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Vaccine 26 (2008) 6577–6586

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

pdating of the correlation between lpELISA titers and protectionrom virus challenge for the assessment of the potency of polyvalentphtovirus vaccines in Argentina

duardo Maradeib,1, José La Torrea,1, Blanca Robioloa, Jorge Estevesb, Cristina Sekia,ndrea Pedemonteb, Marcela Iglesiasa, Ricardo D’Aloiab, Nora Mattiona,∗

Centro de Virología Animal, Instituto de Ciencia y Tecnología Dr. Cesar Milstein, CONICET, Saladillo 2468, C1440FFX, Ciudad de Buenos Aires, ArgentinaServicio Nacional de Sanidad y Calidad Agroalimentaria – SENASA, Av. Fleming 1653, B1640CSI, Martínez, Argentina

r t i c l e i n f o

rticle history:eceived 10 August 2008eceived in revised form2 September 2008ccepted 15 September 2008vailable online 1 October 2008

eywords:oot-and-mouth diseasepELISA

a b s t r a c t

Routine vaccination campaigns are carried out in Argentina twice a year, involving more than 100 milliondoses of foot-and-mouth disease (FMD) vaccine. Although the challenge test in cattle has not been totallyreplaced for the assessment of FMD vaccine potency, Argentine Animal Health authorities have used anindirect alternative method based on specific correlation studies of protection against podal generalization(PPG) tests performed in cattle with a validated liquid phase blocking ELISA (lpELISA). The change ofvaccine formulations that took place after the 2000–2001 outbreaks, generated a gap in the correlationbetween lpELISA titers and PPG for the new FMD virus strains. A reappraisal of the correlation betweenlpELISA titers measured at 60 dpv and virus challenge by the PPG method at 90 dpv, performed for the fourvirus strains presently included in the Argentine vaccine is presented in this work. The data were obtained

accine potencylternative potency test

from 40 bovine challenge trials (647 sera) performed using exclusive batches of commercial vaccine fromthe year 2001 to January 2008 for A24/Cruzeiro, A/Argentina/2001, O1/Campos and C3/Indaial FMD virusstrains. Curves of percentage of expected protection (EPP) versus lpELISA titers were obtained by logitregression for A/Argentina/2001, O1/Campos and C3/Indaial strains, but not for A24/Cruzeiro strain. Theconcordance between the direct and indirect tests using an EPP cut off value of 75% (82%, kappa = 0.62), inagreement with data originating from many years of vaccine control in Argentina, remarks the relevance

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. Introduction

Foot-and-mouth disease (FMD) is one of the economically mostmportant diseases that affect livestock [1–3]. Inactivated foot-and-

outh disease vaccines are used in many parts of the world, andven in countries where vaccination is not currently used, con-entrated inactivated FMD virus antigens are also kept in strategiceserves (antigen banks), which can be rapidly formulated into vac-

ines during an emergency [4].

The measurement of the potency of the vaccines is critical forhe control and eradication of FMDV. Potency testing of inactivatedaccines is performed in the target species using direct or indi-

∗ Corresponding author. Tel.: +54 11 4686 6225; fax: +54 11 4686 6225.E-mail addresses: nmattioncevan@centromilstein.org.ar,

attion@fibertel.com.ar (N. Mattion).1 The first two authors contributed equally and kindly request a joint first co-uthorship.

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264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2008.09.033

ernatives to in vivo potency testing.© 2008 Elsevier Ltd. All rights reserved.

ect methods. Up until now, the “Gold Standard” test is the in vivohallenge of primo-vaccinated animals. There are two direct meth-ds commonly in use: 50% protective dose (PD50) and protectiongainst podal generalization (PPG). The PD50 test consists in a mea-urement of potency by testing the vaccine in groups of at least fiveattle inoculated with different dose volumes of vaccine, so thatotency can be expressed in terms of 50% protective doses [5]. ThePG method, in use in Latin America, is described in this work. It haseen reported that observation of PD50 results indicated a lack ofose–response relationship in a large number of tests, which com-licated the interpretation of the results [6–8]. The PPG method,here the vaccine is used undiluted, proved to be highly reliable

9].However, the challenge methods per se have practical and logis-

ical problems, and many disadvantages from the perspective ofnimal welfare and biosafety. In most countries, official animalealth services as well as the Office International des EpizootiesOIE) experts have supported the use of alternative testing methodsrovided that a statistical correlation can be established between

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um1sdFblesions at the feet) divided by the total number of vaccinated ani-mals × 100.

The trial was considered valid if both non-vaccinated controlanimals showed FMDV-induced lesions on at least one foot. Accord-ing to Resolution no. 351/06 [23], a vaccine batch is approved for

Table 1PPG trials carried out for commercial vaccines in Argentina.

Challenge strain No. of trials No. of serum samples

A24/Cruzeiro 6 94a

A/Arg/01 11 177b

O1/Campos 16 258c

C3/Indaial 7 118d

578 E. Maradei et al. / Vac

hem and the challenge test in the target species. This is also thease in Argentina, where the in vivo PPG test is actually being imple-ented only for vaccine registration or for the introduction of new

trains into an already registered vaccine (vaccine updating).It is generally accepted by FMD research workers that there is

good correlation between the virus neutralization (VN) antibodyiters of primo-vaccinated cattle and their protection from virushallenge [10–14], but several ELISAs have also been validated forhis purpose and have been found to be at least as reliable and pre-ise than the VN tests [13,15–18]. In addition, ELISA can be used withnactivated antigens outside high security laboratories, the data cane obtained earlier and they are more friendly for validation andutomatization. Moreover, ELISA results were found significantlyore reproducible [15,16].In Argentina, we have reported several extensive studies using a

iquid phase blocking ELISA (lpELISA) for the evaluation of protec-ive immunity in cattle vaccinated with commercial oil-adjuvantedaccines. In 1993, potency tests involving 1634 animals wereeported [18], taking advantage of the assay of 102 batches of com-ercial vaccine, representing approximately 100 million doses,ade over a period of 14 months in the years 1991 and 1992.

esults showed that lpELISA serum titers directly correlated withhe percentage of protected animals in vaccinated cattle challengedith FMDV strains O1/Caseros, A/Argentina/79, A/Argentina/87

nd C3/Argentina/85, which represented the prototype strains usedor vaccine production.

