Monoclonal Antibodies against FMDV type Asia 1: preliminary characterization and potential use in diagnostic assays

Transcription

1 Appendix 24 Monoclonal Antibodies against FMDV type Asia 1: preliminary characterization and potential use in diagnostic assays Grazioli S., Fallacara F. and Brocchi E. Istituto Zooprofilattico Sperimentale della Lombardia e dell Emilia Romagna, Brescia, Italy Introduction Foot-and-Mouth Disease (FMD) is economically the most important disease of domestic livestock. Outbreaks with devastating economic consequences still occur; the 21 FMD crisis in the UK was a traumatic experience. The most serious source of the risk of entry of FMD is illegal import of animals or animal products from countries where FMD is endemic. This aspect, associated with increasing immigration from third countries, induces the need that European countries are prepared to recognise and control any of the FMD viruses currently circulating in the world. FMD monitoring systems should ensure that outbreaks are detected at an early stage and that subclinical persistence of infection is promptly recognized. To satisfy these requirements there is the need for updated and new diagnostic tools, which cover the complexity of the FMD diagnostic task. The most significant innovations of the last decade in FMD diagnosis have been the introduction of RT-PCR for sensitive detection of FMDV presence in biological samples and the serological distinction between vaccinated and infected animals, based on the detection of antibodies to viral non structural proteins. Minor advances have been reported concerning reagents and methods for the detection and characterization of FMDV antigens and antibodies. These assays are traditionally performed using polyclonal immune sera, although it has been proven that immunoassays for many infectious diseases, including FMD, can greatly benefit from the use of monoclonal antibodies (MAbs). Furthermore, MAbs represent valuable tools for studying the antigenicity of viruses; this application is particularly important for FMD viruses, which show frequent variability and may thus evade immunity provided by vaccines. Selection of appropriate vaccine strains, that considering their antigenic profile are expected to induce protection against circulating field viruses, is crucial in FMD control based on vaccination strategy. Several reports described the antigenic structure of FMDV and compared the antigenic profile of FMDV isolates using MAbs. Also in diagnostic field, MAbs-based ELISAs for both antigen and antibody detection are being used in our laboratory for almost 2 years on a routine basis (Brocchi et al., 1986, Brocchi et al., 199). However, most MAbs to FMDV used to these purposes were raised against old reference strains of type O, A, C, which are no more representative of viruses currently circulating. Then, one main objective of our laboratory is updating the MAbs collection, taking into consideration either other FMDV types or isolates showing significant antigenic variation. Within this programme, we have produced a new panel of MAbs, raised towards FMDV type Asia 1. Here we describe their partial characterization and a preliminary evaluation of their potential as diagnostic reagents in ELISA assays for both antigen and antibodies detection. 194

2 Materials and Methods Viruses FMDV isolates used were received from the World Reference Laboratory, Pirbright, UK; they are listed in table 1. Viruses were propagated in IBRS-2 cells monolayers and harvested when cytopathic effect was maximum. The strain used for mice immunisation was inactivated with binary ethylenimine and purified by ultracentrifugation through a 25% (w/w) sucrose cushion. Monoclonal antibodies (MAbs) Two Balb/C mice were immunised with 2- doses respectively of 2 µg each of purified FMDV Asia 1, strain Nepal 29/97, at intervals of one month. Cells fusion and cloning of positive hybridomas were performed following procedures standardised in our laboratory (Brocchi et al. 199). Selection was based on results of a trapping ELISA against the homologous virus strain. Ascitic fluids of selected hybridomas were induced in mice. MAbs were purified from ascites by ammonium sulphate precipitation and conjugated with peroxidase using a modification of the method described by Tjissen (1985). Isotype of the MAbs was determined using a commercial ELISA (Boehringer Mannheim isotyping kit). Trapping ELISA The assay used for both screening hybridomas and antigen characterisation was a trapping ELISA (Samuel et al., 1991). Essentially, each MAb was reacted with pre-titrated concentrations of viruses (supernatant of infected cells) which had been trapped using a polyclonal rabbit Asia 1 antiserum. End-point titres of MAbs were determined as the reciprocal dilution that gave optical density readings higher than.2, while the reactivity of mutants and field isolates with each MAb was expressed as a percentage of the corresponding reaction with the parental strain, assumed to be 1%. Sandwich ELISA MAbs-based sandwich ELISA for antigen detection has been carried out as described previously (Brocchi et al., 199). Solid phase competitive ELISA The principle and the procedure of the MAbs-based competitive ELISA, firstly described for the quantification of antibodies specific to the European subtypes O1, A5 and C1 of FMDV (Brocchi et al., 199), and later applied to other infectious diseases (Brocchi et al., 199, Brocchi et al., 1995) was adapted for the detection of FMDV type Asia 1 specific antibodies. The MAb selected for this application was 5E1, used either as antigen catching or as competing, peroxidase-conjugated antibody. Virus Neutralization (VN) test VN test was carried out in microplates against 1 TCID 5 of the homologous FMDV Asia 1 and IBRS-2 as substrate. The final dilution required to neutralise 5% of the inoculated cultures was calculated. MAb neutralization-resistant (MAR) mutants The selection of mutants resistant to neutralization by MAbs was carried out as described previously (Borrego et al. 22). 195

