Formaldehyde enhances BEI-inactivation rates of Foot-and- Mouth Disease (FMD) virus by at least a ten-fold

Transcription

1 27 Formaldehyde enhances BEI-inactivation rates of Foot-and- Mouth Disease (FMD) virus by at least a ten-fold Appendix 34 Simon J. Barteling and Nazeem I. Cassim, Onderstepoort Veterinary Institute, Private Bag X6, Onderstepoort 1, Republic of South Africa Summary For FMD vaccine production, in general, inactivation of the FMD virus is carried out with binary ethylenimine. Under optimal conditions, inactivation rates are in the range of. 1. log per hour. In general, 48 hours of inactivation is required. Formaldehyde, the classical inactivating agent, inactivates at a rate of.3 logs per hour only. Therefore, a significant contribution of formaldehyde to the inactivation of BEI can hardly be expected. However, if formaldehyde is added at the start of the BEI-inactivation process, inactivation rates are augmented to 2 3. logs per hour. This enables overnight inactivation with even higher safety levels of the vaccines. Also, it is known that formaldehyde cross-links viral proteins which stabilizes the antigen. In general, some 146 S antigen is lost during inactivation with BEI. However, for all SAT vaccine strains no antigen losses were observed after the inactivation with the BEI-FA combination. Even in case of the labile SAT2 Zim 7/83 strain, that deteriorates to a great extent if inactivation is carried out with BEI only, no reduction in 146 S yields was observed with the combination of inactivating agents. The consequences for FMD vaccine production (antigen yields), the stability of the antigens (and of vaccines), and the endurance of the immune response will be discussed. Introduction Virus inactivation and safety tests are the most critical steps in the production of inactivated vaccines. In the case of foot-and-mouth disease (FMD) vaccines in particular, guaranteed safety is essential because any occurrence of the disease will have great economic consequences e.g. by a blockade of all export trade of animals and animal products. Classical FMD vaccines as developed by Vallee, Schmidt, and Waldmann (Waldmann et al.,1937) were inactivated by formaldehyde (FA). The nature virus, obtained from diseased cattle was first adsorbed to Al(OH)3 gel and then inactivated with.2 % FA. Safety of vaccines could at first only be tested on cattle tongue and later, in addition, in suckling mice. When titration in tissue culture became possible, inactivation plots could not be made because of the presence of the (toxic) Al(OH)3. Several studies (with non-adsorbed virus) showed that at the FA concentration prescribed by Waldmann, inactivation plots were not linear and often showed tailing off, which may cause incomplete inactivation (Wesslen and Dinter, 197; Graves, 1963; Barteling et al.,1983; Barteling and Woortmeyer, 1984). Consequently, large vaccine batches may contain some surviving virus. However, in the field the vaccines performed quite well, without causing outbreaks that clearly could be associated with vaccination campaigns. However, in 1981 it was shown by King et al. that an outbreak strain isolated in the UK, originating from Brittany (France), could well be vaccine related. By comparing nucleotide sequences of outbreak strains and vaccine strains it was also shown by

2 271 Beck and Strohmaier (1987) that the sequences of European outbreak strains were very similar to those of vaccine strains suggesting that most of the outbreaks in Europe very likely were caused by improperly inactivated vaccines or by viruses that had been escaped from vaccine production laboratories. Linear inactivation plots were obtained with acetyl ethylenimine (AEI) and other aziridins (Brown et al.,1963; Bahnemann 197). Although it was shown that, under proper conditions, FA-inactivation plots - of Al(OH)3-adsorbed virus - were linear as well (Barteling and Woortmeyer, 1984), inactivation with aziridins became the method of choice. Binary ethylenimine (BEI) is used in particular, because this method developed by Bahnemann (197, 199) circumvents the direct handling of the very toxic other aziridins. However, Aziridins lack the cross-linking and fixation activity of formaldehyde that made the old-type Frenkel vaccines stable for years and more (non-published observation). Inactivation with BEI often results in a loss of some 3 % of the 146 S antigen (observation at OVI). Therefore, for labile vaccine strains (like SAT2 Zim 7/83), the antigen is first (partly) inactivated with BEI, fixed with formaldehyde, and then inactivation is completed by the addition of another portion of BEI (Rowlands et al. 1972, Mowat 1973, Rweyemamu 1989). When studying the effect of the simultaneous addition of the two inactivants, we detected a synergistic effect of FA to the inactivation by BEI. For all (SAT) vaccine strains produced at Onderstepoort, inactivation rates were enhanced by more than a -fold. Inactivation reached a safe level within 8 hours and no losses in 146 S were observed. A polyvalent vaccine prepared from the BEI-FA-inactivated antigens induced (in cattle) satisfying levels of neutralizing antibodies and of protection against one of the strains. Materials and methods - Virus antigen production and virus titration Because it was difficult to mimic all aspects of large scale production on the small scale, the new inactivation method was tested for all five vaccine strains on the production scale. This was carried out during working weeks of 4 days when regular production using only BEI was not possible. The strains routinely produced at Onderstepoort are: SAT1-SAR9/81; SAT1-KNP196/91-1; SAT2-KNP19/89-2; SAT2-ZIM7/83; and SAT3-KNP/9-3. Virus was produced on roller bottles as described by Panina et al. (1976) according to a strict weekly schedule. - Inactivation, concentration and storage of the antigen Clarified chloroform-treated virus culture was brought to a temperature of 3ºC and then inactivated with 1 mm of BEI (Bahnemann 199) or with a combination of 1mM BEI and.4% FA (BEI-FA). With BEI only, samples were taken every hour during the first 6 hours and after 24 and 48hours. After 24 hours of inactivation the same quantity of BEI was added according to the same procedure as before. Then, the inactivation mixture was transferred into a second inactivation tank where inactivation was continued. After 48 hours inactivation was stopped by the addition 1/ vol. of a 2 % Na-thiosulfate solution. Samples were collected in 1/ volume Na-thiosulfate (to stop the inactivation by BEI) and 1/ vol. of calf serum, chilled on ice, diluted for titration and/or stored away at 7ºC for back-up (no differences were observed between immediate titration and titration of the frozen samples). Because the BEI-FA combination inactivates FMD very rapidly (> 2 log ID per hour), samples were taken every 2 minutes during the first 3 hours. Sampling was also