In 1995, Robiolo et al. [19] extended the previous study pre-enting the results using lpELISA serum titers of 3920 vaccinatedattle, challenged by the PPG method with the same strains as inhe previous study. In this case, two independent numerical coef-cients were introduced. One of them was the lowest expectedrotection (LEP) calculated from the serum lpELISA titers at 60 daysost vaccination (dpv) of animals challenged with virus at 90 dpv.he LEP evaluation was highly specific (i.e. it was able to predictaccine failure in 100% of the cases), although its ability to pre-ict the challenge approval (sensitivity) comprised only 65% of theaccines that passed the PPG trial. The sensitivity was improved byntroducing an alternative coefficient (Ro) capable of predicting thePG approval of 90% of the vaccines, maintaining acceptable safetyevels (87% specificity) [19].

Later on, these studies were extended by 435 vaccine seriesnvolving a total of 7390 vaccinated/challenged bovines, confirminghe previously reported results (unpublished data).

With the reintroduction of FMDV in Argentina in the years000–2001 several vaccine matching trials were performed forhe emerging strains and there was a total change in vac-ine composition [20]. The regional strains A24/Cruzeiro and1/Campos were introduced together with the emerging local

trains A/Argentina/2000 and A/Argentina/2001. A preliminaryorrelation between serological response and protection from chal-enge with O1/Campos and A/Argentina/2001 FMD virus strainsas published with data derived from experimental and com-ercial vaccine challenge trials [21]. To establish a correlation

f serology with challenge, data from 156 and 138 cattle vac-inated and challenged by intradermolingual inoculation with/Argentina/2001 and O1/Campos FMDV strains, respectively, werenalyzed in order to validate the application of indirect vaccineotency assays and assessment of vaccination efficiency [21].

Recently, restricted in vivo trials were performed for vaccineatching purposes using a well-characterized A24/Cruzeiro mono-

alent experimental vaccine in six homologous (A24/Cruzeiro) andour heterologous (A/Argentina/2001) virus challenge trials per-ormed at 30 days post vaccination [22].

In this work, a correlation of lpELISA titers of sera taken at 60 dpvrom animals challenged by PPG at 90 dpv with each one of the

T

6 (2008) 6577–6586

our strains present in the Argentine vaccine currently in use (A24ruzeiro, A/Argentina/2001, O1 Campos and C3 Indaial) is reported.he study was performed using exclusive batches of commercialaccine and showed that the lpELISA in use in Argentina offeredhigh degree of reliability and safety in predicting the potency of

accines in the absence of live virus challenge.

. Materials and methods

.1. Viruses and vaccines

The four FMDV strains used in this study were24/Cruzeiro/Brazil/55 (A24/Cruzeiro), O1/Campos/Brazil/58

O1/Campos) and C3/Indaial/Brazil/71 (C3/Indaial) (origin: Panmerican Centre for Foot-and-Mouth Disease (PANAFTOSA)), and/Argentina/2001 (A/Arg/01), all of them provided by the Argen-

ine Animal Health Service (SENASA). The serum samples were alsorovided by SENASA and originated from official potency trials forolyvalent batches of commercial vaccine produced by differentanufacturers. The formulation of the vaccines tested was not

xactly the same, considering that the FMDV strain C3/Indaial wasncorporated into the Argentine vaccines in the year 2005 in placef A/Argentina/2000 strain [20,21].

.2. Protection against podal generalization

The vaccine potency trials considered in this study were per-ormed by SENASA from the year 2001 up to January of the year008. Vaccines were from commercial origin, presented to SENASAither for registration or serial control. A total of 40 bovine trialsere performed. In each trial the animals were challenged with

nly one virus strain at a time, as summarized in Table 1.PPG tests were conducted according to SENASA Resolution no.

51/06 [23], as previously described [20-22]. Briefly, 16 individuallyar-tagged cattle, negative for FMDV antibodies, were vaccinatedntramuscularly in the upper part of the neck with a full cattle vac-ine dose (2 ml). Two unvaccinated control animals were includedn each potency trial.

Ninety days post vaccination, 16 vaccinated animals and bothnvaccinated controls were challenged by inoculating intrader-ally 104 suckling mouse lethal doses 50% (SMLD50), equivalent to

04 BID50 (unpublished data), of challenge virus into four differentites on the upper surface of the tongue (0.25 ml per site). Sevenays post challenge (dpc) all animals were clinically checked forMDV-induced lesions on the feet. PPG was calculated as the num-er of vaccinated protected animals (i.e. absence of FMDV-induced

otal 40 647

a Two animals were discarded for outliers.b 17 animals were challenged in one of the trials.c 17 animals were challenged in two of the trials.d 17 animals were challenged in six of the trials.

