Spray-drying of inactivated FMDV antigens for diagnostic use. Amadori M.

Transcription

1 190 Appendix 23 Spray-drying of inactivated FMDV antigens for diagnostic use Amadori M. Istituto Zooprofilattico Sperimentale della Lombardia e dell'emilia, Brescia, Italy, SUMMARY The production of inactivated, spray-dried FMD vaccines has been described by Russian research workers for a long time. We were therefore stimulated to exploit such a technology to obtain preparations of inactivated FMDV antigens for diagnostic use. The latter usually consist of a large proportion of degraded 12S virus particles; yet, they are perfectly suited to work in the established serological tests for Ab to FMDV. Our study was carried out using a laboratory scale spray-drier which includes a main cabinet (control panel, inlet air filter, blower, top chamber, peristaltic pump) and a spray assembly (main chamber, cyclone, collection bottle). The apparatus enables the fine setting of the main working parameters: drying temperature, air flow, compressor air pressure, peristaltic pump speed. The preliminary tests were carried out on an inactivated Cardiovirus preparation as a test model; by using a preserving solution of skim milk + NaCl at different concentrations we could demonstrate almost complete recovery of both EMCV particles, i.e. complete virions and empty capsids. Later on we took to horse serum instead of skim milk, because of the more favourable behaviour of the sugar in the presence of high exhaust air temperatures. As for FMDV, the tests were carried out on BEI-inactivated O 1 Lausanne and A 5 Parma FMDV. In the presence of adequate additions of horse serum and saline, an inactivated virus suspension with high antigen content could keep its usual properties in the serological tests after the spray-drying process, despite an increased concentration of 12S particles. In particular, the dose/response curve of the Ag in ELISA tests for 12S and 146S/12S particles was not significantly different from that of the usual frozen Ag preparations. Furthermore, Ab-negative and Ab-positive bovine sera gave the same results with both conventional and spray-dried antigens. The possible major advantages of this procedure in terms of simplicity and ease

2 191 of use, convenience for the diagnostic laboratory and suitability for a process of stepwise harmonisation of the procedures among the National FMD Laboratories are put forward and illustrated. INTRODUCTION The process of stepwise harmonisation of the procedures among the national FMD laboratories demands the provision of suitable reagents for large-scale ring tests, aimed at evaluating the reliability and the reproducibility of the investigation procedures for both virus and antibody detection. With regard to the latter, the availability of large homogeneous batches of FMDV inactivated antigens is of utmost importance; on the one hand, it is arguable that such large batches should be made available to the regional laboratories in a scenario of extensive serological surveillance in the aftermath of a FMD outbreak; on the other hand, they would certainly conducive to the ongoing accreditation process within a quality assurance scheme ( 8 ). One practical solution is represented by the freeze-drying of FMDV antigens. Infectious FMD viruses, inactivated antigens and even vaccines (6, 10) can be freeze-dried without substantial detrimental effects on their biological properties. Furthermore, shipment of freeze-dried virus antigens is possible without the need for refrigeration, and their reactivity with bovine convalescent antisera in the liquid phase blocking sandwich ELISA is not unduly affected ( 11). Diagnostic kits may also conveniently include reference freeze-dried antisera ( 9 ). However, freeze-drying is a laborious procedure, since it may extend over 2-3 days and demands the filling and plugging of a large number of ampoules. In this respect, spraydrying may represent a convenient alternative. It must be stressed that the production of inactivated, spray-dried FMD vaccines has been described by Russian research workers for a long time; mono- or polyvalent FMD vaccines were obtained, which showed high potency and a shelf life not shorter than 5 years at 4 C; the stability during spray-drying was different between A22 and O1 FMD viruses; both could be also formulated into potent emulsified vaccines for pigs (13,14,15). On the basis of these favourable results, we were therefore stimulated to investigate such a technology to obtain preparations of inactivated FMDV antigens for diagnostic use.

