depth from focus [START_REF] Blaisot | Droplet size and morphology characterization for dense sprays by image processing: application to the Diesel spray[END_REF][START_REF] Fdida | Drop size distribution measured by imaging: determination of the measurement volume by the calibration of the point spread function[END_REF].

Long distance laser correlation velocimeter

This technique is an application to dense liquid sprays of the time-of-flight measurement principle [START_REF] Chaves | High speed flow measurement within an injection nozzle[END_REF]. The one-beam two images configuration [START_REF] Schugger | Influence of spray velocity and structure on the air entrainment in the primary breakup zone of high pressure Diesel sprays[END_REF] has been adapted for the measurement in the pressure chamber with the replacement of the single imaging lens by a long working distance microscope. The setup is composed of one laser beam (HeNe) illuminating the spray and an optical system imaging the two measurement points on photomultipliers coupled to optical fibres. The long distance microscope can image the spray at a reasonable distance between the imagery system and the image plane with the needed magnification. The magnification factor is 40 for a distance between the objective and image plane of 930mm (Fig. 4). The interval between the fibres is 940 µm, giving an interval of 50 µm between the measurement volumes. Since any structure passing in the first measurement volume has a high probability of passing also in the second one, the first signal precedes the second one by a minor delay.

Fig. 4: Experimental apparatus of the laser correlation long distance velocimeter

This time delay is used to calculate the spray velocity. The time delay between the two signals, due to the time-of-flight, is calculated using a cross correlation algorithm between the signals on sliding time windows. A detailed description of the evaluation and validation method can be found in [START_REF] Kirmse | Velocity measurements of dense Diesel fuel sprays in dense air[END_REF], an error-analysis is carried out in [START_REF] Kirmse | Korrelationsvelocimetrische Untersuchungen des Diesel-Einspritzstrahls einer Common-Rail Einspritzung[END_REF].

Fluorescent-Particle Image Velocimetry

Velocity fields are measured using the Particle Imaging Velocimetry (PIV) technique based on fluorescent droplets for the liquid phase or on fluorescent seeds for the ambient gas (Fig. 5). Due to the different dynamic velocities between air and fuel in Diesel jets, the ambient gas and fuel droplets velocity fields are not measured simultaneously. Fluorescence images were preferred to Mie scattering images because liquid and vapour phases can be discriminated and the multi-scattering effect is reduced. The gas phase is seeded with small droplets tagged with pyrromethene 597 (2g/L). This dye offers a relatively narrow emission spectrum around 590 nm for 532 Yag excitation (Wolff et all 2007). The fluorescence signal is detected by an intensified CCD camera (Hamamatsu) through high pass filter (OG550). The Mie scattering of laser light is strongly reduced by this filter. The time delay between the two laser pulses is chosen between 50 µs and 200 µs for measurement on the gas phase, and standard PIV algorithms are used to obtain the ambient gas velocity field. A typical mean ambient gas velocity field is shown in Fig. 5b. The liquid phase velocity is determined by seeding the fuel with the same fluorescent dye species (5mg/L of pyrromethene are fully soluble in dodecane). The addition of this dye does not affect the injector behavior. Liquid phase velocity fields are measured with a time difference between the two exposures of 5 µs. The fuel droplets are well localized in the images because the specific spectral filter avoids the problem of Mie multi-scattering. An in-house post-processing is applied on images to extract each particle and the velocity field is determined on an irregular mesh centred on each particle. The PIV algorithm used to determine velocity fields for the liquid phase was developed in the laboratory [START_REF] Kosiwckuk | Simultaneous velocity field measurements in twophase flows for turbulent mixing of spray by means of two-phase PIV[END_REF]. An example of the mean velocity field is shown in Fig. 5c.

Comparison of nozzle geometries for the stationary phase

The influence of the nozzle geometry on the spray characteristics is investigated for the quasi-steady phase of injection for activation duration of 1000 µs.

Spray cone angle

The cone angle is defined as the angle between both edges of the spray on the first third of the penetration length. The variation of the cone angle versus time is presented in Fig. 6. As expected, the cone angle increases significantly with back-pressure P c and it is larger for the cylindrical nozzle Kf0. This figure also shows that the difference between the cone angles of the Kf0 and Ks1.5 nozzles reduces when P c increases. Indeed, the mean cone angle (determined along the stationary phase of injection) for the Kf0 nozzle is 15.3°, while it is 12.6° for the Ks1.5 for P c =1 MPa (with a standard deviation of 0.7). So the difference in cone angles is 2,7 o for P c =1 MPa, whereas it is only around 1° for Pc = 5 MPa. The computations performed for this case allow explaining these results, as illustrated in Fig. 7, which shows the images of the volume fraction of vapour in both nozzles for the two different back pressures. Indeed, for an injection pressure of 80 MPa, large cavitation zones develop along the cylindrical nozzle for both P c =1 MPa and P c =5 MPa. In the case of 1 MPa back-pressure, however, the cavitation bubbles reach the nozzle exit, while at higher back-pressure, they clearly do not. In the conical nozzle, there is no cavitation for any of these operation conditions. This observation highlights that when cavitation reaches the hole exit, the spray cone angle becomes wider for a given backpressure. When there is no cavitation at the nozzle exit, however, the spray cone angle is assumed to be mainly related to the gas density. At 5 MPa back-pressure, the density of the flow is similar at the exit of both nozzles, hence, the spray cone angles are also very similar.