E. Maradei et al. / Vaccine 26 (2008) 6577–6586 6579

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Fig. 1. Design of the lpELISA microplates. B: Blank; 100%: antigen controls (100

icensing if at least 12 out of the 16 vaccinated animals are foundo be protected (i.e. 75% PPG). A vaccine batch must be retestedf 10–11 vaccinated cattle are protected against challenge (i.e.:2.5–68.8% PPG), and a vaccine batch is rejected if only 9 or less vac-inates show absence of lesions on the feet (i.e.: lower than 62.5%PG).

The cattle used originated from Patagonia (Argentina), a regionfficially recognized by the OIE as free from FMD without vacci-ation [24]. The animals selected were steers, between 24 and 30onths of age and weighing 280–350 kg, healthy, with a good nutri-

ional status and free of parasites. Prior to the study, all cattle wereled and the absence of anti-FMDV antibodies was checked using

pELISA [18,19], and 3ABC-ELISA [25] to confirm the absence of priorontact to structural and non-structural FMDV proteins. Animalsere then bled at 30, 60 and 90 dpv. The serology used for cor-

elation with PPG corresponded to the 60 dpv sera. The challengeith live virus was carried out in the BSL3A facilities of the Insti-

uto Nacional de Tecnología Agropecuaria (INTA) located in Castelar,rovince of Buenos Aires, according to biosecurity and animal wel-

are federal regulations [23].

.3. Liquid phase blocking ELISA

The assay was originally adapted from Hamblin et al. [26–28]nd was carried out with modifications [18–21].

Serum samples were tested in 96-well plates in fourfold dilu-ions (Fig. 1). Four sample dilutions (from 1:32 to 1:2048) werencubated overnight at 4 ◦C with a pre-titrated dose of the corre-ponding virus strain in a saline buffer liquid phase. The mixturesere then incubated for 1 h at 37 ◦C on wells coated with FMDV

train specific rabbit polyclonal antibody, where the virus that didot react with the bovine serum in the previous step was trapped.fter a washing step (five times with PBS containing 0.05% Tween,/v) a second incubation was performed with a saturating con-entration of a pool of monoclonal antibodies (MAbs) specific for

ach of the virus strains being tested. Goat anti-mouse IgG conju-ated with horseradish peroxidase (Jackson InmunoResearch, USA)as added, and colour development was obtained after additionf the substrate/chromophore mixture (H2O2/ABTS (2,2-azino-bis--ethyl-benzothiazoline-6-sulfonic acid diammonium salt, Sigma,

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orbance); 1–16: serum samples; C1–C6: serum controls. C−: negative control.

SA). The optical densities (OD) readings were measured using anutomatic microplate reader (Bio-Rad, Hercules, CA) at a wave-ength of 415 nm. Six control sera of known titers (reference sera ofigh, medium and low titers) were assayed simultaneously as inter-al standards in each ELISA plate in three twofold dilutions from:32 to 1:128. Two negative control sera (unvaccinated animals)ere tested in one dilution (1:16). Eight wells were used for antigen

oncentration control (100% reactivity) and two wells were used aseaction blanks without virus antigen and without serum. Antibodyiters were expressed as the reciprocal log10 of serum dilutions giv-ng 50% of the absorbance recorded in the antigen control wellsOD50%).

Calculations and validation of each plate were performed usingroprietary software (Robiolo et al., unpublished). For validation ofach lpELISA plate, the following performance criteria were applied.

.3.1. BlankThe average absorbance of the two blanks should be <0.300. This

alue was established considering that the mean blank value plusS.D. is always lower than 0.300 for the four FMDV strains. Thelank OD was subtracted from the OD value of every well.

.3.2. Antigen controlThe OD of each replicate should be >0.750 and <1.950, not dif-

ering from each other in more than 0.300 OD. At least seven out ofight replicates had to comply with this condition. The average ofhe eight wells (or seven valid wells) was calculated and OD 50% wasstablished (OD50%). The antigens used as standards were stored in0% glycerol and 0.1% sodium azide.

.3.3. Serum controlsThe titer of each control serum was established as described

bove. The dilutions used corresponded to the linear part of theurves obtained representing OD readings versus the log of theilutions used in the assay. The coefficients of correlation (r) were

alculated. r values should be ≥0.90 for each control serum. Titersf control sera should not differ in more than ±0.200 from the refer-nce values. A correction factor (f) was applied if at least four out ofix control sera presented differences of the same sign, to accountor eventual errors in virus concentration on the plates, which is

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critical assay parameter. ‘f’ values applied were as follows: If theean of the differences from the reference values was lower than

.059 there was no correction; between 0.060 and 0.109, f = 0.05;or 0.110–0.159, f = 0.10; for 0.160–0.205, f = 0.15. After correction,hen corresponded, serum titers should not differ in more than0.13 from the reference values. Four out of six control sera should

omply with all the conditions cited above in order to validate eachlate.

.3.4. Serum samplesTiters were calculated for each sample and the correction factor

f’ was applied when corresponded. The curves representing ODalues versus log10 of serum dilutions of the 16 serum samples wereenerated using the software. The graphic had to show sigmoidhaped curves typical of dose–response in a parallel array in ordero validate each plate. This verification was done visually.

