Appendix 23

Appendix 23

Characterisation of monoclonal antibodies against foot-and-mouth disease vaccine strain C1 Oberbayern and their reactivity with field isolates

N. Aggarwal*, S. J. Cox, R. J. Statham, P. V. Barnett

International Vaccine Bank for FMD, Institute for Animal Health, Pirbright Laboratory Pirbright, Surrey GU24 0NF, U.K.

Introduction

The 2001 epidemic of foot-and-mouth disease (FMD) in the U.K. and mainland Europe was a timely reminder of the devastating consequences of this disease. Although the epidemic was brought under control by slaughter of all infected/in-contact animals and traditional zoosanitary measures, vaccination was considered as an option, at-least in the early stages of the epidemic.

The International Vaccine Bank (IVB) for FMD at Pirbright holds quantities of seven strains of inactivated FMD virus (FMDV) antigen over liquid Nitrogen, ready for immediate formulation into vaccine if required. These will protect against the viruses of serotypes that are most likely to threaten the livestock of the UK or other IVB member countries (i.e. serotypes A, O, C and Asia 1).

The antigenicity of FMDV can change because of frequent mutations in it's genome, and thus evade the immunity provided by vaccines. It is therefore necessary to monitor the field isolates for their relatedness to vaccine strains to ensure that currently available vaccine will provide protection against any new isolate. Currently, an ELISA based on the use of polyclonal hyperimmune serum is used by the WRL for FMD for the antigenic profiling of field isolates. However, the inherent variation in the properties of polyclonal serum batches complicates the interpretation of results. An antigen profiling ELISA, based on monoclonal antibodies (MAbs) has also been described (Samuel et. al., 1991). We aim to refine this approach in order to have a more reliable ELISA that could ultimately alleviate the need for in-vivo testing of selected vaccine strains. As a first step towards this goal, we have started production and characterisation of monoclonal antibodies to cover the entire antigenic spectrum of vaccine strains held by the IVB. In this report results are presented of studies that have been done with the C1 Oberbayern vaccine strain of FMDV.

Materials and Methods

Cells and Viruses

All the strains of FMDV were procured from WRL for FMD at Pirbright and were grown either in BHK-21 or IB-RS2 cells.

Monoclonal antibody production

Six-week old BALB/c mice were immunised by the sub-cutaneous route with 10 µg of C1 Oberbayern antigen that was received from the IVB. Immunisation was repeated at day 21 and 35 after first inoculation. Three days after the last immunisation, spleens were aseptically

removed and fusions performed essentially as described by Aggarwal and Holmes (2000). The resulting hybridomas were screened by an antigen capture ELISA (see below) and cloned twice by limiting dilution before amplification. Isotypes of the MAbs were determined using a commercial kit (Sigma).

Virus neutralisation test

MAbs were tested for their ability to neutralise the homologous virus in a microneutralisation test according to the method of Golding et al., 1976.

Western Blotting

This was essentially done as described by Towbin et. al. (1979) with some modifications. C1 Oberbayern was grown in BHK-21 cells, inactivated with binary ethylenimine and purified by the sucrose density gradient method. Varying concentrations of virus were mixed with Laemmli sample buffer (Bio Rad) and disrupted by boiling at 100°C for 5 mins. Polypeptides were separated by 14% Tris-Tricine gels and the reactivity of the MAbs with FMDV polypeptides detected immunologically.

ELISA

a) Hybridomas were screened using an antigen capture ELISA as described previously (Aktas and Samuel, 2000). For this, sucrose density gradient purified C1 Oberbayern antigen was used at a concentration of 1µg/ml. b) A similar ELISA was used to determine the reactivity of MAbs with trypsin treated virus. For this, 100 µg of sucrose density gradient purified C1 Oberbayern antigen was treated with 50 µl of trypsin (stock solution 2 mg/ml) as described by Crowther et. al., 1993. c) An antigen profiling ELISA, as described by Aktas and Samuel, 2000, was used to determine the reactivity of five field isolates with each of the MAbs.

Results

Five MAbs were produced from two fusions and characterised. Isotypes of these MAbs are shown in Table 1. All of these MAbs except E11.A9 reacted with polypeptides in Western blotting (Table 1) indicating that they recognised a linear epitope. Conversely, E11.A9 recognised a confirmational epitope. Two MAbs, E11.A9 and E2.B4 were shown to be neutralizing. E2.B4, A4.D12 and C2.A3 recognised trypsin sensitive epitopes (Table 1). Field isolate reactivity patterns against the five MAbs are shown in Figure 1.

