Item 8 Closed Session

Dr Nilgün Özdural presented a paper (Appendix 27) on the development of a latex agglutination test kit for the detection of A22 Mahmatli and O1 Manisa FMD antigens which is intended for use in the field. The test is highly sensitive and therefore special attention should be given to the time of observation of agglutination.

Conclusions

• Once fully validated, real-time, automated RT-PCR could support the ELISA tests for the detection of FMDV in epithelial suspensions and largely remove the necessity for virus isolation in cell culture for the confirmation of secondary cases.

• The comparison of the RT-PCR procedures confirmed the high sensitivity of RT-PCR methods. Nested and novel PCR procedures had a higher sensitivity than conventional

RT-PCRs with universal primers, but only quantitative methods scored all samples positive on epithelial suspensions.

• The simplicity and sensitivity of the latex agglutination test (LAT) makes it a good candidate for a pen-side test in endemically infected areas.

Recommendations

• More validation work should be carried out on the automated RT-PCR and the system should be optimised for the testing of probangs and milk.

• A more extensive comparison of RT-PCR procedures should be performed. Thus, more experimental details such as the position of the assay cut-off, the use of positive and negative controls and details of assay validation need to be made available. Also the specificity of the RT-PCR procedures should be further investigated.

• The specificity of the LAT kit should be more thoroughly evaluated.

Item 6: Diagnostics - antibody detection

Dr Kris De Clercq gave an overview of plans for an EU project for the production of FMD reference sera. The recent outbreaks in Europe and the need to develop and validate new assay systems, including those for the detection of NSP antibodies, have highlighted the importance of developing a panel of such reagents as quickly as possible. The project should involve a consortium of laboratories and an agency specialising in the storage, aliquoting and distribution of such materials. The EU Commission will have to tender the work for competitive bids.

Drs David Paton and Bob Armstrong presented results from the FAO Phase XVII serology standardisation exercise (Appendix 28). The reference sera prepared in Phase XV against O Manisa, A22 and C Noville have been adopted by OIE. A new set of reference sera have been prepared against O SKR, A Iran 96 and Asia 1 Shamir. For each strain, a candidate strong, weak and cut-off serum was prepared and distributed, along with a negative serum, to 9 other laboratories. Reagents and a protocol were also supplied for solid phase competition ELISA (SPCE) for sera type O. Results from all the laboratories were presented, including analyses

by virus neutralization test (VNT), liquid phase blocking ELISA (LPBE), SPCE, NSP tests and other in-house assays. The view was expressed that the weak and cut-off sera need to be slightly strengthened.

Dr Esther Blanco presented a paper (Appendix 29) on the engineering of ß-galactosidase enzymatic sensors for the species-independent detection of strain specific antibodies against VP1 of FMDV by way of antibody-concentration dependent modulation of the activity of the enzymes expressing 12 copies of the viral peptide. Research is planned for the engineering of sensors for the detection of antibodies against NSP.

During the 2001 crisis it became evident that quick and high throughput diagnosis of seroconversion was necessary and therefore a quick and simple MAb based ELISA (Ceditest FMDV type O, Cedi-Diagnostics) was developed for the detection of antibodies against FMDV O in all species. The test was evaluated in a number of European FMD laboratories. From the data reported by Dr Liesbeth Jacobs (Appendix 30) it appears that results are highly comparable to SPCE conducted at VAR and IAH-P but further improvement is needed with regard to pig samples. Steps are being taken to develop equivalent tests for other species and other FMD virus strains.

Two papers were presented on studies comparing in-house NSP-tests with a commercially available NSP-test (CHEKIT-FMD-3ABC ELISA, Bommeli).

In the first case Dr Bernd Haas reported on the comparison made on the base of wellcharacterised samples from animals vaccinated and later on infected during an outbreak and of a panel of samples from potency trials (Appendix 31).

As a result of the studies, the in-house competitive ELISA of the Danish laboratory appeared to be superior in sensitivity to the CHEKIT-FMD-3ABC ELISA. However, the sensitivity of the latter could be improved by reducing the cut-off value without significantly reducing its specificity.

A second comparative study was carried out by Mr Robert Armstrong (Appendix 32) on samples including those of vaccinated and challenged cattle and sheep using the CHEKITFMD-3ABC ELISA in comparison with an in-house test of the WRL. It appears that this inhouse test is again superior to the CHEKIT-FMD-3ABC ELISA in sensitivity. However, the data should be interpreted with care and take into account the sampling intervals and the different approaches to deciding on the cut-offs. The in-house test comprises 5 steps and is therefore laborious.

In a field study carried out by Dr Mark Bronsvoort in Cameroon (Appendix 33) endemically infected with multiple serotypes of FMD, the results obtained by testing a statistical number of cattle blood samples with the CHEKIT-FMD-3ABC ELISA and the Danish C ELISA were compared with the VNT. The comparative sensitivity (with VNT) of both NSP tests was low in the studied bovine population where FMD is endemic. Suggestions were made to make a sensitivity comparison between both NSP tests before making final conclusions.