.4. Validation of the lpELISA

Intra-plate and intra-day repeatability were estimated previ-usly (data not shown). The intra-laboratory repeatability precisionas estimated with data obtained by different analysts, selecting at

andom one lpELISA plate from 15 different days, for each of the sixontrol serum of every virus strain being tested (24 control sera),ver a 3 months period (Lab #1). Other estimations of intermedi-te precision were performed in two other laboratories. In Lab #2,elonging to SENASA the same procedure as before was performedor 14 different days over the period of one year. In the Lab #3,elonging to our Institution but in a different location, 34–43 inde-endent testing of the six control sera for each vaccine strain werearried out over a period of 5 months. Standard deviations (S.D.) andoefficients of variation (CV%) were calculated [29]. Reproducibilityas estimated with 14 determinations of lpELISA titers in indepen-ent assays of the 24 control sera in each of the three mentioned

aboratories. S.D.s and CVs% between the three labs were calculated.The specificity data of the lpELISA for each vaccine strain was

stablished, based on assays of negative sera obtained from bovinesf the Patagonia region. In this case only one dilution (1:16) of theerum samples was assayed. Animals were considered as negativesf antibody titer values were ≤1.3.

Thirty-two control charts [29] were used to monitor the perfor-ance of the assay in time (i.e. one per each six control sera of every

erotype, four for each 100% antigen OD and four for each blank).Studies performed to establish the regression model between

he titers obtained in CEVAN lpELISA and the PANAFTOSA ELISAere performed as follows. Forty sera of each FMDV strains24/Cruzeiro, C3/Indaial and O1/Campos, corresponding to 10 dif-

erent antibody levels were titrated by both assays. Each serum wasepeated 16 times, and the mean, maximum and minimum titersere determined. Using previous results from the Rio de La Plataasin Subproject (CEE/Cuenca del Plata/CPFA/OPS [30]) regardinghe correlation of PANAFTOSA ELISA titers with PPG, the percentagef expected protection (EPP) for every CEVAN lpELISA titer in theANAFTOSA logit curves could be established. There were no testsvailable for A/Arg/01 strain in PANAFTOSA.

Further collaborative studies were performed analyzing 621erum samples with both tests in several public and private lab-ratories of Argentina and South America.

.5. Statistical analysis and correlation with PPG

The EPP for each FMDV strain was calculated by comparing theevels of lpELISA antibody titers from sera collected at 60 dpv withesults from cattle protection in challenge tests (PPG) at 90 dpv.

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6 (2008) 6577–6586

The statistical model used to find the correlation between theinary variable (protected/unprotected) and the continuous vari-ble (lpELISA titer) was the logistic regression (Logit), using theormula: Logit(p) = ln(p)/[1 − ln (p)] = ˛ + ˇxi, where x is the lpELISAiter and p is the probability of the event “protected” for a given xiter.

The probabilities were calculated using the logit estimators (˛nd ˇ) derived from the database obtained from the trials. The val-es of p, which indicates the probability of the model adjustmentfitting), were calculated. For the calculation of the theoretical EPPhe formula used was: EPP% = Exp(Logit)/(1 + Exp(Logit) [31].

.6. Concordance between vaccine status by PPG or EPP

The EPP for each individual vaccine was calculated with the logitegression curves using the mean of the lpELISA titers of the 16nimals involved in each PPG trial. These values were used for thestablishment of concordance between both in vivo and in vitroethodologies for vaccine approval.

. Results

.1. PPG trials

The performance of 40 commercial vaccines in the PPG trialsas as follows: 30 approved, 7 were rejected and 3 were sent for

etesting.All 647 sera from vaccinated animals and sera from the unvac-

inated controls collected at 0, 30, 60 and 90 dpv, were assayedy lpELISA for each FMDV strain present in the vaccines. However,nly the 60 dpv titers were considered to study the correlation withPG. The monitoring of the evolution of the lpELISA titers uponime constitutes an overall control of the trials and of the animals’erological status. Usually, most animals seroconverted by 14 dpv.hen, serum titers continue to increase very slowly and most ofhem reach a plateau or even start to decrease by 60 dpv. In thease of high potency vaccines serum titers continue to increaseven after 90 dpv (data not shown). All unvaccinated control ani-als remained negative during the trial and seroconverted at 7 dpc

data not shown) showing clinical signs of the disease (100% assayensitivity).

Although only 16 animals per trial are considered for the cal-ulation of PPG and EPP, it is usual to vaccinate17 animals in ordero prevent eventual losses. Then, the animal with the lowest titers discarded. Given the low number of trials that have been per-ormed, animal number 17 was also challenged in many trails andlthough it was not considered for potency assessment, the lpELISAiter and protection status were included in the database (Table 1).

.2. lpELISA validation

The lpELISA described in this report is officially used inrgentina as indirect potency test for FMD vaccines and was thor-ughly validated. Data on intermediate precision of the test wasbtained from different laboratories (Table 2). CV% values were inll cases <10% when performed by different analysts in differentays, which was the acceptance criteria [29].

lpELISA reproducibility was assessed from 14 determinations ofhe 24 control sera carried out in the three mentioned labs (Table 3)nd CV% values were found to be <10%.

The diagnostic specificity of the lpELISA for each vaccine strainas established, based on assays of negative sera obtained fromovines of the Patagonia region (Table 4). For the selected cut offorresponding to a titer ≤1.3, the specificity of the assay for eachMD virus strain was established as follows: 99.8% for O1/Campos

E. Maradei et al. / Vaccine 26 (2008) 6577–6586 6581

Table 2Intermediate precision of lpELISA assessed in three different laboratories for 24 control sera.

