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Vaccine 25 (2007) 4784–4794

Chimeric tymovirus-like particles displaying foot-and-mouthdisease virus non-structural protein epitopes and its use for

detection of FMDV-NSP antibodies

Masarapu Hema a,b, Singanallur Balasubramanian Nagendrakumar a,Reddivari Yamini a, Dev Chandran a, Lingala Rajendra a, Dorairajan Thiagarajan a,

Satya Parida c, David James Paton c, Villuppanoor Alwar Srinivasan a,∗a Indian Immunologicals Limited, Rakshapuram, Gachibowli, Hyderabad 500032, Andhra Pradesh, India

b Department of Biotechnology, Sri Padmavathi Mahila Viswa Vidyalayam, Tirupati 517502, Andhra Pradesh, Indiac Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, GU24 0NF, United Kingdom

Received 8 January 2007; received in revised form 4 April 2007; accepted 7 April 2007Available online 26 April 2007

bstract

Expression of Physalis mottle tymovirus (PhMV) coat protein (CP) in Escherichia coli (E. coli) was earlier shown to self-assemble into emptyapsids that are nearly identical to the capsids formed in vivo. Aminoacid substitutions were made at the N-terminus of wild-type PhMV CPith single or tandem repeats of infection related B-cell epitopes of foot-and-mouth disease virus (FMDV) non-structural proteins (NSPs) 3B1,B2, 3AB, 3D and 3ABD of lengths 48, 66, 49, 51 and 55, respectively to produce chimeras pR-Ph-3B1, pR-Ph-3B2, pR-Ph- 3AB, pR-Ph-3Dnd pR-Ph-3ABD. Expression of these constructs in E. coli resulted in chimeric proteins which self-assembled into chimeric tymovirus-likearticles (TVLPs), Ph-3B1, Ph-3B2, Ph-3AB, Ph-3D and Ph-3ABD as determined by ultracentrifugation and electron microscopy. Ph-3B1,h-3B2, Ph-3AB and Ph-3ABD reacted with polyclonal anti-3AB antibodies in ELISA and electroblot immunoassay, while wild-type PhMVVLP and Ph-3D antigens did not react. An indirect ELISA (I-ELISA) was developed using Ph-3AB to detect FMDV-NSP antibodies in sera

f animals that showed clinical signs of FMD. Field serum samples from cattle, buffalos, sheep, goats and pigs were examined by using thesehimeric TVLPs for the differentiation of FMDV infected animals from vaccinated animals (DIVA). The assay was demonstrated to be highlypecific (100%) and reproducible with sensitivity levels (94%) comparable to the Ceditest kit (P > 0.05).

2007 Elsevier Ltd. All rights reserved.

tation;

vi[

dtw

eywords: Foot-and-mouth disease; Non-structural protein; Epitope presen

. Introduction

Foot-and-mouth disease (FMD) is caused by foot-and-outh disease virus (FMDV) belonging to Aphthovirus

enus in the family Picornaviridae. FMDV is an icosahe-ral virus of 30 nm size. The viral genome is a positiveense RNA of 8.5 kb with a large open reading frame (ORF)

ncoding 12 viral proteins, L, 1A, 1B, 1C, 1D, 2A, 2B,C, 3A, 3B, 3C and 3D. Structural proteins (SPs), 1A toD self-assemble to form the capsid of the virion. Other

∗ Corresponding author. Tel.: +91 40 23000894; fax: +91 40 23005958.E-mail address: srini@indimmune.com (V.A. Srinivasan).

aodacy

264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2007.04.023

Physalis mottle tymovirus; VLPs; DIVA

iral proteins are non-structural proteins (NSPs) that partic-pate in replicatory and other functions within the host cell1,2].

FMD is controlled by culling and vaccination programsepending on the epidemiological situation. Current conven-ional vaccine is a chemically inactivated partially purifiedhole-virus preparation. Identification of FMDV-infected

nimals is important because they act as potential carriersf the virus and become a source for new outbreaks [3]. The

ifficulty of differentiating FMDV-infected from vaccinatednimals (DIVA) has kept many non-endemic countries fromonsidering vaccination as primary control measure for manyears [4].

cine 25

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M. Hema et al. / Vac

FMD-infected animals produce antibodies to both SPs andSPs while vaccinated animals produce antibodies princi-ally to SPs of FMDV. Therefore, NSP antibody assays aresed to differentiate infected animals from those that haveeen vaccinated [4]. Use of diagnostic tests for DIVA basedn NSP antigens like 3ABC, 3AB, 3A or 3B, produced inifferent expression systems has been described earlier [5].everal other reports have shown the use of NSPs for theIVA [2,6–10]. The use of various recombinant NSPs in

he commercial ELISAs gave a few false positives due toon-specific reactions [11,12]. FMDV-NSP infection-related-cell epitopes and T-cell epitopes were mapped by analyzingverlapping peptides which were used in ELISAs as syntheticeptides [2,4,11,13]. But synthetic peptides are expensive andoorly antigenic for use in the ELISA based tests. Therefore,here is a need to produce novel antigens which can help in theevelopment of sensitive and specific tests for the accurateetection of anti-NSP antibodies.