3 192 MATERIALS AND METHODS Apparatus. Our study was carried out using a laboratory scale spraydrier (LAB-PLANT model SD/05), which includes a main cabinet (control panel, inlet air filter, blower, top chamber, peristaltic pump) and a spray assembly (main chamber, cyclone, collection bottle). The apparatus enables the fine setting of the main working parameters: drying temperature, air flow, compressor air pressure, peristaltic pump speed. Antigens. Preliminary experiments were carried out on a BEI-inactivated Cardiovirus preparation. Subsequent experiments were performed on BEIinactivated O 1 Lausanne and A 5 Parma FMD viruses. These were supplemented with either skim milk or horse serum in the presence of variable final concentrations of NaCl. The ph of these preparation varied between 7.4 and 7.8.Small aliquots were dried under vacuum for a rough estimate of the total dry content; this enabled us to calculate an expected recovery for each sample. All antigens had been supplemented with 0.5% sodium thiosulfate (final) after BEI treatment. Spray-drying. 200 to 500 ml of inactivated antigen were used at a time. After repeated preliminary testing, the following range of conditions was adopted:! Sample temperature: 4 to 20 C (excursion during the process).! Process temperature: 130 or 150 C! Exhaust air temperature: 80 to 90 C! Pump speed: 500 to 700 ml/hour! Compressor pressure: 1.8 to 2.0 bar! Air-flow: 60 to 65 m 3 /hour Tests on dried antigens. At the end of each experiment, the powder was weighed and the obtained recovery was compared with the expected one. Aliquots of O 1 Lausanne FMDV were reconstituted with distilled water at a precise weight to volume ratio and tested by sandwich ELISA with couples of monoclonal antibodies reacting with 146S and/or 12S particles ( 1 ). After reconstitution with water, both O 1 Lausanne and A 5 Parma FMDV spray-dried antigens were employed for antibody detection by a monoclonal antibody-based competition ELISA, as previously described (

4 193 5 ); tests were carried out on reference post-vaccination and post infection bovine sera in parallel with the usual antigens, stored in small aliquots at - 70 C. RESULTS Cardiovirus. This was chosen as a test model in the preliminary phase of this study. The inactivated antigen was obtained from a high-titred virus preparation ( 10 9 TCID 50 /ml). It was supplemented in the 1 st experiment with 10% skim milk and 3% NaCl and dried at 130 C. Due to the low recovery in the collection bottle, the two following experiments were carried out at 150 C in the presence of 10% skim milk + 0.5% NaCl (2 nd test) and 8% skim milk (3 rd test). The three dried Ag preparations were reconstituted with distilled water to the initial volume and tested by sandwich ELISA with couples of monoclonal antibodies recognising both complete and empty capsids; the two antigenic forms were well preservedin the 3 tests without an appreciable loss of reactivity, the OD/Ag dilution curves being very similar to that of the control untreated antigen. A slightly better recovery of complete virions was observed in the 1 st test. FMDV. On the basis of these favourable results, we decided to undertake the experiments on FMDV. All of these were carried out at 150 C with the addition of horse serum instead of skim milk because of problems linked to early clogging of the nozzle and coagulation of lactose. We reasoned that optimal recovery of 146S particles was not a priority, since the usual ELISAs for detection of Ab to FMDV can work in the presence of antigens characterised by a large degradation of 146S particles into 12S particles. The 1 st set of experiments were carried out on a batch of O 1 Lausanne FMDV antigen, showing a high initial infectious titre (8.4 TCID 50 /ml, log 10 ). The best results in terms of both dry content and Ag recovery were obtained in the presence of 10% (final) horse serum and a final Ag dilution 1:2 in saline (0.42% NaCl, final). It must be stressed that the OD/Ag dilution curves in the ELISAs for total Ag (figure 1a) and for 12S Ag (figure 1b) were very similar to those of the reference standard antigen kept at -70 C. Furthermore, the results of the Ab tests on reference bovine

5 194 sera were almost identical by using these same two antigens (see figures 2a and 2b). The 2 nd set of experiments was carried out on a batch of A 5 Parma FMDV, showing a much lower initial infectious titre (7.5 TCID 50 /ml, log 10 ). In this case, the antigen preparation before spray-drying had a dramatically reduced reactivity in the test system and gave rise to Ab titres of some post infection bovine sera, which were lower than those obtained with the reference antigen preparation; an equal or even lower reactivity was shown by the spray-dried preparations; the best results were obtained in the presence of 12.5% horse serum and 0.42 to 0.85% (final) NaCl, in close agreement with the above results on O type FMDV. DISCUSSION Spray-drying is largely used in the pharmaceutical industry; the applications in the field of microorganisms are instead scanty but anyway significant; spray-drying has been described for microorganisms as diverse as Streptococcus bacteriophages (7) and lactic acid bacteria ( 17 ). Interesting experiences have been also described in the field of vaccinology; in particular, spray-drying can be successfully used to prepare microparticles containing entrapped protein antigens ( 2,3 ); such microparticles were very efficient e.g. for immunoglobulin delivery to the respiratory tract (4) and for inducing a strong immune response in guineapigs to diphteria toxoid ( 12 ). There is in practice a large body of evidence that proteins can retain stability and antigenic properties during spraydrying, thus giving rise to a product with an extended shelf life. In our laboratory, a notable concentration of 146S particles was shown e.g. in a dried A22 FMD vaccine prepared 11 years before at ARRIAH, Vladimir (Amadori M., unpublished results). In this respect, the experience of the Russian research workers in the FMD field suggests that temperature and residual humidity play a major role: in the presence of a residual humidity 3% the shelf life is not less than 5 years at 2-8 C and the product can work better than the same one kept at room temperature for one year only (16). A further reduction of residual humidity may be obtained by vacuum dehydration of concentrated powders: in this case the moisture content can