Strain Lab #1 Lab #2 Lab #3

Mean S.D. CV (%) Mean S.D. CV (%) Mean S.D. CV (%)

A24/Cruz. n = 15 n = 14 n = 43C #1 2.06 0.07 3.59 2.11 0.12 5.50 2,06 0.08 3.88C #2 1.85 0.07 3.54 1.90 0.12 6.21 1.85 0.04 2.38C #3 1.68 0.10 6.20 1.72 0.10 6.05 1.66 0.07 4.35C #4 2.14 0.09 4.03 2.15 0.11 5.12 2.14 0.11 5.12C #5 1.90 0.04 2.37 1.92 0.08 4.44 1.93 0.14 7.10C #6 1.68 0.01 5.99 1.78 0.09 5.17 1.72 0.09 5.41

A/Arg/01 n = 15 n = 14 n = 37C #1 1.97 0.08 3.97 1.95 0.08 4.13 1.98 0.06 2.83C #2 1.76 0.09 5.14 1.79 0.10 5.61 1.78 0.05 2.95C #3 1.55 0.12 7.65 1.53 0.14 9.50 1.55 0.08 5.01C #4 2.02 0.06 3.05 1.99 0.09 4.37 2.07 0.08 3.84C #5 1.85 0.10 5.24 1.79 0.07 3.94 1.86 0.05 2.78C #6 1.72 0.08 4.66 1.57 0.12 7.42 1.63 0.07 4.18

O1/Cam n = 15 n = 14 n = 43C #1 2.26 0.06 2.82 2.23 0.15 6.71 2.20 0.10 4.52C #2 1.96 0.05 2.41 1.99 0.08 3.90 1.94 0.06 3.11C #3 1.83 0.05 2.92 1.85 0.09 4.83 1.80 0.05 2.99C #4 2.30 0.12 5.09 2.28 0.13 5.82 2.28 0.13 5.77C #5 2.00 0.03 1.54 2.02 0.09 4.58 1.98 0.06 3.25C #6 1.82 0.04 2.04 1.87 0.07 3.49 1.81 0.06 3.20

C3/Ind n = 15 n = 14 n = 34C #1 2.12 0.07 3.32 2.17 0.12 5.41 2.22 0.12 5.61C #2 1.83 0.04 2.37 1.89 0.09 4.62 1.86 0.06 3.08C #3 1.73 0.07 3.82 1.77 0.08 4.33 1.73 0.07 4.12C #4 2.04 0.07 3.39 2.06 0.09 4.37 2.07 0.10 4.66C #5 1.83 0.07 3.65 1.83 0.06 3.45 1.81 0.06 3.48

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Table 4Diagnostic specificity of lpELISA for FMDV strains A24/Cruzeiro, A/Arg/01,O1/Campos and C3/Indaial.

Strain lpELISA titers Freqa % Acc %b

O1/Campos (n = 1045) ≤0.9 987 94.4 94.41.0 34 3.3 97.71.1 16 1.5 99.21.2 4 0.4 99.61.3 2 0.2 99.8

C #6 1.60 0.05 3.31 1.60

: number of assays; S.D.: Standard deviation; CV: Coefficient of variation.

n = 1045); 99.9% for A24/Cruzeiro (n = 799); 99.7% for A/Arg/01n = 779) and 99.6% for C3/Indaial (n = 785).

Thirty-two control charts were used for monitoring the per-ormance of the assay in time. The values obtained for the serumontrols usually remained within ±2 S.D. Three of these charts, rep-esenting one control serum of each serotype, are shown in Fig. 2.he titers obtained are plotted as points, the dotted line representshe mean titer and the solid lines represent ±1 S.D., ±2 S.D. and3 S.D. from the mean. The mean and S.D. were initially found

fter 15 determinations of each serum (C #5 for A/Arg/01, C #2or O1/Campos and C #3 for C3/Indaial (mean ± S.D.: 1.85 ± 0.097,.96 ± 0.047 and 1.73 ± 0.066, respectively)). In this case, the chartepresents 50 independent runs of the assay, performed within 5

able 3eproducibility of lpELISA assessed in three different laboratories.

train Meana S.D. CV (%) Strain Mean S.D. CV (%)

24/Cruz. O1/CamC #1 2.07 0.09 4.37 C #1 2.22 0.11 4.89C #2 1.87 0.08 4.34 C #2 1.96 0.06 3.17C #3 1.70 0.09 5.20 C #3 1.82 0.06 3.32C #4 2.15 0.11 4.96 C #4 2.32 0.16 6.70C #5 1.93 0.14 7.14 C #5 2.01 0.08 3.77C #6 1.74 0.08 4.49 C #6 1.83 0.05 2.97

/Arg/01 C3/IndC #1 1.98 0.07 3.55 C #1 2.19 0.10 4.77C #2 1.79 0.07 4.13 C #2 1.87 0.07 4.00C #3 1.57 0.11 6.84 C #3 1.74 0.06 3.41C #4 2.05 0.13 6.32 C #4 2.06 0.08 4.07C #5 1.83 0.08 4.10 C #5 1.81 0.05 2.93C #6 1.60 0.10 6.28 C #6 1.58 0.09 5.56

.D.: standard deviation; CV: coefficient of variation.a Mean of 42 determinations (14 in each Lab).

1.4 2 0.2 100≥1.5 0 0.0 100

A24/Cruz. (n = 799) ≤0.9 695 87.0 87.01.0 64 8.0 95.01.1 24 3.0 98.01.2 12 1.5 99.51.3 3 0.4 99.91.4 1 0.1 100

≥1.5 0 0.0 100

A/Arg/01 (n = 799) ≤0.9 627 80.5 80.51.0 62 8.0 86.41.1 52 6.7 95.11.2 27 3.5 98.61.3 9 1.2 99.71.4 2 0.3 100

≥1.5 0 0.0 100

C3/Indaial (n = 785) ≤0.9 621 79.1 79.11.0 121 15.4 94.51.1 26 3.3 97.81.2 11 1.4 99.21.3 3 0.4 99.61.4 1 0.1 99.71.5 2 0.3 100

≥1.5 0 0.0 100

n: number of serum samples tested.a Frequency.b Accumulated frequency.