3 272 carried out at 6 hours, when the inactivating mixture was transferred into a second inactivation tank, and at 24 hours, when the inactivation was stopped by the addition of Na-thiosulphate. At the end of the inactivation (and for the FA-BEI also at 6 hours) a large sample of 2 ml was taken for an additional safety control on BHK- and IBRS-roller bottles with 2 subsequent blind passages each after 48 hours (modified after Anderson et al. 197). The inactivated antigen was chilled to 8ºC and then transferred to a cold room where it was concentrated by ultra-filtration. The concentrate was stored away above liquid nitrogen. - Preparation and testing of (sample) vaccines An experimental vaccine was prepared from a mixture of all five FA-BEI-inactivated vaccine strains. At the time of formulation, the (computerized) 146 S analysing system valued antigen concentrations approximately 4 times too high. Unfortunately, this was detected only when the vaccine was already injected in cattle. Therefore, the vaccine contained per (3 ml) dose.4 µg (approx.) of each of the vaccine strains with the exception of SAT3 KNP9/3 of which.8 µg was incorporated. A quarter of a vaccine dose (.7 ml) was injected intramuscularly in cattle. Two cattle were left as controls. Serum samples were taken weekly. At 2 weeks p.v. the serum samples were immediately tested in a micro-neutralization test. At 3 weeks p.v. the cattle were challenged with the strain showing the lowest antibody levels at 2 weeks p.v.(sat 1 KNP 196/91-1). Challenge was carried out by injecting. ID intra-dermolingually. Cattle were observed for clinical disease for the following 8 days when they were slaughtered and post mortem inspection was carried out for signs of clinical disease. Results and discussion A representative inactivation plot obtained with BEI is given in fig 1a. In our experience, with BEI alone, inactivation rates vary between.4 and 1. log per hour (see also Bahnemann, 199). At OVI inactivation is carried out for 48 hours. At 24 hours a second portion of BEI is added. Thus, even at the lowest inactivation rates, sufficient safety can be guaranteed. The BEI-FA plots obtained with the vaccine strains that are routinely produced at OVI are given in fig1(b-f). The inactivation rates were varying from 2. (SAT1-SAR9/81, fig 1b) to more than 3 logs per hour (SAT2-ZIM 7/83 and SAT2 KNP-19/89/2, fig 1d and 1e resp., table 1) Thus, sometimes inactivation is over a -fold faster if FA is added. It is difficult to say what causes this synergistic effect. Under optimal conditions, FA alone inactivates at a rate of approx..3 log/hr only (Barteling, 1984). Thus hardly any addition is to be expected to the much faster inactivation of BEI. Aziridins attacks the nucleic acid. It seems that the cross-linking action of FA makes the virus particle more accessible for the BEI enabling a faster attack of the nucleic acid. From these graphs it is clear that by the FA-BEI combination linear plots were obtained and virus titers were found to be reduced at least more than 2 logs per hour. Thus already within 8 hours sufficient inactivation was reached for acceptable safety. In accordance, no surviving virus could be detected in a large (2 ml) sample taken after 6 hours, as was the case for the 24 hr samples. It should be noted that in the micro-titer system undiluted and -fold diluted samples were causing a cpe-like effect but this was due to the toxicity of the formaldehyde and no virus could be propagated from these cups. Thus inactivation can be carried out within the time span of a working day or just overnight in stead of the 48 hours required for the inactivation with BEI alone. This gives greater flexibility in weekly production schedules. E.g. in Onderstepoort a production batch could just