A novel approach with potential biological applicationnvolves the translational fusion of small foreign peptides toiral proteins. The sites of insertion were chosen so that theesulting peptides were displayed on the viral particle [14,15].everal plant viruses/viral capsid proteins were used as effi-ient presentation systems for viral antigenic peptides orpitopes to use as potential vaccine candidates [14–18]. Rodhaped plant virus-like particles were used for the presenta-ion of heterologous epitopes and used as vaccine [19–21].ubella VLPs and calcivirus capsids were used for the detec-

ion of rubella and porcine enteric calcivirus, respectively22,23]. Physalis mottle tymovirus (PhMV), a spherical plantirus consists of ssRNA genome of size 6.3 kb encapsidatedn an icosahedral shell of 180 coat protein (CP) subunitsrranged at T = 3 symmetry. PhMV CP expressed in E. colias found to self-assemble into capsids in vivo [24]. Further,either deletion nor addition of amino acids at the N-terminusf PhMV hinders the assembly of the capsids, indicating theccessibility of the N-terminal arm of PhMV CP for manip-lations [24,25]. This could potentially facilitate the displayf immunodominant epitopes of heterologous antigens on theurface of PhMV CP.

In this paper, we report the design and display of FMDVnfection-related B-cell epitopes of NSPs 3A, 3B and 3D4,11] on PhMV CP to produce chimeric tymovirus-like parti-les (TVLPs) and the use of these chimeric TVLPs as antigenn an indirect ELISA (I-ELISA) for the detection of FMDV-SP antibodies in sera of cattle, buffaloes, sheep, goats andigs. Specificity and sensitivity levels of I-ELISA test wereompared with a commercial kit.

. Materials and methods

.1. Experimental sera

The bovine convalescent sera (BCS) were collected fromaıve control cattle challenged with virulent FMDV belong-

2

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(2007) 4784–4794 4785

ng to serotypes O, A, C and Asia 1 used in routine potencyests. The carrier status of these animals were confirmed withirus isolation from probang samples. Negative sera includedn the test originated from cattle sheep and goats free fromMD. Adult bovine serum (Selbourne, UK) certified as freerom anti-FMDV antibodies was also used as known neg-tive control. The seronegative status of these samples wasonfirmed by virus neutralization test (VNT) and liquid phaselocking (LPB) ELISA using standard protocol [3]. In addi-ion FMDV-NSP antibodies positive and negative cattle seraere obtained from Institute for Animal Health, Pirbright,nited Kingdom for evaluation. Polyclonal rabbit and mouse

era against E. coli expressed 3AB protein were used as con-rol positive sera. Non-immunized rabbit and mouse sera weresed as control negative sera. Sera of cattle (n = 89), buf-aloes (n = 65), sheep (n = 61) and pig (n = 39) collected fromifferent herds and abattoirs in India were used.

.2. Commercial NSP test

Ceditest FMDV-NS (Cedi Diagnostics BV, Lelystad, Theetherlands), a competitive ELISA that can be used for cattle,

heep and pigs was used as a standard for comparison.

.3. Construction of wild-type PhMV coat protein andhimeric constructs

Synthetic wild-type PhMV CP [26] (EMBL accessionumber: S97776) was made with XhoI and HindIII sites at′ and 3′ ends, respectively and with a stop codon at the endf the reading frame (GENEART, Regensburg, Germany). ApnI site was introduced as silent mutation at 44 and 45 aminocids of the CP for the insertion of heterologous sequences.his synthetic wild-type PhMV CP of 564 bp was cloned athoI and HindIII sites of the pRSET-A vector (Invitrogen,SA) (pR-Ph-CP). As the reading frame started at the ATGithin the NdeI site of the vector, the wild-type PhMV CP

onstruct (pR-Ph-CP) was expressed with an N-terminus con-aining additional 39 amino acids from the vector backbone.