3 Western Blot analysis Hybridoma supernatants were assayed against the purified homologous FMDV, resolved by SDSpolyacrilamide gel electrophoresis 12% and transferred to nitrocellulose filters, following standard procedures (Harlow & Lane, 1988, Towbin et al., 1979). Results and Discussion Characterization of monoclonal antibodies Twenty-four MAbs were raised against FMDV type Asia 1, strain Nepal 29/97, in two fusion experiments. Table 1 outlines results of a preliminary characterisation, including the following details: immunoglobulin class, ability to neutralize viral infectivity, identification of linear epitopes in viral proteins, specificity with respect to the homologous type Asia 1 and the heterologous types O, A and C, profile of reactivity with field isolates; analysis of these results lead to the classification of the MAbs in 6 separate groups. Concerning immunoglobulin class, the MAbs panel include 21 IgG1, 1 IgG2a and 2 IgM. Results of VN test revealed that 1 MAbs neutralized viral infectivity, showing variable neutralising titres, whilst 24 MAbs were not neutralising. According the different pattern of reactivity shown, the 14 non-neutralizing MAbs could be clustered in three separate subgroups: group 4, including two type-specific MAbs, group 5, including 11 MAbs with different degree of cross-reactivity with two or even three heterologous FMDV types examined, and group 6 including one single MAb, directed against a linear epitope in VP2 (as demonstrated by reactivity in Western Blot), trypsin-sensitive (not shown) and shared by the three heterologous FMDV types examined. In contrast, all the neutralizing MAbs were type-specific; end-point titres of hybridoma supernatants in trapping ELISA did not necessarily correlate with VN titres, since several factors may influence this aspect, like antibody concentration, antibody affinity or mechanism of neutralisation. Results of Western blot test indicated that three of the neutralising MAbs react with VP1; as virus trypsin treatment prevented this reactivity, the target site is likely the linear trypsinsensitive sequence corresponding to the G-H loop (site A) in VP1. A further and accurate way to distinguish neutralizing MAbs and to map the relevant sites is the selection and the characterisation of MAb neutralization resistant mutants. So far, of the neutralizing MAbs were used to select escape mutants. The pattern of reactivity of each mutant with the 1 neutralizing MAbs in a trapping ELISA proved evidence that these MAbs identify at least different antigenic sites (figure 1 and table 1). One site, already distinguishable on the basis of its location in a linear sequence of VP1, was defined by the MAbs 5C12, 4E1, 4F1: mutations induced in any of the relevant escape mutants annulled the reaction of the three MAbs, but did not alter the reactivity of the other seven MAbs. Another independent site includes three additional MAbs: in fact, the mutant resistant to MAb 5E1 lost reactivity not only with 5E1 itself, but also with two further MAbs, C6 and 1F1, while maintaining full reactivity with other MAbs. Finally, the remaining four MAbs were not affected by mutations in the two previous sites: this reactivity indicates that they correspond to a distinct site. Figure 1. ELISA reactivity of FMDV Asia 1 MAR mutants with the panel of neutralizing MAbs 196