Dr Lucas Schalch presented additional data (Appendix 34) to further validate the test at the established cut-off value, and to provide a method for monitoring the operational performance of the test. The additional data presented concerned in particular sera from experimentally and

challenged cattle and sheep, from single and multiple vaccinated cattle and pigs and various samples of antibody negative animals. Validation results on specificity, sensitivity, repeatability and reproducibility were presented. The kinetic of antibodies against NSP was followed and certain infected animals were scored negative by 133 dpi. Dr Lucas Schalch also explained the charting method as an essential tool for day to day performance validation of laboratory tests.

Following on from earlier developments of peptide based ELISAs for the detection of 3B NSP, Dr Scott Liu, presented a confirmatory system including 3-ELISA using peptides from NSP 3A and 3B (Appendix 35). There is a confirmatory 3B blocking ELISA and a confirmatory 3A ELISA which were established in order to increase sensitivity of the 3B NSP-ELISA and simultaneously to reduce the number of false positive results. However, true sensitivity of the test needs to be established especially using sera from vaccinated and challenged animals of various species and of known status.

Conclusions

• Plans will be finalised for an EU project for the production of FMD reference sera.

• New candidate reference sera have been assessed under phase XVII but some strengthening of weak positive and cut-off sera are required.

• The development of enzymatic sensors for FMD diagnosis is at an early state of development but deserves further investigation.

• The commercially available test kit “Ceditest®FMD” for the detection of antibodies against O1 FMDV is promising.

• The CHEKIT-FMD-3ABC ELISA may be used with increased sensitivity in ruminants when read with modified cut-off. Further work is necessary for the validation.

• Animals vaccinated with a highly potent vaccine and challenged at the peak of immunity and which become infected do not all necessarily develop antibodies to NSP. These findings confirm that NSP testing can only be carried out for surveillance purposes on a herd level.

• Based on European data, the competitive NSP-ELISA developed in Denmark has a very high sensitivity. The Pirbright in-house NSP test performs well but is laborious.

• The peptide-based ELISA (UBI) has sufficient specificity but has incompletely characterised sensitivity.

Recommendations

• Laboratories are encouraged to develop a consortium to undertake the EU FMD reference serum project.

• The constitution of the new reference sera tested under phase XVII should be finalised.

• Future FAO serology standardisation should make use of reagents developed from the above-mentioned EU project. They should also look closely at the standardisation and internal quality control practiced within participating laboratories.

• Development of enzymatic sensor should be pursued in particular with regard to NSP.

• The ELISA test kit “Ceditest” should also be developed for other serotypes and strains.

• In relation to NSP tests more data on sensitivity in all target species, including sheep and pigs, are needed from animals vaccinated and subsequently challenged.

• Consideration should be given to increase sensitivity in the 3ABC-Chekit test, possibly by modifying inter alia the cut-off value.

• The transfer into commercial production of the Danish in-house SND test is strongly encouraged.

• Laboratories are encouraged to implement the charting methods for day by day performance check.

Item 7: FMD vaccines and vaccination

Dr Kris De Clercq presented a report (Appendix 36) of the progress made by the Committee for Veterinary Medicinal Products (CVMP) ad hoc group, a working group comprised of members of the Immunological Working Party of the CVMP, of the Research Group of the EUFMD, OIE, Pharm.Eur., EU and at a later stage the FMD vaccine manufacturers tasked with preparing guidelines on the requirements for FMD vaccines. Recent recommendations for FMD control strategy make vaccination more likely and recent experience suggests that the general public and the farming industry would expect any vaccine that is used to be fully authorised. The guidelines currently being prepared propose possible solutions to many of the technical challenges presented by FMD vaccines and it is to be hoped that after the consultation period they will provide valuable advice to manufacturers.

Dr Chris Griot reported that in Switzerland (Appendix 37) a batch of A Iran 96 antigen stored in the vaccine bank had been formulated into a vaccine lot and tested for potency in cattle with strain A Iran 99 as challenge. The experiment showed that the vaccine was able to protect against A Iran 99 which differs antigenically from A Iran 96.

Dr Gülhan Aynagöz showed how the efficacy of FMD vaccine batches was assessed by testing serum samples taken from cattle in the field (Appendix 38). Blood sampling was done before and 21 or 28 days after vaccination. Based on LPB ELISA results it was estimated that more than 80% of vaccinated animals were protected against FMD homologous strains. The study is ongoing.

Along with the application of assays able to detect antibodies against non-structural proteins (NSP) of FMDV it is important that antigens prepared for vaccines are free from contaminating NSP. Remarks on this matter were presented by Dr Fuat Özyörük (Appendix 39) who used an amplified Western Blot method to detect NSP 3A, 3AB and 3ABC in fluids

eluted from Al (OH)3 adjuvanted vaccines. Anti 3A monoclonal antibody (2C2) produced in IZSLER, Brescia was used to demonstrate the presence of 3A, 3AB and 3ABC NSP at a detection limit of 11.5 ng/lane. Contaminating NSP 3 AB was sedimented together with cellular debris and could not be detected in vaccines tested.