Cylindrical nozzle

Liquid phase velocity

The velocities in the liquid phase close to the nozzle exit were measured using the long distance Laser Correlation Velocimeter (LCV). For the case of 80 MPa injection pressure and 1 MPa back-pressure, radial profiles of the axial velocity normalized by the frictionless velocity are shown in Fig. 8 for two positions: 10 mm (a) and 25 mm (b) downstream of the nozzle exit. The geometry of the nozzle does not seem to affect the normalized profiles, which are very similar for both the Kf0 and the Ks1.5 holes. The maximum velocity in the centre of the jet reaches about 35% of the frictionless velocity.

The mass flow rate measured using an EFS EMI2 mass flow meter is higher for the Ks1.5 nozzle, although its orifice exit diameter is smaller than the cylindrical one. Indeed, it has a higher discharge coefficient of 0.88, against 0.74 for the Kf0 nozzle. The discharge coefficient is defined as the product between the area-contraction coefficient C a and the velocity coefficient C v . Since the velocity profiles for both geometries are similar, it seems reasonable to assume that the velocity coefficients are very close for the two nozzles. Hence, the areacontraction coefficient C a , which is an indicator of the effective flow area section compared to the orifice section, is smaller for the cylindrical nozzle, due to the presence of the cavitation bubbles at the exit. Indeed, it is known that the effective area can be reduced because of vapor bubbles reaching the orifice exit [START_REF] Chaves | Experimental study of cavitation in the nozzle hole of Diesel injectors using transparent nozzles[END_REF].

LCV measurements did not indicate that there is an effective section reduction, probably due to the fact that velocity is measured on every fluid structure passing through the LCV probe, whatever its nature, i.e. a droplet, a ligament or a cavitation bubble.

Drop size distribution

The size of the droplets is estimated using the image processing of over 100 pictures for each point. The mean diameters measured 10 mm downstream of the nozzle exit and at 2 mm from the jet axis are reported in Table 2. As expected, the injection pressure has a significant influence on the reduction of the droplets size in the spray, whatever the nozzle. Rising the ambient pressure from 1 MPa to 5 MPa has also an effect on the drop size distribution, though only for the Kf0 nozzle, as illustrated in Fig. 9 and Fig. 10. Indeed, the droplets size distributions for the Ks1.5 nozzle are very similar for both back-pressures, and Fig. 10 shows that for 5 MPa back-pressure, the size distribution of the cylindrical nozzle is also very similar to that of the conical nozzle. However, for the cavitating case, Kf0 and 1 MPa back-pressure, it differs significantly from the others: the cavitation results in a production of larger droplets close to the nozzle exit, and smaller droplets further downstream. [mm] Ks1.5 nozzle Kf0 nozzle the two nozzles. For two-phase flows, [START_REF] Cossali | LDV Characterization of air entrainment in transient diesel spray[END_REF] proposed to estimate the mixing performance of nozzles by the entrainment coefficient Ke given by equation 2.

Gaseous Phase velocity and air entrainment

   dξ ξ U R π ρ z m z r a a   0 2  Eq. 1 a f m dz m d Ke   f a d    Eq. 2
This coefficient is greater for the Kf0 nozzle (see Erreur ! Source du renvoi introuvable.b) because the orifice diameter is greater and the fuel flow rate is smaller for this nozzle.

The mixing rate, defined as the ratio between cumulated air mass flow rate and fuel mass flow rate, is shown in Erreur ! Source du renvoi introuvable.c. The maximum mixing rate is located at the vortex centre location. As the velocity field measurements begin 8 mm downstream of the nozzle exit, the mixing rate is underestimated.  

1 16 1 2 1 2 f a    Z m m   Eq. 3   2 .tan c d ~a    a z Z a f 
with a=0,66 Eq. 4

Behavior of injector during transient phases for multi-injection condition

Penetration length

Penetration lengths obtained for a single injection (500 µs) or a pilot (200 µs) and a main injection (500 µs) are reported in Fig. 13 for two injection pressures: 80 MPa and 150 MPa. The influence of the pilot injection on the main injection is more significant for P inj = 80 MPa. In the case of a pilot injection occurring 5°CA before the main injection, the lapse of time between closing and opening is 216 µs only, which is too short to obtain a complete closing of the needle at the end of the pilot injection. When the delay between the pilot and main injections is greater than 10°CA, the effect of the first injection disappears. In this case, the time between the two injections is long enough to obtain a complete closing of the needle before the second activation. For high injection pressure (150 MPa), the closing and opening times are shorter and there is sufficient time for the needle to close before the

Liquid phase velocity

The velocity measured by VLC shows that the main jet is faster at the beginning of the main injection when there is a pilot injection than when there is a single injection. This effect persists for the Kf0 nozzle up to Z = 25 mm although it disappears at this distance for the Ks1.5 nozzle (see Fig. 14). The longer penetration lengths obtained for the double injection confirm this.

