Detection of antibodies against a conserved capsid epitope as the basis of a novel universal serological test for foot-and-mouth disease

Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral non-structural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus serotype and their sensitivity performances may be affected by antigenic variability within each serotype and mismatching between tests reagents. As a consequence, FMD Reference Laboratories need to maintain contingency to employ multiple type-specific assays for large-scale serological surveillance and post-vaccination monitoring in the event of FMD outbreaks. In this study, a highly conserved region in the N terminus of FMDV capsid protein VP2 (VP2N) was characterised using a panel of intertypic-reactive monoclonal antibodies. This revealed a universal epitope in VP2N which could be used as a peptide antigen to detect FMDV-specific antibodies against all types of the virus. A VP2-peptide ELISA (VP2-ELISA) was optimised using experimental and reference antisera from immunized, convalescent and negative animals (n=172). The VP2-ELISA is universal, simple and provided sensitive (98.6 %) and specific (93%) detection of antibodies to all FMDV strains used in this study. We anticipate that this SP test could have utility for sero-surveillance during virus incursions in FMD-free countries and as an additional screening tool to assess FMD virus circulation in endemic countries.


Introduction
that are known to be infected or vaccinated with FMDV. Selection of the positive samples 106 was based up on more than 7days post vaccination or infection to ensure a positive response. 107 See supplementary table (1) for more details.

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Production of mAbs 109 The following FMD viruses were used as immunogens to produce mAbs in mice and for the Fisher Scientific, UK) in blocking buffer for 45 min at RT. After washing, the cells were 139 mounted using Vectashield mounting medium with DAPI (4,6-diamidino-2-phenylindole) 140 (Vector Labs) and the coverslips sealed with nail varnish. All data were collected sequentially 141 using a Leica SP8 confocal laser scanning microscope.

SDS-PAGE and western blot
144 Initial tests to verify the reactivity in western blot of each mAb with the homologous partially 145 purified strain were performed as previously described [21]. Later on, the cross-reactivity of 146 one representative mAb (4A3) with all FMDV serotypes was confirmed as follows.

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The frequency distribution of values generated by various serological assays for the negative

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Characterisation of an FMDV-VP2 conserved epitope by cross reactive mAbs 189 Among the multiplicity of mAbs generated from mice independently immunized with four  (Table 1).

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Previous studies have identified the conserved N-terminus of VP2 as a site for recognition by 196 cross-reactive mAbs [15,16,17]. We therefore tested the reactivity of the seven mAbs 197 against peptides equivalent to the first 15 (VP2N15), 30 (VP2N30) or 45 (VP2N45) amino 198 acids of the N-terminus of VP2 from FMDV O1K (Fig.1a). The N-terminus of VP2 is known 199 to be most highly conserved within the first 15 amino acids. The five mAbs (4D1, 1D6, 4A3, 200 5B2 and 5F10) identified as VP2-specific by Western blots also reacted strongly with the 201 VP2 peptides in ELISA (Fig.1b). Among them, two mAbs (4A3 and 5B2) showed an 202 equivalent reactivity with the three peptides, while the three remaining mAbs recognized the 203 VP2N15 peptide with lower intensity (Fig.1b). The mAb 4A3 was taken forward for further 204 characterisation. In particular, fine mapping using 15mer peptides with 10 amino acids 205 overlaps ( Fig.1a) showed that mAb 4A3 reacted with the 15mer peptide that corresponded to 206 the N-terminus of VP2 and not with a 15mer starting at amino acid 6, confirming the 207 presence of an epitope at the N-terminus of VP2 (Fig.1c). The mAb 4A3 specifically detected An indirect ELISA using peptides VP2N15, VP2N30 or VP2N45 was used to assess the 216 presence of antibodies against the N-terminus of VP2 in a representative serum from an animal 217 infected with type O FMDV. All three peptides captured antibodies, with the longer peptides 218 producing a slightly higher signal (Fig.2a). A control peptide equivalent to a capsid sequence 219 from the related picornavirus human rhinovirus gave a low signal consistent with background.

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The longer peptide VP2N45 was then used to test monovalent sera from different animals 221 vaccinated against the seven serotypes of FMDV; this showed that the same peptide was able 222 to detect antibodies against all the serotypes (Fig.2b).