4 Antigenic sites defined by neutralising MAbs Site 1 Site 2 Site 5C12 4E1 4F1 5E1 C6 MAR mutants 1F1 2C 2G1 5G4 4D8 5C12-a 5C12-b 5C12-c 5C12-d 4F1-a 4F1-b 5E1-a parental MAR mutants are named according to the MAb used to select them. Reactivity is expressed as percentage of binding with respect to the parental strain, FMDV Asia Nepal 29/97, assumed to be 1%: white cells, no reactivity; light grey, 25% reactivity; black, 1% reactivity. The level of conservation of the sites identified by the MAbs was analysed by testing their reactivity in a trapping ELISA with 11 isolates representative of a thirty-year period, being the oldest virus of this panel originated in 197 (Asia 1 Turkey 15/7) and the most recent one in the year 2 (Asia 1 Greece 1/2). The antigenic profiles (table 1) show that non-neutralizing MAbs displayed a broad reactivity with the isolates; the antigenic stability of the relevant sites was expected, mainly concerning sites 5 and 6, which were common even to heterologous FMDV types. Also sites 1 and 2 involved in neutralisation were proven to be conserved in the majority of isolates, whilst the neutralising site was shown to be almost exclusive for the homologous strain, being mutated in all other isolates. With respect to this particular panel of MAbs, FMDV Asia 1 isolates showed limited antigenic variation, with the exception of one isolate, Cambodia /9, which was not recognised by any of the MAbs specific for the Asia type. Evaluation of MAbs performances in diagnostic assays Preliminary studies to investigate the potential of the characterised MAbs as diagnostic reagents were undertaken. Figure 2. Sandwich ELISA for antigen detection: results obtained with representative pairs of catching and conjugated MAbs 197

5 Optical Density 2,5 2 1,5 1,5 catch. Mab - 4F1 type specific conj. Mab - 5F1 cross reactive a) Asia 1 Asia 1 12S O A C Optical Density 2,5 2 1,5 1,5 catch. Mab - 2A4 cross reactive conj. Mab - 5F1 cross reactive Asia 1 Asia 1 12S O A C b) 1/5 1/15 1/45 1/15 1/45 1/1215 1/5 1/15 1/45 1/15 1/45 1/1215 Antigen dilution Antigen dilution Optical Density 2,5 2 1,5 1,5 catch. Mab - 4F1 type specific conj. Mab - 5F1 cross reactive c) Asia 1 Asia 1 12S O A C 1/5 1/15 1/45 1/15 1/45 1/1215 Optical Density 2,5 2 1,5 1,5 catch. Mab - 2A4 cross reactive conj. Mab - 5E1 type specific d) Asia 1 Asia 1 12S O A C 1/5 1/15 1/45 1/15 1/45 1/1215 Antigen dilution Antigen dilution Titration curves of supernatants of FMDV infected cultures Asia 1 : strain Nepal 29/97 homologous to MAbs, infectious titre TCID/ml Asia 1 12S : same as before, treated at 56 C for min. O : O1 Switzerland 1965; A : A5 Italy 1962; C : C1 Noville For antigen detection, performances of a sandwich ELISA carried out with several combinations of different MAbs, used as catching and conjugated antibody, were evaluated using supernatants of infected cells as source of antigen: titration curves for either the homologous virus type, Asia1, and the heterologous types O, A and C, were performed; the reactivity with 12 S viral particles, obtained by heat treatment (56 C, min.) was analysed also. A strong signal was observed with most MAbs, with a detectability limit of 1 5 TCID 5 /ml, expected from immunoassays which do not benefit from a preliminary amplification step (figure 2). In particular, when combinations of two type-specific MAbs were used as catching and conjugated antibody, only the homologous type was detected (figure 2a), whilst combinations of two crossreactive MAbs did recognise the homologous as well as the heterologous types with similar profile and sensitivity (figure 2b). This result offers the possibility of choosing a universal assay for FMDV detection or a type-specific one, depending on requirements. Furthermore, figures 2c and 2d report antigen titration curves obtained by combinations of one type-specific with one cross-reactive MAb: in both cases the reactivity was specific for the homologous type only: then, virus typing can be achieved by combining a universal catcher with a type-specific conjugated MAb or vice versa. This result further extends the possibility to modulate and adapt antigen detection assays to different needs. All MAbs pairs reacted with 12S particles, showing a slightly lower signal in the case of neutralizing MAbs and slightly higher reactivity when non-neutralizing MAbs were used. 198