The paper presented by Dr Eliana Smitsaart (Appendix 40) described the development and performance of oil adjuvanted FMD vaccine used in recent emergency and mass vaccination campaigns in Argentina. Newly emerged virus strains of type A were characterised by in-vitro neutralisation assays and in-vivo protection studies, in addition to 2 dimentionally CFT, partial sequencing and MAb profiling. These studies lead to the incorporation of two new field strains of type A in the O1 Campos – A24 Cruzeiro vaccine. Dose-response studies including challenge in cattle were performed in order to determine the required amount of antigen of the new field strains in the vaccine. After two rounds of vaccination of the whole herd with the finally composed vaccine, immunity levels above 80% protection were achieved. The potency was reflected by the dramatic decrease in the number of FMD outbreaks reported. The contribution of the national vaccine bank was recognised.

Dr Scott Liu described progress towards the development of a peptide vaccine based on the VP1 G-H loop and incorporating a T helper epitope. The peptide has now been evaluated in pigs and, to a lesser extent, in cattle.

Dr Ismet Gürhan presented data on the production of a synthetic peptide vaccine combined with synthetic polymeric adjuvants (Appendix 41). Two amino acid sequences of VP1 protein of strain A Aydin 98 were conjugated to four locally synthesised polymeric complexes to prepare vaccines. Following preliminary studies such as in vitro cytotoxicity, in vivo toxicity in mice and guinea pigs, peptide specific antibody response in mice, and guinea pig potency tests, a so-called VAC2 was selected as a candidate vaccine for further studies. The results indicated that VAC2 is able to protect guinea pigs against type A FMDV challenge, but it did not induce sufficient specific antibody against either the synthetic peptides or intact virus in cattle. The antibody level estimated with both LPB ELISA and NT was not at an acceptable level for protection experiments. Quantitative as well as qualitative modifications such as addition of T-cell epitope sequences or increasing the quantity of the peptide in the vaccine may be useful to induce a stronger immune response in cattle.

Conclusions

• The Research Group fully supports the important initiative of the CVMP ad hoc group on the regulations of FMD vaccines which, when it achieves its objectives, will be a major step forward in supporting FMD disease control not only in the EU but world-wide.

• Modern vaccine batches with a strong potency are able to cover only partially related field strains which have recently emerged.

• Vaccines applied in the field which contain antigens of recent field strains have a higher potential to be effective than heterologous antigens of type A after single vaccination.

• Non-structural proteins can be sufficiently removed during processing from antigenic preparations so that there is only a minimal risk that NSP have an antigenic potential in vaccinated animals.

• A synthetic peptide vaccine developed in Turkey, adjuvanted with synthetic polymer is capable of inducing an immune response in laboratory animals and to protect against homologous challenge. However, so far, it has not induced sufficient specific antibodies in cattle. The result of the UBI synthetic peptide vaccine looks promising.

Recommendations

• The RG supports the effort to harmonise the activities of the CVMP and other organisations and to supplement the monograph of the European Pharm. With guidelines in order to cover the specific requirements for FMD vaccines.

• In view of the limited capacities in the FMD institutes or laboratories it is recommended that the results of vaccine potency tests which include heterologous challenge be reported, where possible, to EUFMD and further be distributed to other FMD vaccine laboratories.

• Studies with synthetic peptide vaccines should be continued if it can be expected that a specific immune response in ruminants and/or pigs is achieved.

• A simple procedure such as should be developed to detect low levels of residual NSP in antigen preparations.

Item 8: Closed Session

The members of the Research Group, Prof. Dr Reinhard Ahl, the Representative of the EC Scientific Committee, Dr Alf Füssel, the Representative of the European Commission and Dr David Paton, the Head of the WRL Pirbright met in a closed meeting on 19 September. Dr Kris De Clercq, Belgium, chaired the meeting. The EUFMD Secretariat was represented by Keith Sumption. The agenda circulated at the start of the meeting was approved. It was agreed that the issue of procedures to reduce risk from sausage casings should be re-considered at a later stage. The following items were examined at the meeting:

1. Information on recent and future activities relating to the Caucasus, Turkey, Greece and Bulgaria

The Secretary reported that the joint proposal to FAO from Turkey, Greece and Bulgaria for a TCP for strengthening surveillance and control of FMD and other exotic diseases had been received by FAO and that a follow-up meeting is planned for 25 October 2002, in Athens. The timing is also opportune for a meeting of the Tripartite (EUFMD/OIE/EC) to discuss the situation in the region and particularly the plans for sero-monitoring in Turkish Thrace. The current Session of the Research Group had provided a good opportunity to discuss seromonitoring post-vaccination in Turkish Thrace in 2003 and guidelines for this would be prepared by the Secretariat before the Tripartite meeting.

The Secretary reported that it is anticipated that OIE would organise a follow-up meeting of the Tripartite (EUFMD/OIE/EC) Group in Paris on 4 November 2002 to discuss the plans for the vaccination campaigns for Spring 2003, and associated sero-surveillance activities. A longer term vision and strategy for the control of FMD in the region was required and this