DNA coding for single and tandem repeats of infection-elated B-cell epitopes of 3A, 3B and 3D [4,11] linked byGS linker sequence of lengths 144, 198, 147, 153 and 165ucleotides were synthetically made with Nde1 site at 5′ end,ollowed by a part of PhMV sequence until KpnI site at the 3′nd (Table 1). These heterologous sequences were swappedn-frame into the NdeI and KpnI digested pR-Ph-CP (Fig. 1).lasmid DNA isolations were carried out by the alkaline lysisethod [27]. The inserts in the chimeric constructs were con-rmed by automated DNA sequencing. The chimeras wereamed as pR-Ph-3B1, pR-Ph-3B2, pR-Ph- 3AB, pR-Ph-3Dnd pR-Ph-3ABD.

.4. Expression and purification of chimeric proteins

Expression and large scale purification of wild type andhimeric proteins were done as described by Sastri et al.

4786 M. Hema et al. / Vaccine 25 (2007) 4784–4794

Table 1List of infection related epitopes according to Hohlich et al. [11]; Sun et al. [4] in FMDV 3ABD nonstructural protein with respect to each chimera

FMDV-NSP epitopes Detail of sequences with linkers

3B1 GPYAGPLERQKPLKVRAKLPQQEGPYAGPMERQKPLKVKAKAPVVKE3B2 GPYAGPLERQKPLKggsPMERQKPLKVKAKAggsGPYAGPLERQKPLKggsPMERQKPLKVKAKA3AB NEYIEKANITTDDKggsGPYAGPLERQKPLKggsPMERQKPLKVKAKA3 TKLAP3 LETOK

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D MRKTKLAPTVAHGVFggs MRKABD NEYIEKANITTDDKggsGPYAGP

lycine–glycine–serine linker has been represented in small case between e

24]. Protein content was estimated using standard proce-ures [28].

.5. Preliminary characterization of chimeric proteins

.5.1. Indirect ELISAThe reactivity of Ph-CP, Ph-3B1, Ph-3B2, Ph-3AB, Ph-

D and Ph-3ABD with different sera containing FMDV-NSPntibodies was tested by indirect ELISA (I-ELISA). Ph-CPnd an E. coli expressed recombinant 3AB (r-3AB) werencluded as negative and positive antigen controls, respec-ively (Table 2).

.5.2. Electroblot immunoassayThe purified wild-type and chimeric proteins were sep-

rated on 15% SDS-PAGE [29] and transferred ontoitrocellulose membranes (Millipore Corporation, Bedford,A) [30]. r-3AB was used as positive control. The assay was

arried out using polyclonal 3AB rabbit antiserum (1:2000)aised against r-3AB. Horseradish peroxidase labeled goatnti-rabbit IgG (Sigma, USA) at 1:1000 dilution was used asecondary antibody. The blot was developed using diaminoenzidine (Sigma, USA) and hydrogen peroxide in 0.05 Modium citrate buffer (pH 4.8).

.5.3. Electron microscopyThe wild-type and chimeric proteins Ph-CP and Ph-

B1, Ph-3B2, Ph-3AB, Ph-3D and Ph-3ABD, respectively

aiPl

Fig. 1. Schematic representation of FMDV-NSP chimeric constructs pR

TVAHGVFggs MRKTKLAPTVAHGVFggs MRKTKLAPTVAHGVFggsPLKggsMRKTKLAPTVAHGVF

tope.

0.5 mg/ml) were applied onto carbon-shadowed, formvaroated grids and visualized by negative staining with uranylcetate (1%, w/v). These grids were examined in a high res-lution Transmission Electron Microscope (Hitachi H 7500)t a magnification range of 60,000–80,000×.

.6. Development of an indirect ELISA using knownositive and negative samples

The optimal concentration of antigen and dilution ofhe serum were determined by performing a checkeroard titration of the Ph-3AB antigen and a known pos-tive and known negative cattle serum. ELISA platesNunc® MaxisorpTM,The Netherlands) were coated witherial dilutions of purified chimeric proteins (4 �g to.95 ng/50 �l/well) in carbonate-bicarbonate coating bufferpH 9.0). After 1 h of incubation at 37 ◦C the plates wereashed with phosphate buffered saline with 0.05% Tween 20

PBST). The wells were blocked with 3% Skim Milk Pow-er in PBST (S-PBST). Known positive serum and knownegative serum diluted in S-PBST were added as serial dilu-ion of 1:5 to 1:320 completing the checker board titration.fter incubation at 37 ◦C for 1 h the plates were washedith PBST and 50 �l of HRP conjugated anti-bovine IgG at

ppropriate dilution in S-PBST was added to the plates. Afterncubation at 37 ◦C for 1 h and the plates were washed withBST and 50 �l of Chromogen/Substrate mix (orthophene-

ine diamine/hydrogen peroxide (Sigma, USA) was added to

-Ph-3B1, pRPh-3B2, pR-Ph-3AB, pR-Ph-3D and pR-Ph-3ABD.