6 percentage inhibition Neg Cut off Weak Strong 1/1 1/ 1/9 1/27 serum dilution Figure. Evaluation of a monoclonal antibody-based competitive ELISA (SPCE) for the assessment of antibody to FMDV type Asia 1 (catching and conjugated MAb 5E1) A) Results obtained with PHASE XVII sera (candidate reference sera) 4 threshold? n negative sera 2 1 neg. cattle sera neg. pig sera B) Frequency distribution of percentage inhibition observed with negative sera (n 17 cattle, n 74 pigs) percentage inhibition For antibody assessment, a solid phase competitive ELISA was designed, determining the capability of sera to block the binding of a specific MAb to the virus. The virus was preliminarily captured onto microplates wells by the catching MAb 5E1, that allowed the use of a non-purified preparation as source of virus. Competition between test sera and the peroxidase-conjugated MAb 5E1 was then evaluated. Epitopes target of neutralizing, type specific MAbs are expected to be more immunogenic, then the selection of 5E1, satisfying these requirements, as competing antibody should direct the detection of specific sets of serum antibodies. Sensitivity and specificity of this experimental assay were preliminary evaluated by testing a panel of field negative sera from cattle and pigs and the Asia 1 specific sera of phase XVII exercise, aimed at selecting candidate reference sera, and including a cut-off, a weak positive and a strong positive serum, together with a negative one. Results, obtained using pre-determined optimal concentration of antigen and competing MAb, are shown in figure, expressed as percentage inhibition produced by tested sera. In particular, figure A shows titration curves of the 4 candidate reference sera: the 4 sera were distinguishable each other and gave, at the screening dilution 1/1, percentage inhibition coherently 199

7 distributed along the whole scale: the negative serum presented inhibition close to %, the cut-off, weak and strong positive sera scored inhibition of 5%, 65% and 92 % respectively. Figure B shows frequency distribution of percentage inhibition produced by negative sera at the screening dilution 1/1: pig sera displayed a normal distribution, characterised by a restricted percentage range (-4%), with mode in the class 11-2% and none out of 74 samples examined over 4%; differently, cattle sera showed a wider distribution, extended up to 8% inhibition in exceptional cases, although maintaining the mode in the class 11-2%. Selecting 6% inhibition as threshold, the test was able to detect weak and strong positive reference sera, but missed the cut-off serum; with regard to specificity, the assay presented approximately % of false-positive results ( out of 17) in the cattle population but none in the pig species. Although assay standardisation is still in course, the SPCE for the detection of Asia 1 specific antibodies proved to work in principle. Several assay parameters will be evaluated to improve specificity and sensitivity, in particular: modulation of antigen/antibody concentration, more stringent buffer conditions and the assessment of other MAbs and competitive designs, including washing steps and changing from a solid phase to a liquid phase competitive ELISA. Conclusion Within the programme of developing/updating MAbs suited for the study and the diagnosis of recent FMDV isolates, we have produced a panel of 24 MAbs against FMDV type Asia 1. Their preliminary characterisation demonstrated that 1 type specific MAbs identify at least different antigenic sites involved in neutralisation, two of them conserved in different isolates and the remaining restricted to the homologous strain; 14 non-neutralizing MAbs showed high antigenic stability among Asia 1 isolates and some of them presented different degrees of heterotypic reactivity also. Studies to investigate the potential of these MAbs as diagnostic tools lead to the development of prototype assays for both antigen and antibody detection. In particular, sandwich ELISAs for either the specific detection of Asia 1 FMD virus or a universal detection of FMD viruses have been developed, showing a detection limit of 1 5 TCID 5 /ml. A SPCE for the specific detection of Asia 1 antibody is under evaluation: the assessment of few positive and negative sera provided promising results, although assay standardization is not yet finalized and performances may be improved. Acknowledgments We wish to thank Dr D. Gamba for the work in MAbs production, Dr M. Bugnetti for growing viruses, Dr. B. Haas for providing rabbit sera used in capture ELISAs and Dr. F. De Simone for his valuable advice. Work was supported by national grant IZSLER 7/98 from National Ministry of Health and by Concerted Action grant FAIR CT98 42 from the European Union. References Borrego B, Carra E, Garcia-Ranea JA and Brocchi E. (22). Characterization of neutralization sites on the circulating variant of Swine Vesicular Disease virus: a new site is shared by swine vesicular disease and the related Coxsackie B5 virus. J Gen Virology. 8: 5-44 Brocchi E, Capucci L, De Simone F, Panina GF (1986). Potential of monoclonal antibodies (MAbs) for FMD diagnosis and characterization of the isolates. Report of Sess. Res. Gr. St. Tech. Committee of the Europ. Comm. Control FMD, Italy, Rome, 1986, p -1. 2