6 195 be reduced to less than 1%, while the structure and properties of FMD virus are retained ( 18 ). The substantial effectiveness of spray-drying for obtaining suitable preparations of FMDV antigens for diagnostic use has been confirmed in our study, although further data are needed before reaching final conclusions. It must be stressed that the laboratory spray-drier used in this study does not allow for a complete recovery of the powders; hence qualitative rather quantitative data should be taken into account. In our experience, the real bottleneck of the procedure was represented by the process temperature: at 150 C the flow rate should not exceed ml/hour, provided that the compressor pressure and the air flow are within the aforementioned limits; if not, condensed moisture accumulates in the main chamber, which is an adverse prognostic factor for the recovery and the final quality of the product. Another crucial point is the NaCl concentration; on the one hand, a certain addition of salt is badly needed to increase the recovery of powder in the cyclone; on the other hand, there is evidence that high NaCl concentrations are detrimental to the stability of inactivated 146S particles and, to a lesser extent, of 12S particles as well. This may be due to the inverse relationship between ionic strength and isoelectric point of FMD virus particles ( 19 ); notice that ionic strength was also increased in our experiments by the addition of sodium thiosulfate after the BEI treatment. Finally, the initial Ag concentration could possibly play a major role: good results were obtained on high-titred virus suspensions only, and it is arguable that a preliminary 5 to 10-fold concentration before drying could be actually of use; anyhow, a preliminary ELISA on the inactivated virus suspension could be predictive of the final results of the spray-drying procedure. An adequately standardised procedure could be very convenient: 10 litres/day of inactivated FMDV antigen could be easily processed by one technician using a combined ultrafiltration and spray-drying scheme with a simple laboratory apparatus; then, the powder should be stored at 2-8 C protected from humidity; at the beginning of each week, a certain amount of powder could be reconstituted with cold distilled water at a defined weight to volume ratio and the antigen used in the next 4 5 days. Such a working scheme could be very convenient and conducive to the reproducibility of the Ab tests; therefore, it would be instrumental to the process of accreditation and quality assurance ongoing in many national FMD laboratories ( 8 ).

7 196 ACKNOWLEDGEMENTS The skilful technical assistance of G. Passador, I. Reda and M. Scaramuzza, as well as the precious co-operation of the serology laboratory of the Italian National Reference Centre for Vesicular Diseases are gratefully acknowledged. REFERENCES 1. Amadori M. et al. (1994), Vaccine, 12, Baras B. et al. (2000), J. Microencapsul., 17 (4), Baras B. et al. (2000), Vaccine, 18 (15), Bot A.I. (2000), Pharm. Res., 17 (3), Brocchi E. et al. (1990), Rpt. Sess. Res. Grp. Stan.Tech.Eur.Comm. Cont. FMD, Lindholm, Denmark, June, Appendix Butchaiah G. and Rao B.U. (1988), Rev. Sci. Tech. Off. Int. Epiz., 7 (2), Chopin M.C. (1980), J. Dairy Res., 47 (1), De Clercq K. And Donaldson A.I. (1997), Rpt. Sess. Res. Grp. Stan.Tech.Eur.Comm. Cont. FMD, Poiana-Brasov, Romania, Appendix Ferris N.P. et al. (1988), J. Virol. Methods, 19 (3-4), Ferris N. P. et al. (1990), J. Virol. Methods, 19 (1), Ferris N.P. et al.(1990), J. Virol. Methods, 30 (2), Johansen P. et al. (1999), Vaccine, 18 (3-4), Kravchenko V.M. et al. (1991), Proc. Int. Conf. "Towards a new strategy to combat FMD", Vladimir, Russia, pp Kravchenko V.M. et al. (1991), Proc. Int. Conf. "Towards a new strategy to combat FMD", Vladimir, Russia, pp Kravchenko V.M. et al. (1991), Proc. Int. Conf. "Towards a new strategy to combat FMD", Vladimir, Russia, pp Kurlova N. P. et al. (1991), Proc. Int. Conf. "Towards a new strategy to combat FMD", Vladimir, Russia, pp Mauriello G. et al. (1999), J. Food Prot., 62 (7), Ponomarev A.P. et al. (1996), Vopr. Virusol., 41 (5), Vande Woude G.F. (1967), Virology, 31 (3),