M. Hema et al. / Vaccine 25 (2007) 4784–4794 4787

Table 2Differentiation of vaccinated and infected animals (DIVA) using TVLP antigens

Experimental sera Antigens

Ph-3B1 Ph-3B2 Ph-3AB Ph-3ABD Ph-3D Ph-CP E. coli r-3AB

Type O BCS 0.258 0.275 0.164 0.154 0.230 0.237 0.279 0.261 0.109 0.125 0.046 0.045 0.479 0.448Type A BCS 0.186 0.217 0.093 0.115 0.102 0.120 0.168 0.179 0.082 0.084 0.042 0.044 0.336 0.271Type C BCS 0.298 0.297 0.182 0.172 0.229 0.236 0.291 0.280 0.253 0.285 0.046 0.046 0.486 0.493Type ASIA1 BCS 0.279 0.262 0.144 0.146 0.220 0.225 0.234 0.248 0.122 0.119 0.045 0.046 0.246 0.415r-3AB IMS 0.298 0.323 0.144 0.138 0.183 0.188 0.194 0.178 0.201 0.205 0.049 0.054 0.460 0.462r-3AB IRS 0.307 0.337 0.113 0.127 0.147 0.126 0.116 0.107 0.135 0.134 0.059 0.060 0.097 0.100r-MVA 3AB IRS 0.424 0.415 0.147 0.148 0.176 0.165 0.151 0.130 0.155 0.155 0.052 0.055 0.083 0.091Normal BS 0.047 0.050 0.046 0.053 0.047 0.049 0.047 0.048 0.045 0.043 0.043 0.043 0.059 0.056Normal MS 0.049 0.047 0.050 0.060 0.056 0.046 0.043 0.041 0.042 0.041 0.042 0.044 0.055 0.051Normal RS 0.076 0.054 0.050 0.050 0.064 0.053 0.057 0.042 0.043 0.042 0.054 0.073 0.060 0.056CGM control 0.056 0.058 0.054 0.068 0.060 0.056 0.058 0.049 0.048 0.044 0.041 0.041 0.088 0.096P 8 0.

N ouse serc

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BS control 0.065 0.062 0.071 0.063 0.056 0.05

ote: BCS, bovine convalescent serum; BS, bovine serum; IMS, immune mell growth medium; PBS, phosphate buffered saline.

he plates. The plates were incubated in dark for 10 min atoom temperature. The reaction was stopped with 1 M Sul-uric acid (Emerck, Germany) and the plates were read at92 nm using ELISA plate reader (Multiscan®TitertekTM,inland). The optimal concentration of the chimeric proteinntigens and the optimal dilution of the serum for clear differ-ntiation of positive and negative samples were decided usinghis ELISA. Similarly a known positive and a known negativeig serum was used to arrive at the optimal concentration ofhe antigen and the serum dilution.

.7. Establishing cut-offs for negative samples

Known negative samples from cattle, goats and sheep sam-les were tested to derive cut-off value. The mean of the ODalues was calculated and the cut-off was fixed as the sum ofhe calculated mean and twice the standard deviation. Afteretermining the cut-off value, known positive samples andeld samples were tested using the chimeric antigens.

.8. Kinetics of antibody response to Ph-3AB antigen inattle, buffaloes, sheep and goats

The kinetics of the antibody response to Ph-3AB antigenn experimentally infected animals of different susceptiblepecies was evaluated. The animals were naıve unvaccinatednimals but challenged with either FMD Type O virus. Theamples were collected at different intervals on days 0, 5, 10,5, 21 and 28 or 35 post challenge. The samples were usedn a dilution of 1:50 and the Ph-3AB antigen as used at aoncentration of 50 ng (arrived after standardization of theLISA).