Gaseous phase velocity and air entrainment

The effect of the pilot injection on air entrainment is reported for the Kf0 nozzle only, at P inj =80 MPa and P c =1 MPa. The effect of a pilot injection 5°CA before the main injection on the entrainment coefficient is mainly related to the longer penetration length, whatever the delay after the main injection activation. This is illustrated in Fig. 15 (a) and (b). As the penetration length is greater with a pilot injection 5°CA before the main injection, the zone of induced gas flow oriented toward the spray axis is also longer. The maximum mixing rate is thus greater with a pilot injection 5 °CA before main, just because the spray is longer in this case. However, since the mixing rate slopes are the same for the three types of injection, it seems that the pilot injection does not improve the momentum exchange induced by the spray (Fig. 15c).

Conclusions

The investigations presented here for different nozzle geometries (cylindrical and conical) demonstrate that the cavitation plays a major role on the atomization process and on the interaction between the liquid spray and the surrounding gas. Calculations have shown that the cavitation bubbles may reach the cylindrical nozzle exit, especially when the back-pressure is relatively low. When the levels of cavitation are low and do not reach the nozzle exit, measurements show that the two nozzles behave similarly. For example, at moderate injection pressure and high back pressure, the spray cone angles as well as the drop size distributions of the two tested nozzles are very similar. Differences occur when cavitation is generated in the cylindrical nozzle. The measurements have shown that the cavitation favours a wider spray angle and so improves the air entrainment, and promotes the production of smaller droplets. However, the cavitation does not seem to influence the spray velocities: indeed, the velocity profiles and the penetration lengths are similar for the two nozzles. These are more sensitive to the needle dynamics.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Silicone mould of injector

Fig. 5 :

 5 Fig. 5 : Fluorescent particle image velocimetry: geometrical configuration (a), mean velocity for the gas phase (b) and mean velocity field for the liquid phase (c)

Fig. 6 :

 6 Fig. 6 : Development of spray cone angles, measured at 80 MPa of injection pressure

Fig. 7 :

 7 Fig. 7 : Cavitation pattern for the cylindrical and conical nozzle at 80/1 and 80/5 MPa. Color scale: 0-1

Fig. 8

 8 Fig.8Radial profiles of the axial spray of the velocities Kf0 and Ks1.5 nozzle, Pc=1MPa, Pinj=80Mpa.

Fig. 9 Fig. 10

 910 Fig. 9 Droplets size distribution for Pinj=80 MPa and Pc=1 MPa Fig. 10 Droplets size distribution for Pinj=80 MPa and Pc=5 MPa

  Fig. 11 Mean air velocity fields for Pinj=80 MPa and Pc=1 MPaThe air entrainment has been calculated on the average velocity fields following the method of[START_REF] Cossali | LDV Characterization of air entrainment in transient diesel spray[END_REF]. A reference surface limited by a cylinder of radius R around the spray is defined for each field. The air mass flow rate a m  cumulated through this reference surface is determined by equation 1. The local air flow rate

Fig. 12

 12 Fig. 12Air entrainment performances of nozzles for Pc = 1 MPa and Pinj = 80 MPa, 950 µs after SOA However, the slope is very similar to that predicted by Siebers model[START_REF] Siebers | Scaling liquid-phase fuel penetration in Diesel sprays base on mixing-limited vaporization[END_REF]) expressed by equations 3 and 4. It is worth noting that the wider cone-angle of the Kf0 nozzle improves the performance of this nozzle in terms of momentum transfer from droplets to ambient air.

  Fig. 13 : Penetration length versus time

  Fig. 14 velocity versus time for the 200+500µs 5°AC

Fig. 15 .

 15 Fig. 15. Kf0 nozzle, Pinj=80 MPa Pc=1 MPa

Table 1 .

 1 Experimental test conditions

	Parameters	Values
	Discharge pressure Pc	1 MPa, 5 MPa
	Injection pressure Pinj	80 MPa, 150 MPa
	Single injection duration	500 µs, 1000µs
	Double injection duration	200 µs (pilot), 500 µs (main)
	Dwell angle (double injection)	5°CA, 10°CA @ 2000 rpm

Table 2 .