8 Brocchi E., De Simone F., Bugnetti M., Gamba D., Capucci L. (199) Application of a monoclonal antibodybased competition ELISA to the measurement of anti-fmdv antibodies in animal sera. Report of Sess. Res. Gr. St. Tech. Committee of the Europ. Comm. Control FMD, Lindholm, Denmark, June , Appendix 14. Brocchi E, Gamba D, Poumarat F, Martel JL, De Simone F. (199). Improvements in the diagnosis of contagious bovine pleuropneumonia through the use of monoclonal antibodies. Rev Sci Tech Off Int Epizoot. 12: Brocchi E, Berlinzani A, Gamba D, De Simone F. (1995). Development of two novel monoclonal antibodybased ELISAs for the detection of antibodies and the identification of swine isotypes against swine vesicular disease virus. J Virol Methods. 52: Brocchi E, Gamba D, Bugnetti M, De Simone F (1998). Monoclonal antibodies profiling of FMD viruses type A currently used for FMD control. Report of the Sess. Res. Gr. St. Tech. Committee of the Europ. Comm. Control FMD, UK, Sept. 1998, p Harlow E. & Lane D. (1988). Immunoblotting protocols. In Antibodies: a Laboratory manual, p Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Samuel, A.R., Knowles, N.J., Samuel, G.D. and Crowther, J.R. (1991) "Evaluation of a trapping ELISA for the differentiation of FMDV using MAbs." Biologicals 19: Tjissen P. (1985). Preparation of enzyme-antibody or other enzyme-macromolecule conjugates. In Laboratory techniques in biochemistry and molecular biology. Practice and therapy of enzyme immunoassays (R.H. Burdon & P.H. van Knippenberg, eds). Elsevier, Amsterdam,

9 Table 1 Pattern of reactivity of 24 monoclonal antibodies raised against FMDV type Asia 1, strain Nepal 29/97 Ig class MAb VNT titre culture supernatant asc.fluid Western Blot Asia (titre) homologous ELISA Trapping O1 Swtz.1965 A5 Italy 1962 C1 Noville Ag site Nepal 29/97 (homologous) percentage reactivity (ELISA) with 11 FMDV isolates - type Asia 1 Cam 9/8 India 1/82 Pak 2/98 Pak /98 Iran 58/99 SAU 9/94 Gre 1/2 Kuwait 2/81 Tur 15/7 Nepal 58/88 Cam /9 NEUTRALISING MAbs NON NEUTRALISING MAbs IgM 4E VP IgM 4F1 > 2 >248 VP IgG1 5C12 > 2 >248 VP IgG1 5E1 24 nd IgG1 C IgG1 *2 1F1 > 2 nd nd not done not done IgG1 2C 12 nd IgG1 2G1 24 nd IgM 5G4 6 nd IgG2a 4D8 1 nd IgG1 4G IgG1 *2 D8 - nd nd not done not done IgG1 4B1 - nd /- - +/ a IgG1 4B2 - nd /- - +/ IgG1 2F7 - nd - 2 (+) (+) IgG1 2G (+) (+) - 5b IgG1 *2 2B11 - nd nd 625 (+) (+) - 1 not done not done IgG1 G - nd IgG1 2A IgG1 B6 - nd c IgG1 5H5 - nd IgG1 4G2 - nd IgG1 *2 H12 - nd nd not done not done IgG1 5F1 - - VP *2 : MAb originated from the second fusion; (+) : reduced signal with respect to the homologous type