.9. Differentiation of FMDV infected from vaccinated

nimals (DIVA) using chimeric proteins

ELISA plates (Nunc® MaxisorpTM,The Nether-ands) were coated with purified chimeric proteins

tts1

056 0.050 0.052 0.049 0.049 0.068 0.052 0.065

um; MS, mouse serum; IRS, immune rabbit serum; RS, rabbit serum; GM,

50 ng/50 �l/well) in carbonate-bicarbonate coating bufferpH 9.0). All test and control samples were blocked at7 ◦C for 2 h by preparing pre-dilutions (1:50) in phosphateuffered saline containing 3% Skim Milk Powder and 0.05%ween 20 (S-PBST). The plates were washed with phosphateuffered saline with 0.05% Tween 20 (PBST). 50 �l ofach pre diluted blocked serum was added to the platesnd incubated at 37 ◦C for 1 h. The plates were washedith PBST and 50 �l of HRP conjugated anti-species

gG at appropriate dilution in S-PBST was added to thelate. After incubation at 37 ◦C for 1 h the plates wereashed with PBST and 50 �l of Chromogen/Substrate mix

orthopheneline diamine/Hydrogen Peroxide (Sigma, USA)as added. The plates were incubated in dark for 10 min

t room temperature. The reaction was stopped with 1 Mulfuric acid (Emerck, Germany) and read at 492 nm usingLISA plate reader (Multiscan®TitertekTM, Finland). The

est samples were declared as positive or negative based onhe cut-off value arrived as mentioned previously.

.10. Statistical analysis

The I-ELISA results were statistically analyzed and com-ared with Ceditest for sensitivity and specificity [31].

. Results

.1. Expression in E. coli and purification of chimericroteins

Wild-type and chimeric constructs were expressed intrain BL21 (DE3) pLys S. All the constructs showed high

otal cell extracts (Fig. 2A). Fig. 2B shows the solubiliza-ion of wild-type and chimeric proteins. Purification of theseoluble chimeric proteins resulted in yields varying from0-15 mg/litre of culture.

4788 M. Hema et al. / Vaccine 25 (2007) 4784–4794

F ced (UIP B, 3Dt pR-Ph3 presents

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The mean of the OD values of the known FMDV-NSP anti-bodies negative samples was 0.21 and the S.D. was 0.03. Thecut-off for differentiation of infected animals from vaccinatedones was fixed as 0.270.

Fig. 3. Western blot analysis of chimeric TVLPs Ph-3B1, Ph-3B2, Ph-3AB,

ig. 2. Expression analysis of chimeric constructs by SDS-PAGE. (a) Uninduh-CP (wild type), 3B1—pR-Ph-3B1, 3B2—pR-Ph-3B2, 3AB—pR-Ph-3A

otal (T) and soluble (S) fractions of pRSET-A (NC)—negative control, WT—D—pR-Ph-3D and 3ABD—pR-Ph-3ABD expressed in E. coli. Lane M re

.2. Characterization of chimeric TVLPs

.2.1. Indirect ELISAIn the I-ELISA for assessing the reactivity of the chimeric

ntigens, Ph-3B1, Ph-3AB and Ph-3ABD reacted well withnown positive samples except Type A BCS and r-3AB IMS.h-3B2 did not react with any of the positive sera. Ph-3Deacted only with Type C BCS and r-3AB IMS. Ph-CP did nothow any reaction with the tested samples. All the tested anti-ens did not show any reaction with known negative samples.he reactivity of chimeric antigens with BCS were consistentith Ph-3AB and Ph-3ABD. This indicated that the epitopesere displayed on the capsid and were functional.

.2.2. Electroblot immunoassayPh-3B1, Ph-3B2, Ph-3AB, Ph-ABD and r3AB showed

eaction with the recombinant 3AB rabbit antiserum whileh-CP and Ph-3D did not react. This indicates the fusion ofMDV-NSP epitopes in the PhMV-CP (Fig. 3).

.2.3. Electron microscopyElectron microscopy study revealed the presence of spher-

cal particles of diameter in the range of 26–28 (±2.0) nmndicating that the chimeric proteins were indeed capablef self assembly into TVLPs (Fig. 4, panels B to F). Thesehimeric TVLPs had diameters identical with that of wild-ype PhMV TVLP (Fig. 4, panel A).

.3. Development of Indirect ELISA for DIVA

Results of checker board titration of the antigen and theerum indicated that Ph-3AB antigen at concentrations of

Pbrwm

) and induced (IN) fractions of pRSET-A (NC)—negative control, WT—pR-—pR-Ph-3D and 3ABD—pR-Ph-3ABD expressed in E. coli. (b) Induced-CP (wild type), 3B1—pR-Ph-3B1, 3B2—pR-Ph-3B2, 3AB—pR-Ph-3AB,the standard molecular weight markers (MBI fermentas).