 2 Mean diameters of droplet size distributions

		0,6							0,6
		0,5							0,5			Kf0
	Velocity/VBernouilli	0,1 0,2 0,3 0,4						Velocity/VBernoulli	0,1 0,2 0,3 0,4			Ks1.5
		0							0
		-15	-10	-5	0 X/d	5	10	15	-25 -20 -15 -10 -5	0 X/d	5	10 15 20 25
						Kf0		Ks1.5
	P inj =80 MPa
		P c =			1 MPa	5 MPa	1 MPa	5 MPa
		D30 [µm]		7.4		9.1	9	8.5
		D32 [µm]		8.4		10.6	10.2	10.3
		D43 [µm]		9.6		12.2	11.4	11.9
	P c =1 MPa
		P inj =		80 MPa 150 MPa 80 MPa 150 MPa
		D30 [µm]		7.4		6.3	9	7.2
		D32 [µm]		8.4		7.1	10.2	8
		D43 [µm]		9.6		7.9	11.4	8.9

Acknowledgments

Part of this work has been performed in the framework of the PREDIT program "EMPhASE", supported by the French Environment and Energy Management Agency (ADEME).


Christina B Planitzer

Maria R Farcet

Richard I Schiff

Hans D Ochs

PhD Thomas R Kreil
email: thomas_kreil@baxter.com

Baxter Bioscience

Global Pathogen

Safety Farcet

Neutralization of

Keywords: Intravenous Immunoglobulin, human -IVIG, Echoviruses, virus neutralization, antibody functionality, replacement therapy Intravenous immunoglobulin, human -IVIG, echoviruses, virus neutralization, antibody 2 functionality, replacement therapy

information on neutralization capacity variability of individual IVIG lots against the different 6 echovirus serotypes is available.

7

Infectivity assays were established for the most prevalent echovirus serotypes (E 9, E 11, 8 E 13 and E 30) circulating in the United States (US) and the European Union (EU). The 9 echovirus serotype specific neutralization titers of 41 IVIG lots manufactured from either whole 10 blood (Recovered) or collected by apheresis (Source) and from either the US or EU, were 11 determined.

12 Significantly higher (P < 0.0001) neutralization titers against E 11 and E 30 were found in 13 IVIG lots manufactured from US Source plasma compared to US Recovered plasma.

14 Geographically, IVIG lots made from US plasma contained significantly (P < 0.0001) higher 15 neutralization titers against E 9 and E 11 than lots manufactured from EU plasma, whereas lots 16 made from EU plasma showed significantly higher neutralization of E 30. 17 To conclude, IVIG lots differ in their neutralizing antibody content against different 18 echovirus serotypes, depending on plasma collection practices and geographic origin.

19

Based on these results, an informed choice in selecting IVIG lots with the highest available 20 neutralization titer against the specific echovirus serotype would seem to be beneficial during 21 treatment of break-through infections. or EU plasma, for E 9 (P = 0.0002), E 11 (P < 0.0001) and E 30 (P = 0.0008) (Fig. 2). IVIG lots 19 derived from US plasma had significantly higher NT 50 values for E 9 and E 11, whereas 20 EU plasma derived IVIG had significantly higher neutralization capacity for E 30. For E 13, no 21 difference (P = 0.05) in neutralization was observed between the IVIG lots tested (Fig. 2).

22

F

221 12 [

 12 IntroductionEchoviruses belong to the human Enterovirus B group (family Picornaviridae) and 2 45 serotypes can be distinguished[CDC, 2006]. They are distributed worldwide and echovirus 3 infections have been associated with diverse clinical outcomes, ranging from asymptomatic 4 infection to minor febrile illness or persistent and severe, fatal conditions [Abzug, 2004]. More 5 recently, an involvement of echovirus in the development of chronic diseases such as 6 type 1 diabetes has been suggested [Viskari et al., 2004]. It has been estimated that echovirus is 7 responsible for 10 to 15 million symptomatic infections in the US each year [CDC, 2006]. 8 Clinically severe manifestations of echovirus infections are reported most frequently in two 9 patient groups: (i) infants and young children (up to 4 years of age), accounting for approximately 10 60% of all echovirus infections [CDC, 2006], who are prone to develop severe echovirus 11 infection with significant morbidity from sepsis, meningoencephalitis or myocarditis Abzug et al., 1993; Abzug et al., 1995; Abzug, 2004; McKinney et al., 1987] and, (ii) people 13 with antibody deficiency, particularly those with X-linked agammaglobulinemia, are at high risk 14 to develop severe and / or chronic echovirus infections, particularly 15 meningoencephalitis [McKinney, 1987]. 16 While no specific therapy for echovirus infection is available, current clinical treatment 17 regimen still include pleconaril [American Academy of Pediatrics, 2006; Abzug et al., 2004, 18 Desmond et al., 2006]. Also, as antibodies are known to play an important role in the defense 19 against enteroviral infections [Abzug et al., 1995], treatment of echovirus infection with 20 intravenous immunoglobulin (IVIG) preparations has been suggested as beneficial [Abzug et 21 al., 1995; Erlendsson et al., 1985; Galama et al., 2000; Johnston and Overall, 1989; Mease et 22 al., 1981; Nagington, 1982; Nagington et al., 1983]. IVIG is a therapeutic preparation that 23 contains the pooled antibodies from thousands of healthy plasma donors. Two different plasma

2 3Fig. 1 .Fig. 2 .