Discussion

The antigen profiling ELISA results indicate that the five MAbs recognise four different epitopes. MAbs A4.D12 and C2.A3 appear to recognise an identical epitope where as the rest of the MAbs recognise individual epitopes. We are currently defining the critical residues for these MAbs. Antigen profiling studies suggest that all the five field isolates tested share some epitopes with the vaccine strain C1 Oberbayern. However, none of the field isolates tested showed an identical reactivity as the vaccine strain.

In any outbreak of FMD, if vaccination is to be implemented as a control policy, one main issue is to find a vaccine strain that is antigenically similar to the field isolate in question. Our observations in this report and those of various previous workers (Barteling et. al., 1986; Brocchi et. al., 1998; Samuel et. al., 2000) indicate that it is possible to measure antigenic relatedness between vaccine strains and field isolates in an ELISA based on MAbs. The panel of C MAbs used in this study was very limited but it is hoped that when complete panels of MAbs against this and other vaccine strains are available, it will be possible to define the

antigenically important homologous epitopes between field and vaccine strains. However, the significance of each antigenic epitope in relation to protection against disease still remains to be determined. Once more complete information is available, it will help to define epitopes which must be matched to identify vaccine strain that will offer greatest protection against a field isolate. Given the scarcity of such data at present the use of MAb based antigen profiling ELISAs for vaccine strain selection as a replacement for more traditional methods, reducing the use of animals, is a distant prospect.

The importance of continually producing more MAbs and delineating the significance of each epitope in protection against disease and analysing such information in conjunction with data generated from polyclonal based serological tests is essential to make further progress. Given the amount of work required to produce and characterise MAbs, it would be highly desirable for different research groups to co-operate in these studies to speed up the work and to avoid duplication of the efforts.

Acknowledgment

We would like to thank Dr. N. Ferris (IAH, Pirbright) for providing rabbit and guinea pig sera used in capture ELISAs.

References

Aggarwal, N. and Holmes, M.A. (2000) “Production and characterization of two monoclonal antibodies reactive with equine IgG Fc receptors.” Vet. Immunol. Immunopathol. 73:63-71 Aktas, S. and Samuel, A.R. (2000) "Identification of antigenic epitopes on the foot and mouth disease virus isolate O1/Manisa/Turkey/69 using monoclonal antibodies." Rev. Sci. Tech. Off. Int. Epiz. 19(3), 744-753 Barteling, S.J., Boerke, J. and Woortmeyer, R. (1986) "Use of MAbs for surface scanning of different field and vaccine strains of A-type FMDV new approach of subtype identification?" Rpt. Sess. Res. Grp. Stand. Tech. Comm. Eur. Comm. Control of FMD, Madrid, Spain. 32-34 Brocchi, E., Gamba, D., Bugnetti, M. and De Simone, F. (1998) "Monoclonal antibody profiling of recent FMD type A field viruses and comparison with reference virus strains." Rpt. Sess. Res. Grp. Stand. Tech. Comm. Eur. Comm. Control of FMD, Pirbright, U.K. 56-61 Crowther, J.R., Farias, S., Carpenter, W.C. and Samuel, A.R. (1993) "Identification of a fifth neutralizable site on type O FMD virus following characterization of single and quintuple monoclonal antibody escape mutants." J. Gen. Virol. 74: 1547-1553 Golding, S.M., Hedger, R.S. & Talbot, P. (1976). "Radial immuno-diffusion and serum neutralisation techniques for the assay of antibodies to swine vesicular disease." Research in Veterinary Sciences 20: 142-147 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: 299-310 Samuel, A.R., Turner, L.S. and Knowles, N.J. (2000) "Antigenic and genetic characterisation of FMD type O viruses from the far East." Rpt. Sess. Res. Grp. Stand. Tech. Comm. Eur. Comm. Control of FMD, Borovets, Bulgaria. 58-66 Towbin, H., Staehelin, T. and Gordon, J. (1979) "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA. 76: 4350-4354.

Table 1: Characteristics of C1 Oberbayern MAbs

MAbs Isotype Reactivity in Western blota Neutralising activityb Trypsin sensitive

E11.A9 IgG2a - + No

A4.D12 IgG1 + - Yes

D7.G2 IgG1 + - No

C2.A3 IgG2a + - Yes

E2.B4 IgG2a + + Yes

a – No reactivity; + reactivity b – No activity; + activity

C1 Ober C Phil 9/94 C Phil 4/90 C Nep 1/94 C Bhu 2/91 C Ban 1/92 E11.A9 A4.D12 C2.A3 D7.G2 E2.B4

< 10 % reactivity

10-50 % reactivity

50 – 100 % reactivity

Figure 1: Reactivity of vaccine strain C1 Oberbayern and field isolates with MAbs raised against IVB C1 Oberbayern vaccine strain