6582 E. Maradei et al. / Vaccine 26 (2008) 6577–6586

F 5 for1 runs oc ation

ms

a

Fcb

ig. 2. Control charts representing one control serum of each FMDV serotype (C #.96 ± 0.047 and 1.73 ± 0.066, respectively)). The charts represent 50 independentorrection factor. The dotted lines represent the mean titer (found after 15 determin

onths. The CV% were 5.24%, 2.41% and 3.82%, respectively, for theerum controls mentioned above.

Parallel determinations of serum titers using the CEVAN lpELISAnd the PANAFTOSA ELISA, performed on the same set of sera at

PFuf

ig. 3. Correlation of titers obtained using the CEVAN lpELISA and PANAFTOSA ELISA, foollected from assays performed in CEVAN, SENASA and PANAFTOSA laboratories. (B) They technicians belonging to different public and private laboratories following the protoc

A/Arg/01, C #2 for O1/Campos and C #3 for C3/Indaial (mean ± S.D.: 1.85 ± 0.097,f the assay, performed within 5 months. (�) lpELISA titer after application of the

s of each serum) and the solid lines represent ±1 S.D., ±2 S.D. and ±3 S.D.

ANAFTOSA (Brazil) showed a good correlation for the regionalMDV strains A24/Cruzeiro, O1/Campos and C3/Indaial, with R2 val-es of 0.9804 for O1/Campos, 0.7851 for A24/Cruzeiro, and 0.9903or C3/Indaial. The range of variation was satisfactory, presenting

r O1/Campos, A24/Cruzeiro and C3/Indaial FMDV strains. (A) The data shown wasdata shown was collected from assays performed in parallel with both ELISA kits

ols of CEVAN and PANAFTOSA.

cine 26 (2008) 6577–6586 6583

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Table 6Concordance of lpELISA and PPG for vaccine approval.

PPG

Approved Rejecteda Total

lpELISAApproved 19 1b 20Rejected 5 9 14Total 24 10 34

Concordance 82%

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E. Maradei et al. / Vac

ood parallelism and the expected tendency for the different titerevels (not shown). Statistically, the capacity of both assays to dif-erentiate sera with different antibody levels was demonstrated.ispersion diagrams are shown in the upper panel of Fig. 3 for eachMD virus type (O1/Campos, C3/Indaial and A24/Cruzeiro). A lin-ar relationship between titers determined with both assays can bebserved.

Further collaborative studies were performed analyzing 621erum samples with both tests. Data was collected from severalublic and private laboratories of Argentina and South AmericaBrazil and Colombia). The results are shown all together in theower panels of Fig. 3, with the corresponding R2 values for eachtrain, displaying a good correlation of the titers obtained with bothssays at different laboratories.

Preliminary data from assay robustness was obtained runninghe assay with control antigens heated at 56 ◦C for 2 min, in ordero degrade 140S virus particles into 12S protomers. Untreated anti-ens were used as controls. All the serum controls were validatedimilarly in both cases (data not shown). A similar experiment waserformed after storage of the antigens at 37 ◦C for 40 days. We arearrying out systematic experiments in order to further study thetability of the antigens and its impact on the performance of thessay.

.3. Correlation between protection against challenge andpELISA titers in vaccinated cattle

Data on in vivo protection (PPG) and lpELISA titers for each virustrain analyzed (A/Arg/01, O1/Campos, C3/Indaial A24/Cruzeiro)re shown in the four left panels of Fig. 4(A–D). In each case theumber of sera analyzed is shown. On the left side of the figures,he PPG data is depicted as an histogram ordered per fixed lpELISAiter interval. The bar sizes account for the percentages of protectionorresponding to a specific titer interval. The total number of ani-als whose serum titers are included in each titer interval is shown

t the top of each bar. A close correlation can be observed betweenhe serum lpELISA titers and the percentage of in vivo protected ani-

als for A/Arg/01 and O1/Campos FMDV strains, whereas a goodut less close correlation was established for C3/Indaial strain, forhich only seven PPG trials were carried out.

Using these databases, the logit estimators ˛ and ˇ were calcu-ated and were found highly significant (p < 0.01) for the four FMDVtrains (Table 5). The fitting of the models, given by the value of p-alue (probability of model adjustment) was obtained for three ofhe FMDV strains as follows: A/Arg/01 = 0.8186, O1 Campos = 0.8819nd C3 Indaial = 0.9768. In the case of A24/Cruzeiro strain, theegression could not be obtained with the data available in thisork (p > 0.05). The theoretical EPPs were calculated from the logit

ransformation with the formula EPP% = Exp(Logit)/(1 + Exp(Logit).In the right side of each panel of Fig. 4, the EPP curves for lpELISA

iters extended from 0.60 to 4.1 obtained by logit regression arehown for A/Arg/01, O1/Campos and C3/Indaial FMDV strains. Theut off of 75% EPP is shown as a dotted line. Three tables were

able 5arameters of logit transformation and model fitting.

train ˛ ˇ p-reg p-tit p-value

/Arg/01 −5.9346 3.2019 < 0.01 < 0.01 0.81861/Campos −4.9111 2.8474 < 0.01 < 0.01 0.88193/Indaial −5.1759 2.8965 < 0.01 < 0.01 0.976824/Cruz. −1.7292 1.8064 0.3728a 0.0444 0.9999

and ˇ: logit coefficients; p-tit: probability of significant differences between titersf protected/not protected animals (should be <0.05); p-reg: probability of regres-ion (should be <0.05). p-value: adjustment.

a p-regression > 0.05.

rAa

4

aaotitloAi

Kappa 0.62

a Vaccines to be retested were considered as rejected.b Status: retesting by PPG, approved by ELISA (EPP = 76%).

erived from this transformation were the EEP% value for every pos-ible lpELISA titer for each of the strains can be found. These tablesre not shown in this report for space problems but are availablen-line as a Supplemental file.