 212 Fig. 1. Echovirus neutralization titers 50% (NT 50 ) of individual IVIG lots manufactured from

Fig. 1 .Fig. 2 .

 12 Fig. 1. Echovirus neutralization titers 50% (NT50) of individual IVIG lots manufactured from US plasma were determined and analyzed separately by plasma collection practice, i.e. Recovered (R) from whole blood donations or Source (S) collected by apheresis. Error bars indicate standard error of the means; asterisks indicate significant difference in mean titers (P < 0.001). N = number of IVIG lots tested. 149x96mm (300 x 300 DPI)

Titration of echovirus infectivity and determination of echovirus neutralization titers 1

  Neutralization assays were performed at least in duplicate by two different operators and each

	Page 7 of 24	Journal of Medical Virology
		7	Planitzer et al.
	2
	3
	3	assay included internal controls for the verification of virus suspension infectivity. Equal volumes
	4 4	[Abzug et al., 1995; Erlendsson et al., 1985; Galama et al., 1997; Galama et al., 2000; Johnston containing 2,000 tissue culture infectious doses 50 (TCID 50 ) echovirus and serial twofold dilutions
	5 5	and Overall, 1989; McKinney et al., 1987; Mease et al., 1981; Nagington, 1982; Nagington et of IVIG were mixed, incubated for one hour at room temperature and eightfold replicates per
	6 6 6	al., 1983] or protect those at risk of infection, especially during the echovirus season from June to dilution were transferred to a microtiter plate containing a Vero cell monolayer. After 7 days of
	7 7 8 9	October [CDC, 2006]. Although some of these studies have reported echovirus antibody titers for IVIG preparations and corresponding clinical efficacy, the specific results obtained are of limited value for current clinical use, since none of the products analyzed are available anymore [Abzug F o F o IVIG preparations F o incubation at 37°C, cells were assessed for the presence of a cytopathic effect. The echovirus F o r r r r
	10 11 12 13	et al., 1995]. A number of echovirus serotypes have been associated with chronic enteroviral P P P P meningoencephalitis in agammaglobulinemic patients and are therefore of greatest concern for the Primary Immune Deficiency community (an estimated 250,000 children and adults in the US e e r e e r e e r e e r
	14 15 16 17 18	alone) [Boyle and Buckley, 2007]. The most frequent echovirus serotypes producing chronic enteroviral meningoencephalitis are: E 11 > E 9 > E 30 and > E 13 [McKinney, 1987]. In addition, E 13 has recently caused re-occurring severe disease in children with primary immune deficiency (R. Schiff, pers. communication). Echovirus serotypes show distinct epidemiologic characteristics and temporal patterns of circulation [Strikas et al., 1986]. Throughout the last R e v i e w R e v i e w R e v i e w R e v i e w
	19	decades E 9, E 11 and E 30 were reported most frequently in the general US [CDC, 2006] and
	20 19	EU [Ortner et al., 2009; RKI, 2010] populations, whereas E 13 has been circulating in the EU for
	21	some time but has only recently been associated with disease in the US [Mullins et al., 2004].
	20
	22	Previous efforts to link echovirus occurrence in the general population with echovirus antibody
	23	content of IVIG were not successful [Galama, 2000]. Other studies that investigated IVIG
		antibody specificities for different pathogens in IVIG lots suggested that antibody content varies
		John Wiley & Sons

J.Med.Virol. DOI xx.xxxx/jmv collection practices are utilized, where 'Recovered' plasma is collected from whole blood 1 donations and 'Source' plasma is obtained from donors by apheresis. Previous work has shown 2 that administration of IVIG with high echovirus antibody content improves clinical outcome J.Med.Virol. DOI xx.xxxx/jmv according to plasma donor age [Modrof et al., 2008], vaccination status [Audet et al., 2006; 1 Farcet et al., 2010] and virus epidemiology [Planitzer et al., 2007]. Because anecdotal evidence 2 suggests that understanding the echovirus antibody variation in IVIG lots could be of clinical 3 relevance for the treatment of persons with primary immune deficiency [Abzug et al., 1995; 4 Erlendsson et al., 1985; Galama et al., 2000; Johnston and Overall, 1989; Mease et al., 1981;