2.5 to 31.25 ng of the antigen and serum at a dilution of 1:50as found to be optimum for the use in the test (Fig. 5; data onh-3ABD not shown). The pig samples also showed similaresults. Based on these initial titrations 50 ng of antigen and:50 serum dilution was used in all further ELISAs.

.4. Establishing cut-offs for negative samples

h-3D, Ph-3ABD and Ph-CP. r3Ab used as positive control. Gel was electro-lotted and then probed with rabbit 3AB polyclonal antiserum (1:2000)aised against r3AB and HRP labeled anti-rabbit goat antiserum (1:1000)as used as secondary antibody. Lane M represents the pre-stained proteinolecular weight marker (NEB).

M. Hema et al. / Vaccine 25 (2007) 4784–4794 4789

Ph-CP

3c

a

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Fig. 4. Electron micrographs of wild-type and chimeric TVLPs. (A)

.5. Kinetics of antibody response to Ph-3AB antigen inattle, buffaloes, sheep and goats

Positive reactions could be detected in all infected animalss early as 10 days post infection in all the species (Fig. 6a–d).

.6. Testing of other experimental FMD infected and

accinated samples

The OD values of the negative samples (Pirbright samples,= 100; in-house samples, n = 90) were between 0.05 and

trpd

Fig. 5. Standardization of concentration of chimeri

; (B) Ph-3B1; (C) Ph-3B2; (D) Ph-3AB; (E) Ph-3D; (F) Ph-3ABD.

.25. The positive samples showed OD values above 0.50.hirty six out of 40 positive samples obtained from Pirbright

urned positive, while all the 100 known negative samplesid not show reactivity with Ph-3AB (Table 3). The assaylearly indicated that I-ELISA was highly specific based onhe negative and positive samples.

Seventy four and 67 samples out of 77 in-house posi-

ive samples showed reactivity with Ph-3AB and Ph-3ABDespectively. Ph-3D antigen could detect only 52 out of 77ositive samples. All 90 in-house negative samples wereeclared negative by all the three antigens. The results of

c Ph-3AB antigen using control cattle sera.

4790 M. Hema et al. / Vaccine 25

Table 3Comparison of different chimeric TVLPs using known positive and negativesera

Sera of known status Ceditest® Ph-3AB Ph-3ABD* Ph-3D*

Sera from carriersdeclared as positive

117 110 67 52

Sera from carriersdeclared as negative

0 7 10 25

Sera from non-carriersdeclared as negative

190 190 90 90

Sera from non-carriers 0 0 0 0

IEsw

ia

3

3

PPsn

3

Fs

declared as positive

* Only in-house sera tested.

-ELISA with Ph-3AB were comparable with Ceditest. I-LISA results using the other two antigens and Ceditesthowed minor variation. Between the three antigens Ph-3ABas more sensitive and hence it was used for further screen-

C3C

ig. 6. FMDV-NSP antibody kinetics with Ph-3AB antigen. (a) O1 Manisa challeheep. (d) O1 Manisa challenged goats.

(2007) 4784–4794

ng and comparison with Ceditest while the use of the otherntigens was discontinued.

.7. Testing of field samples using Ph-3AB

.7.1. Cattle samplesOut of 89 samples tested 23 were declared positive by

h-3AB, while 25 were declared positive by Ceditest. Bothh-3AB and Ceditest declared 64 samples as negative. Twoamples that were declared positive by Ceditest were declaredegative in Ph-3AB (Table 4).

.7.2. Buffalo samples

Thirty five out of 65 buffaloe samples were positive by

editest, while 37 out of 65 samples were positive when Ph-AB antigen was used. Eleven samples declared positive byeditest were declared negative by Ph-3AB ELISA while 5

nged cattle. (b) O1 Manisa challenged buffalos. (c) O1 Manisa challenged

M. Hema et al. / Vaccine 25 (2007) 4784–4794 4791

(Contin

sb

3

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Ep

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S

C

B

S

S

Fig. 6.

amples that were negative by Ceditest were turned positivey Ph-3AB ELISA (Table 4).

.7.3. Sheep samplesThirty were declared positive by Ceditest, while 33 sam-

les were declared positive by Ph-3AB antigen. Six samplesositive by Ceditest were declared negative by Ph-3AB

3

Pa

able 4omparison of performance of Ph-3AB ELISA with Ceditest® on field sera

pecies Ph-3AB ELISA Ceditest® Ceditest

(+) ve (−) ve Specific

attle (n = 89) (+) ve 23 0 92.00(−) ve 2 64

uffalo (n = 65) (+) ve 31 6 88.57(−) ve 4 24

heep (n = 61) (+) ve 27 6 87.10(−) ve 4 24

wine (=39) (+) ve 13 1 92.86(−) ve 1 24

ued ).