.4. Concordance of lpELISA titers and PPG performance forpproval or rejection of commercial vaccines

Vaccine status according to lpELISA titers was derived from theogit regressions curves shown in Fig. 4 for A/Arg/01, O1/Camposnd C3/Indaial. Out of 34 PPG vaccine trials carried out for1Campos, C3 Indaial and A/Arg/01, 20 vaccines were approved,were rejected and 10 scheduled for retesting according to EPP

alues. In the case of A24/Cruzeiro strain (6 PPG trials), EPPs wereerived from PANAFTOSA’s correlation curve [30] and thereforehey were not considered in this analysis.

A two-way table was assembled with the data of both the inivo trials and the in vitro assays (Table 6). For this exercise, vac-ines sent for retesting were considered as rejected. Using a 75% cutff value for EPP, the concordance between both methods (PPG andpELISA) was 82%, associated with a Kappa index of 0.62 (good con-ordance). Kappa values vary between 0 (not concordance) and 1full concordance) representing degrees of concordance excludingandom events [32,33].

Nineteen out of 34 vaccines were approved by both methodshile 9 out of 34 were rejected by both methods, 5 were approved

y PPG but rejected by ELISA and 1 was approved by lpELISA andejected by PPG (retesting). Fig. 5 shows the EPPs calculated frompELISA titers of 34 batches of commercial vaccine series, where thetatus of approval or rejection by PPG challenge is represented byymbols. Vaccine series that did not pass PPG but may be admittedor a new potency test are represented as triangles. For an EPP cutff of 75% (dotted line) only one vaccine sent to retesting by PPGas approved based on their lpELISA titers (EPP = 76%).

Although the EPP data was not derived from Argentine cor-elation curves, the potency data on vaccines formulated with24/Cruzeiro strain are in agreement with the results mentionedbove (gray filled circles in Fig. 5).

. Discussion

According to OIE regulations, indirect tests may be used tossess the potency of a vaccine provided that a statistical evalu-tion has established a satisfactory correlation between the resultsbtained by the indirect test on the relevant vaccine serotype andhe potency test in cattle [5]. Although the most explored area inndirect potency methods has been the correlation between pro-

ection and neutralizing antibody, our group and others have had aong term experience in studying the correlation of potency basedn ELISA titers either with experimental or commercial vaccines.s described in Section 1, one of the main strengths of this work

s the fact that it was performed using commercial vaccine series

6584 E. Maradei et al. / Vaccine 26 (2008) 6577–6586

F ams ws Camp( ection

fap(

ig. 4. Correlation of protection by PPG versus lpELISA titers. To the left, the histogrhown. The logit transformation curves are shown to the right for A/Arg/01 (A), O1/p > 0.05) (D) with the data generated in this work. EPP, expected percentage of prot

or which both data, lpELISA titers and PPG outcome, were avail-ble for particular FMDV strains. We had reported in two previousublications the correlation of direct and indirect potency testslpELISA) involving 5554 vaccinated/challenged bovines with the

A[vd

ith data of in vivo percentage of protection (PPG) for each lpELISA titer interval areos (B), and C3/Indaial (C). No logit regression was obtained for A24/Cruzeiro strain; n, number of animal sera tested.

rgentine strains (A/Arg/87, A/Arg/79, O1/Caseros and C3/Arg/85)18,19]. Later on, these studies were extended to another set of 7390accinated/challenged bovines with the same strains (unpublishedata), reaching a total 12,944 vaccinated animals analyzed.

E. Maradei et al. / Vaccine 2

Fig. 5. Concordance of lpELISA and PPG for 40 Argentine commercial vaccines,shown as vaccine status in PPG trials versus percent expected protection (EPP) cal-culated by lpELISA titers. (�) Approved by PPG; (♦) rejected by PPG; (�) retestingbdc(

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y PPG. Gray area: EPP retesting area. EPP = 75% is depicted by a dotted line. Theata corresponding to six A24/Cruzeiro vaccines approved by PPG whose EPP wasalculated using PANAFTOSA logit regression curves [30] are shown by gray circles

).

Statistical analysis of this large database had been performedn the past in order to establish the correlations (EPP logit regres-ion curves) used by SENASA for vaccine approval (Zanelli et al.,npublished). This work definitely denoted that the lpELISA was an

ndirect test which corresponded to protection in the target speciesn vivo, and was used in Argentina until FMD eradication in 1999.

Vaccines manufactured after the year 2001 were composed ofifferent virus strains and have been approved mostly by PPG car-ied out in BSL3A animal facilities, or by lpELISA titers based onhe existing PANAFTOSA correlation curves for A24/Cruzeiro and1/Campos strains and on preliminary data for A/Arg/01 strain

21]. This work reports a reappraisal of the correlation between inivo challenge and indirect potency testing in the target speciesor FMDV strains considered (O1/Campos, C3/Indaial, A/Arg/01,24/Cruzeiro). This is of great importance to countries with vac-ination programs like Argentina. Multivalent commercial vaccineesting is performed nowadays by PPG challenge only for registra-ion of new products (at least for the three first commercial series)r in the case of an introduction of a new strain in an alreadyxisting vaccine. Another important consideration is that in mul-ivalent FMD vaccines containing four different strains of FMDVt is not possible to undertake a challenge test with more than aingle strain of virus at a time. Usually, sanitary authorities selectne of them for a challenge trial and the other strains are testedy serology with lpELISA. The same criterion is applied for regularommercial vaccine series after the registration process is com-leted, where only sporadic at random in vivo tests are performedfterwards.