Development of a VP2 ELISA for universal detection of FMDV antibodies 224
A VP2 ELISA using peptide VP2N45 was developed using reference sera. The optimal 225 concentration of peptide and dilution of sera to be used in the test was first evaluated by checkerboard titrations using bovine sera known to be negative or strongly positive or weakly 227 positive for antibody by existing tests. The best signal to noise ratio (positive: negative) was 228 obtained using a serum dilution of 1 in 100 and peptide concentration of 2μg/ml (Fig S.1 antigenic match between reagents used (virus/antigen and antibodies) and the serum sample 251 being tested. Therefore, the data from VNT and LPBE were subdivided into groups carried 252 out with homologous (same virus used to vaccinate or infect the animal) or heterologous 253 (same serotype but strain different than those used to vaccinate or infect the animal) reagents.

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The data obtained with PrioCHECK kits was only available for samples from infections with 255 serotypes O, A and Asia 1.

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Overall these results show that the VP2 ELISA detected antibody to all serotypes and the OD 269 values may provide an estimate of the level of antibodies. The sensitivity of the new test 270 resulted equivalent to or better than PrioCHECK kits and SPCE; sensitivity was significantly 271 higher than LPBE and VNT when such assays are carried out with heterologous reagents.
This study describes the development of a novel assay for the detection of antibodies against 274 the FMDV capsid that can be used to test for seroconversion in infected or vaccinated animals.

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The benefits of this assay are that FMDV-specific SP antibodies from all seven serotypes can 276 be detected without the requirement for individual specific antigen or antibody reagents that 277 are required for existing tests such as VNT, LPBE, SPCE.  Results demonstrated that the VP2 ELISA detected antibody to all serotypes with a diagnostic 295 specificity of 93% and sensitivity of 98.6%. The sensitivity of the new ELISA was equivalent to or better than existing tests, such as PrioCHECK kits and SPCE; sensitivity was significantly 297 higher than LPBE and VNT carried out with heterologous reagents.

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The VP2 ELISA is suitable for detection of antibodies against the capsid of FMDV either post 299 vaccination or post infection. The capture antigen contains a universally conserved viral 300 epitope that is expected to be present on any isolate of FMDV, this ensures that the VP2-ELISA 301 is able to detect FMDV antibodies regardless of the viral strain. In contrast to the biological 302 reagents necessary in many other ELISA, the VP2 capture antigen is a synthetic peptide, greatly 303 facilitating standardisation, continuity of supply and reproducibility. More importantly, it does 304 not require the optimisation and re-validation when serum from antigenic distant strains needs 305 to be tested.

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Serological testing is a suitable tool for FMD surveillance. Detection of NSP antibodies 307 currently offers the advantages of a DIVA and cross-serotype test. However, the VP2 ELISA 308 can be used as a complementary or confirmatory test to the NSP ELISA, which is especially 309 useful in obtaining FMDV free status after an outbreak. As for the NSP ELISA, the VP2 ELISA 310 can also be used as (1) a front-line sero-surveillance assay in areas which are normally free 311 from FMD without vaccination, (2) for areas conducting surveillance to achieve free from 312 vaccination status, and (3) at the point of import and export to confirm the freedom of animals 313 from FMDV antibodies. The test may also provide a simple approach for evaluating vaccine 314 efficacy in experimental and field trails, although additional studies would need to be carried 315 out to determine the cut-off that correlates to protection.

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In conclusion, the results suggest that the VP2 ELISA developed for the detection of antibodies 317 to FMDV has potential applications as a rapid, simple and inexpensive test in the sero-diagnosis 318 of FMDV and in sero-surveillance programmes. Further validation and standardisation will be 319 required to confirm the potential benefits of the VP2 ELISA.  326 The authors declare that there are no conflicts of interest.  Fig 1. FMDV heterotypic-reactive mAbs recognise the N terminus of VP2. (a)

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Overlapping peptides representing the VP2 N-terminal 45 amino acids. The (K)6 denotes to 334 addition of 6 lysine residues at the C-terminus of the peptide to increase peptide solubility.  Reactivity with VP2N45 peptide at 2μg/ml of different dilutions of a strong responder serum 368 sample (type C) and a weak responder serum sample (type SAT3). The asterisk denotes the 369 best conditions of peptide at 2μg/ml and sera diluted 1:100.