5 Nagington, 1982; Nagington et al., 1983], neutralization capacity of 41 IVIG lots against 6 echovirus serotypes E 9, E 11, E 30 and E 13 were determined. These serotypes are amongst the 7 most relevant for individuals with primary immune deficiency who are at increased risk of severe 8 systemic disease. This study presents the echovirus neutralization capacity of currently available 9 IVIG preparations and highlights important differences between IVIG lots manufactured from 10 plasma collected in different geographies and from different plasma donor population segments. J.Med.Virol. DOI xx.xxxx/jmv Methods 1 Viruses and cells 2 Virus isolates were obtained from the American Tissue Culture Collection (ATCC; E 9 3 strain 'Hill', ATCC VR-39; E 11 strain 'Gregory', ATCC VR-41; E 13 strain 'Del Carmen', 4 ATCC VR-43 and E 30 strain 'Bastianni', ATCC VR-322) and propagated and titrated on Vero 5 cells (European Collection of Cell Cultures, ECACC no. 84113001). 8 KIOVIG TM (Baxter AG, Vienna, Austria) and Gammagard Liquid TM (Baxter Healthcare Corp., 9 Westlake Village, United States), are 10% liquid IVIG preparations produced by the same 10 manufacturing process. The licensed shelf life for these products is two years when stored at 11 2-8°C and the IgG subclass distribution is within the physiological range, i.e. IgG1 > 56.9%, 12 IgG2 > 26.6%, IgG3 > 3.4% and IgG4 > 1.7% [Teschner et al., 2007]. 13 A total of 41 IVIG lots were analyzed in 2008. 23 lots were made from US plasma (12 lots from 14 Recovered and 11 lots from Source plasma) and 18 lots fractionated from EU plasma (2 lots from 15 Recovered and 16 lots from Source plasma). The dates of manufacture ranged from 16 December 2006 -November 2007 for US and from May 2006 -July 2007 for EU plasma derived 17 IVIG lots. The limited number of IVIG lots manufactured from EU Recovered plasma did not 18 allow a differential analysis between EU Source and Recovered plasma.

7 neutralization titer, expressed as the test article dilution that resulted in 50% virus 8 neutralization (NT 50 ), was calculated from the number of negative wells and the initial sample 9 dilution according to the method of Reed and Muench [Reed and Muench, 1938]. Virus 10 concentrations were calculated according to the Poisson distribution and expressed as 11 log 10 TCID 50 per mL [Planitzer et al., 2009].

12 13 Statistical analysis 14 Statistical analysis was done using GraphPad Prism v5.0 software (San Diego, CA, USA). 15 Differences in IVIG antibody content were analyzed using the Student's t-test 16 (unpaired, two-tailed) and comparison of variance was done using the F-test.

Results 1 Echovirus neutralization titers of IVIG lots derived from Recovered versus Source plasma 2 collected in the US

	3
	4	There was a significant difference in neutralization titers for E 11 and E 30 (Fig. 1). IVIG lots
	5	made from Source plasma had significantly higher (P < 0.0001) NT 50 values for E 11
	6	(mean ± SEM, N; Source: 180 ± 7, 11; Recovered: 135 ± 5, 12) and E 30 (mean ± SEM, N;
		Source: 611 ± 17, 11; Recovered: 422 ± 17, 12), as compared to lots derived from Recovered

7 plasma. The NT 50 values were approximately 3-fold higher for E 30 than for E 11 and the 8 difference between mean NT 50 values of Recovered versus Source plasma derived IVIG lots was 9 similar within the same echovirus serotype, i.e. Source NT 50 values were approx. 40% higher than 10 Recovered NT 50 values, for E 11 as well as for E 30. No significant difference in neutralization 11 titers for E 9 (P= 0.48) and E 13 (P= 0.48) was observed between Recovered and Source plasma 12 derived IVIG lots (Fig. 1). 13 Significant variation in NT 50 values obtained for IVIG against E 9 (P= 0.02) and E 13 (P= 0.003) 14 was seen.

15 16 Echovirus

neutralization titers of IVIG lots derived from US or EU plasma 17

  Significant differences in echovirus neutralization were observed for IVIG lots derived from US

	18

2

  Even though IVIG lots are manufactured from the pooled plasma donations of several thousand The statistically higher E 11 and E 30 neutralization titers observed for Source plasma as 1 compared to Recovered plasma-derived IVIG lots produced by the same manufacturing 2 procedure during the same period of time were surprising, as earlier studies had revealed the[START_REF] Ortner | Epidemiology of enterovirus types causing neurological disease in Austria 1999-14 2007: detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent 15 genotypes[END_REF] RKI, 2010], results outlined in the presented study suggest that this echovirus 1 serotype is more prevalent in the US, as evidenced by a significantly greater E 11 antibody 2 content in US plasma derived IVIG. The relative infrequent occurrence of E 13 in the These results, in conjunction with the information gained from the analysis of Recovered 17 versus Source plasma derived IVIG lots, are of importance to health care professionals who are The neutralization capacity of individual lots of IVIG against the most prevalent echovirus 2 serotypes varies up to threefold. These differences are linked to plasma collection practices, i.e. Abzug MJ. 2004. Presentation, diagnosis, and management of enterovirus infections in neonates.