LISA, while 4 samples negative by Ceditest were declaredositive by Ph-3AB ELISA (Table 4).

.7.4. Pig samplesThirty nine pig samples were subjected to Ceditest and

h-3AB ELISA wherein 13 were positive while 24 were neg-tive. However, one sample declared positive by Ceditest was

® Vs Ph3AB ELISA Ph-3AB ELISA Vs Ceditest®

ity (%) Sensitivity (%) Specificity (%) Sensitivity (%)

100 100.00 96.97

80.00 83.78 80.00

80.00 87.10 80.00

96.00 92.86 96.00

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792 M. Hema et al. / Vac

eclared negative by Ph-3AB while another sample declaredegative by Ceditest was declared positive by Ph-3AB ELISATable 4).

The results indicated that Ph-3AB based ELISA is verypecific (100%) based on the negative samples tested and wass sensitive as Ceditest. When compared with the Ceditest theensitivity of Ph-3AB ELISA was 95.80%, while the sensi-ivity of the Ceditest when compared with Ph-3AB ELISAas 98.21. However, it is pertinent to mention that few buf-

alo, sheep and pig samples which were declared negative byeditest were positive in Ph-3AB ELISA (Table 4).

. Discussion

Antibodies to FMDV-NSPs are considered to be reliablendicators of FMD infection and are useful for DIVA [5,8].urrently, many diagnostic techniques based on detectionf antibodies to the NSPs have been developed for DIVA4,5,8,10,32]. Non-endemic countries are seriously debatinghe advantage of vaccinating animals so as to reduce the needo stamp out susceptible in contact as well as infected animalsnder the vaccinate-to-live policy [33]. Though four com-ercial test kits and a few in-house NSP tests are available

t the moment, their sensitivity and specificity are insuffi-ient to demonstrate absence of infection with a high levelf confidence in small herds [34]. The recent internationalSP test validation meeting at Brescia has suggested valida-

ion of more than one NSP test kit to increase the efficiency ofIVA [9,10]. Keeping these recommendations in mind, otherMDV-NSPs 2B, 2C or assays using infection-specific epi-

opes of NSPs to remove cross reactivity are being explored2,4,11,32,35].

The NSPs expressed in E. coli and baculovirus expressionystems [5,36,37] may sometimes create problems in thenterpretation of the results on account of non-specific reac-ions. This may be due to the presence of antibodies againsthese expression vector antigens that are likely to get co-urified with the recombinant products. Also the number ofpitopes found on such a long recombinant protein may inter-ere with antibodies to other Picornaviruses [4,11,38,39].hemically synthesized synthetic peptides of NSPs weresed for DIVA [4,11,32,35,39]. Difficulty in synthesizingong peptides and poor antigenicity of short peptides affectedhe specificity and sensitivity of solid-phase immunoassays4]. Many commercial kits were developed using suchecombinant proteins or synthetic peptides. However, thepecificity and sensitivity of these kits vary greatly [34,40].oreover, such kits are expensive and need to be imported

y the developing countries for effective use in DIVA.ence, there is a need to develop low cost diagnostic kits.In the present study, we explored one such option of

xpressing the FMDV-NSP infection-related linear B-cellpitopes [4,11] on plant virus-like particles (VLPs) that areeterologous to the mammalian system thereby reduce theross contamination. Epitope presentation was successfully

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mployed with viruses whose structures were well defineduch as Tobacco mosaic virus (TMV), Cowpea mosaic virusCPMV) and Tomato bushy stunt virus (TBSV). The manip-lations were done at genome level to produce recombinantiruses in plants [14,15,17]. Recombinant PhMV CP isnown to self-assemble into empty capsids in E. coli. Byhe extensive mutational analysis of the recombinant PhMVP, the structural dynamism of the N-terminus of the coatrotein was demonstrated [24,25]. The crystal structures ofative and recombinant PhMV capsids were reported [41].he flexibility of N-terminus, which was mobile and canssume different conformations, suggested the use of PhMVP as carrier molecule for the epitope presentation.