The CV% of the lpELISA described in this work for intermediaterecision and reproducibility (Tables 2 and 3) was below 10%, which

s a suitable accepted value [29] and it showed diagnostic specifici-ies over 99.5% and sensitivities of 100%, for the four studied FMDVtrains (Table 4). Several further collaborative studies have beenerformed involving public and private laboratories of Argentina,razil and Colombia. Professionals of these labs have been capaci-ated at CEVAN in every case, before transference of the techniquend its reagents (kits). The same assay was adapted for use in the

creening of herd immunity status in the field, using a single serumilution (not shown in this work).

In this work a statistically suitable correlation was establishedy logit regression for strains A/Arg/01, O1/Campos and C3/Indaial,ut not for A24/Cruzeiro (regression p > 0.05). A24/Cruzeiro was the

u2bar

6 (2008) 6577–6586 6585

train less tested by PPG in commercial vaccine trials due to the facthat the emerging strain A/Arg/01 was prioritized in potency testsn vivo. It is worth mentioning that many experimental PPG testsave been carried out with A24/Cruzeiro, including the six recentlyescribed by Goris et al. [22]. C3/Indaial strain was also tested only

n seven PPG tests because it was not incorporated into the vaccinentil the year 2005. The strength of the correlations was shown

n this work (Table 5 and Fig. 4). The logit estimators ˛ and ˇ wereound highly significant (p < 0.01) for three out of four FMDV strainsnd the fitting of the model was given by the probability of signifi-ant differences between titers of protected/not protected animalsnd the probability of regression, which were <0.05 in both casesor all strains except for A24/Cruzeiro. More tests in vivo involv-ng commercial vaccines should be performed with A24/Cruzeirohallenge in order to establish a suitable correlation for this strain.

When the analysis with a two-way table was applied (Table 6),t was clear that for the EPP cut off considered (75%) there wereo vaccines rejected by PPG that were approved based on their

pELISA titers, except one vaccine that was approved by EPP withPPG status of retesting. Two vaccines rejected and five vaccines

pproved by PPG were considered for retesting by ELISA. The otherwo vaccines whose potency might be retested according to PPGFig. 5) were considered in the same status according to lpELISAiters.

It is worth mentioning from the point of view of the valid-ty of indirect methods, that although the serum titers for24/Cruzeiro were determined with our lpELISA and the corre-ponding PANAFTOSA EPP correlation curve, the data obtainedt into the same rationale: The six A24/Cruzeiro vaccines werepproved by PPG and five of them were also approved by ELISA,hile the sixth was in the retesting area by lpELISA (Fig. 5).

There is still a concern from the point of view of vaccine manu-acturing companies based on the fact that five vaccines that haveassed PPG are considered for retesting by lpELISA. In this worke have used 75% EPP as an example, because it is the value beingowadays considered by SENASA in Argentina. For instance, lower-

ng the EPP cut off to 70%, would mean that four more good vaccinesre approved by lpELISA, but also two rejected vaccine and two thatad been assigned for retesting by PPG would be approved by EPP.he cut off level to be used may be modified in order to meet theesired vaccine potency needed for control of FMD. In any case, san-

tary authorities should establish the cut off values of the indirectest as it exceeds the scope of this work.

Finally, we would like to emphasize that the reliability ofpELISA to evaluate the potency of FMD vaccines as well as itsase of adaptability for many different virus serotypes could leadn the near future to the complete replacement of the challengerocedures not only in Argentina but in other regions of the world,llowing the elimination of the massive use of in vivo challengeests for regular vaccine testing and avoiding the use of live virusor that purpose. Efforts for the replacement of the in vivo potencyests performed for aphtovirus vaccines have been in course for aong time at several public and private laboratories [17,34] becausef ethical and economical reasons. In the lpELISA described inhis work, pools of well-characterized MAbs are used as detectors.his reagents are available for multicenter collaborative studies ifhey are required, where the best pool composition and workingonditions can be set up for use with other FMDV strains. Morehan 80 MAbs have been developed in our laboratory (Seki et al.,npublished data) and a selection of the pools has been made for

se with every different strain present in South America in the last0 years. Many specific reagents (polyclonal and monoclonal) haveeen also reported by other labs [35,36]. Although internationalcceptance and production of reference materials might be aesponsibility of the OIE, it will probably also depend on particular

6 cine 2

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overnmental decisions made in each country. The data collectedn this work will hopefully help to contribute in this direction.

cknowledgements

This work was supported by the Consejo Nacional de Investi-aciones Científicas y Técnicas (CONICET) of Argentina, FONCYTPID2003-00330) and the Animal Virology Studies FoundationFEVAN). CEVAN and SENASA are members of the Inter-institutionalMD Research and Development Network (RIIDFA) of Argentina. Wehank Miss Silvia Rojana, Maria Rodriguez and Carmen Devincenzoor their technical assistance.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at doi:10.1016/j.vaccine.2008.09.033.

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36] Crowther JR, McCullough KC, Brocchi E, De Simone F. Monoclonal antibod-ies against FMDV: use and potential application. In: Session of the ResearchGroup of the Standing Technical Committee of the European Commission forthe Control of FMD. Rome: FAO; 1984. p. 40.