		11 13 21	Planitzer et al. Planitzer et al. Planitzer et al.
	1	Conclusions
	3 3 3 3
	4 4 4 4	donors, there is still a certain level of variation in antibody content between individual lots of exact opposite, i.e. higher measles virus [Audet et al., 2006] and parvovirus B19 (B19V) [Modrof EU [RKI, 2010] and the US [CDC, 2006] is reflected by a low NT 50 observed in IVIG whole blood versus apheresis donations, as well as geographic origin of the plasma, i.e. US
	5 5 5 5 5	IVIG [Lejtenyi and Mazer, 2008]. Previous studies have shown that antibody specificities in et al., 2008] antibody titers in Recovered plasma-derived IVIG preparations. In both these cases, preparations from both geographic areas. The high incidence of E 30 reported from the EU versus EU. contributions of the whole Global Pathogen Safety team, particularly Karin Berka, Elisabeth
	6 6 6 6 6	IVIG depend on the plasma donor population segment from which the plasma was however, the generally older age of Recovered plasma donors (approx. two thirds of Source [RKI, 2010] correlates well with the results obtained. However, the high antibody titers detected Despite IVIG substitution therapy, rare cases of infection by different echovirus serotypes occur Pinter, Bettina York and Claudia Schwarr (virus propagation and cell culture) are gratefully
	7 7 8 6	o r F o r collected [Modrof et al., 2008], their vaccination status [Audet et al., 2006; Farcet et al., 2010] or virus epidemiology [Planitzer et al., 2009]. These variations are linked to plasma collection F o r plasma donors are below 30 years of age, whereas the same proportion of Recovered plasma F o r for E 30 in US plasma do not correlate well with E 30 incidence rates reported by the National F o r F o r in people with primary antibody deficiencies. Knowledge of the echovirus serotype neutralization titers of individual IVIG lots would enable targeted selection of higher titer lots against a specific F o acknowledged. F o infection: virology, serology, and effects of intravenous immune globulin. Clin Infect Dis. 20:1201-6. F o F o F o F o echovirus serotype for substitution therapy during an infection episode. r r r r r r
		P P P P P P P P P P P P
		e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r e e r
	18 15 12 13 14 15	R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w Galama JM, Gielen M, Weemaes CM. 2000. Enterovirus antibody titers after IVIG replacement in agammaglobulinemic children. Clin Microbiol Infect. 11:630-2 Johnston JM, Overall JC, Jr. 1989. Intravenous immunoglobulin in disseminated neonatal echovirus 11 infection. Pediatr Infect Dis J. 8:254-6. R e v i e w R e v i e w R e v i e w
	19 19 19 16 16	treating patients with severe echovirus infection. By choosing an IVIG lot with the highest immunoglobulins. J Infect Dis. 194:781-9. Lejtenyi D, Mazer B. 2008. Consistency of protective antibody levels across lots of intravenous
	20 20 20 17 17	from severe echovirus infections [Abzug et al., 1995; Erlendsson et al., 1985; correlation should exist between echovirus incidence rates and echovirus neutralization capacity possible echovirus serotype specific antibody titer, it is possible to increase the amount of Boyle JM, Buckley RH. 2007. Population prevalence of diagnosed primary immunodeficiency immunoglobulin preparations. J Allergy Clin Immunol. 121:254-5.
	21 21 21 18	Galama et al., 2000; Johnston and Overall, 1989; Mease et al., 1981; Nagington, 1982; in individual lots of IVIG. A significantly greater E 9 antibody content in administered echovirus neutralizing antibodies by up to 3-fold, thereby maximizing the possible diseases in the United States. J Clin Immunol. 27:497-502. McKinney RE, Jr., Katz SL, Wilfert CM. 1987. Chronic enteroviral meningoencephalitis in
	22	Nagington et al., 1983], echovirus serotype specific antibody content in 41 IVIG lots, US plasma derived IVIG correlates well with an approximately 6-fold higher incidence of E 9 therapeutic benefit, as has previously been shown [Abzug et al., 1995; Erlendsson et al., 1985; agammaglobulinemic patients. Rev Infect Dis. 9:334-56.
	23	Galama et al., 2000; Johnston and Overall, 1989; Mease et al., 1981; Nagington, 1982;
		Nagington et al., 1983]. Based on the findings of the presented surveillance study, a selection of

J.Med.Virol. DOI xx.xxxx/jmv 1 Discussion 8 practices, i.e. whole blood versus apheresis donations (Fig.

1

), as well as geographic origin of the 9 plasma, i.e. US versus EU (Fig.

2

)

[Galama et al., 1997]

.