In this report, we attempted to fuse the FMDV-NSPnfection-related B-cell epitopes [4,11] to the PhMV CP car-ier molecule and display them on the surface of the capsids.he results clearly demonstrated that the cloning of syntheticild-type PhMV CP gene into pRSET-A vector resulted inrotein with an additional 39 amino acids at the N-terminusf the CP which self-assembled into tymovirus-like particlesTVLPs). Further, the tandem repeats of FMDV 3B, 3AB,ABD and 3D epitopes were swapped at the N-terminus ofhe wild-type PhMV CP. These substitutions did not hinderhe expression and assembly of chimeric CP subunits, but ledo the formation of chimeric TVLPs. Earlier research [24,25]ad shown deletion of 30 amino acids and addition of 41mino acids in two different constructs. In our study, we wereble to delete and add upto 66 amino acids of the heterolo-ous protein at the N-terminus in the independent constructsithout affecting the assembly process. Further, the presen-

ation of NSP epitopes in tandem repeats with short linkersGGS) gave scope to display one or more epitopes on theurface of the TVLPs.

The fusion and display of epitopes on the PhMV TVLPsere confirmed by electroblot immunoassay and I-ELISAmong the several chimeric TVLPs generated, Ph-3B1,h-3AB and Ph-3ABD reacted well with BCS and IRSaised against r3AB. The I-ELISA results using chimericVLPs with the tested experimental samples encouraged us

o develop a simple, easy to use and cost effective ELISAor DIVA. The reactivity of chimeric TVLPs with the posi-ive samples and negative samples showed that the chimericVLPs were able to detect FMDV-NSP antibodies in I-LISA. The specificity was indicated by the non- reactivitygainst the known negative samples. Among the current NSPests available commercially, Ceditest had the highest speci-city and sensitivity [34]. The specificity and sensitivity ofSP antibody assay using Ph-3AB was determined by com-aring the reactivity of negative and positive samples witheditest kit as standard. The sensitivity of Ph-3AB ELISAas around 96% while the specificity was 100%. Based onLISA results with the different control samples, Ph-3AB

as selected as a candidate antigen for use in I-ELISA forIVA of different species from the field. When comparedith Ceditest, the specificity of I-ELISA with Ph-3AB was00% with FMDV negative sheep and goat samples.

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The proportion of sero-positive cattle detected were 100%nd 94% in Ceditest and Ph-3AB ELISA, respectively andhere was no significant difference between the two testsP > 0.05). Of the total 552 field samples tested using the I-LISA with Ph-3AB and Ceditest 268 and 278 samples wereeclared positive, respectively and 278 and 274 samples wereeclared negative, respectively. The sensitivity and specificityf the two tests varied when random samples collected fromhe field and the abattoir were tested. Two way comparisonf both the tests showed that for cattle samples the sensitivityas 92% and 100% and specificity was 100% and 96.97%

or Ceditest and I-ELISA with Ph-3AB antigen, respectively,hile it was varied for other species of animals. The study onig ELISA reveals the need to test more number of pig sam-les. Also the kinetics of the FMDV-NSP antibodies of pigso the Ph-3AB antigen needs to be studied so that the ELISAan be extended to the pig samples as well. However, the pre-iminary results have been encouraging. Hence it is proposedhat the PhMV 3AB antigen can be used for detection ofntibodies to NSP antigen and employed for DIVA. It can bemployed for DIVA of various domestic susceptible animals.

In conclusion, we were successful in displaying morepecific infection-related epitopes on the surface of PhMVVLPs and there by reducing the problems of cross-reactivitynd non-specific reactions. Advantages of PhMV TVLPased epitope presentation system are high yields of thehimeric TVLPs, minimal batch to batch variation, simplend economical downstream processing. This study givescope to apply this technology to present the other eco-omically important animal/human pathogen epitopes foriagnostic/vaccine purpose. Further, Ph-3AB ELISA has theotential to identify FMDV infection in susceptible herds.he sensitivity is comparable to the Ceditest.

Further work is underway to use the Ph-3AB I-ELISA tocreen more number of samples from the field to have theequired levels of confidence.

cknowledgements

We thank Prof. H.S. Savithri (Indian Institute of Science,angalore, India) for her advice, Dr. David Paton, and Dr.atya Parida (Institute for Animal Health, Pirbright, UK) foriving the positive and negative samples samples of cattle. Wehank our colleagues Dr. Rajan Sriraman, Dr.S.Rajalakshmind Dr. V. Maroudam for providing r-3AB antigen and r-AB antiserum. We also thank Dr. Y. Naga Malleswari (Srienkateswara Veterinary University, Hyderabad, India) forer help in electron microscopy work.

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