[START_REF] Nagington | Use of normal immunoglobulin in an echovirus 11 11 outbreak in a special-care baby unit[END_REF]

To date, only four studies have evaluated the neutralizing antibody levels against the most 11 common echovirus serotypes and reported highly variable levels in different IVIG 12 products

[START_REF] Abzug | Neonatal enterovirus Planitzer et al[END_REF] Galama et al., 1997; Galama et al., 2000;[START_REF] Mease | Successful treatment of echovirus 1 meningoencephalitis and myositis-fasciitis with intravenous immune globulin therapy in a 2 patient with X-linked agammaglobulinemia[END_REF]

. This 13 variability in reported antibody levels is, at least partially, explained by the use of different virus 14 neutralization assay formats as well as different IVIG products, which are produced using different manufacturing procedures. Of note, these investigations were done prior to 2000 and 16 included a limited number of IVIG lots no longer available, thus no information on echovirus 17 antibody content in current IVIG preparations is available.

18

As it has been suggested that this information might be of vital importance to patients suffering 22 manufactured from plasma of different geographic and demographic origin, were determined. J.Med.Virol. DOI xx.xxxx/jmv 7 donors are older) has been shown to result in higher virus antibody titers. In the case of measles, 8 higher titers are elicited after wild-type virus infection as opposed to exposure through a vaccine 9 [Audet et al., 2006], and for B19V, higher prevalence of antibody-positive donors is found in 10 advanced age groups [Modrof et al., 2008]. For echoviruses, however, exposure occurs uniformly 11 by infection with wild-type virus as no vaccine exists, but the likelihood of infection is greater for infants and young children [CDC, 2006]. Of note, the overall level of circulation increased since 13 1975 for E 30 and since 1979 for E 11 [CDC, 2006] and thus titers can be expected to be highest 14 for younger donors, and then declining during adolescence of donors towards the average age of 15 Recovered plasma donors. However, with the limited information on the epidemiological 16 occurrence of those viruses prior to 1970, the association of significantly higher titers with the 17 younger age of Source plasma donors remains speculative. 18 As IVIG products reflect the immune status of a population subset, a certain degree of 22 infections in the US [CDC, 2006] as compared to the EU [Ortner et al., 2009; RKI, 2010]. While 23 the incidence rates reported for E 11 are similar for the US [CDC, 2006] and EU J.Med.Virol. DOI xx.xxxx/jmv [7 Enterovirus Surveillance System (NESS), which indicate that E 30 is the third most prevalent 8 echovirus serotype in the US, after E 9 and E 11 [CDC, 2006]. The E 30 antibody content 9 detected in US IVIG was by far the highest for the echovirus serotypes included in this study, 10 which may suggest that E 30 infections occur more frequently in the US than reported by the 11 NESS, possibly reflecting underreporting due to the generally milder illness associated with this 12 serotype [CDC, 2006], or even asymptomatic infections.

13

The presented results indicate that the surveyed echovirus serotypes occur at similar 14 frequencies in both geographies, i.e. E 30 > E 9 > E 11 > E 13, and that, with the exception of 15 E 30, the echovirus serotype specific antibody titers were always higher in US plasma derived 16 IVIG. 7 8 Paediatr Drugs. 6:1-10. 9 American Academy of Pediatrics. 2006. Red Book: 2006 Report of the Committee on Infectious 10 Diseases. In: Pickering LK, Baker CJ, Long SS, McMillan JA, editors. Enterovirus 11 (Nonpoliovirus) Infections. Elk Grove Village, IL: American Academy of Pediatrics, p 12 284-85. 13 Audet S, Virata-Theimer ML, Beeler JA, Scott DE, Frazier DJ, Mikolajczyk MG, Eller N, Chen 14 FM, Yu MY. 2006. Measles-virus-neutralizing antibodies in intravenous 18 Centers for Disease Control and Prevention. 2006. Enterovirus Surveillance -United States, J.Med.Virol. DOI xx.xxxx/jmv 1

Table I .

 I Selection of IVIG lots for substitution therapy during echovirus infection episodes

	Page 21 of 24	Journal of Medical Virology
	2
			EU -IVIG	US -IVIG
			Source	Recovered
		E 9		x	x *
		E 11		x
		E 13	x	x *	x
	3	E 30 *NT 50 values varied significantly within the group x x * F o
	4	EU: European Union, E: echovirus, NT 50 : 50% virus neutralization; IVIG: Intravenous r
	5	immunoglobulin, US: United States P
			e e r
				R e v i e w
			John Wiley & Sons

J.Med.Virol. DOI xx.xxxx/jmv 1

J.Med.Virol. DOI xx.xxxx/jmv

John Wiley & SonsJournal of Medical Virology

Acknowledgements 2

We thank P. Noel Barrett, PhD for his long-term support and encouragement, and 3 Don A. Baker, PhD for his unconditional support and strategic vision for the project. The

Desmond RA, Accortt NA, Talley L, Villano SA, Soong S-J, Whitley RJ. 2006