4 273 be completed within working days. Now, the rapid inactivation by FA-BEI allows production within 4 days and production is no longer hampered by e.g. national holidays. With BEI alone, antigen yields (of SAT-strains) were often reduced from to 3 percent during the 48 hours of inactivation. No reduction in 146 S antigen concentration was observed after the 24 hr of inactivation with FA-BEI. Where conditions were otherwise identical, a reduction with approximately half that observed with BEI could be expected. The optimal yields are probably due to fixation of the antigen by the cross-linking action of FA (Barteling, 1984). Considering the low quantities of antigen that were incorporated in the vaccine, the vaccine performed quite well. At 2 weeks p.v., the lowest neutralising antibody response was against SAT 1 KNP 91/1(with a mean titre of 1.8 log) and, therefore, this strain was selected as challenge strain (table 2). Two animals were protected and one was partly protected, indicating a protection level of approximately % (1 PD ). Because the injected vaccine dose contained approximately.2 µg per FMD virus type, these results indicate that (for the weakest antigen) per µg a protection level of approximately PD can be expected which in our experience is quite good. Stability of the 146 S antigen is an important parameter in the selection of new vaccine strain. By the cross-linking action of FA, which stabilizes the 146 S antigen, this parameter becomes less critical, and, therefore the BEI-FA inactivation method will make the rapid introduction of new vaccine strains more easy. Because of the fixation of the antigen by the cross-linking action of FA, we expect the vaccines to be of superior stability. This is particularly important for developing countries (e.g. in Africa), where maintenance of cold chain conditions is not always possible. Whether these vaccines induce a relatively long lasting immune response (as for the Frenkel vaccines) remains to be confirmed. Acknowledgement We thank Mrs Erika Kirkbride, Mrs Brenda Botha, Mr. Anton Barnard, Mr. Philemon Dolo, Mr. Lourens VanStaaden, and Dr. Comfort Piri greatly for their technical assistance and for performing the animal experiments. References 1.Waldmann O, Pyl G, Hobohm KO, Mohlmann H: Die Entwicklung des Riemser Adsorbatimpfstoffes gegen Maul- und Klauenseuche und seine Herstellung. Zbl Bakt I Orig 148: 1, Graves JH: Formaldehyde inactivation of foot-and-mouth disease virus as applied to vaccine preparation. Am J Vet Res 24: 1131, Weslen, T. and Dinter, Z. Then inactivation of foot-and-mouth disease virus by formali. Arch. Ges. Virusforsch. 197, 7, Barteling SJ, Woortmeijer R, Visser N: Innocuity testing of foot-and-mouth disease vaccines. I. Formaldehyde-inactivated alhydrogel vaccines. J Biol. Stand : 297,. Barteling SJ, Woortmeijer R: Formaldehyde inactivation of foot-and-mouth disease virus. Conditions for the preparation of safe vaccine. Arch Virol 8: 3, 6. King AMQ, Underwood BO, McCahon D, Newman JWI, Brown F: Biochemical identification of viruses causing the 1981 outbreaks of foot-and-mouth disease in the U.K. Nature 293: 479, Beck E, Strohmaier K: Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence determination. J Virol 61: 1621, Brown F, Hyslop NSG, Crick J, Morrow AW: The use of acetyl-ethyleneimine in the production of inactivated foot-and-mouth disease vaccines. J Hyg (Camb) 1963b, 61: Bahnemann HG: Binary ethylenimine as an inactivant for foot-and-mouth disease virus and its application for vaccine production. Arch Virol., 197, 47: 47-6

5 274. Bahnemann, H.G.: Inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethylenimine. 1999, Vaccine 8, Rowlands et al. () Stabilizing the immunizing antigen of foot-and mouth disease virus by fixation with formaldehyde. Arch. Ges. Virusforsch. 1972, 39, ; 12. Mowat, G.N. Enhancement of immunizing potency of foot-and-mouth disease vaccine for cattle by treatment of the antigen with formaldehyde. Arch.ges.Virusforsch. 1973, 41, M M Rweyemamu et al. Effect of formaldehyde (FA) and binary ethyleneimine (BEI) on the integrity of foot-and-mouth disease virus capsid. Rev. sci. tech. Off. Int. Epiz , Panina, G.F. Animal cell culture and FMD virus production in a large scale insuatrial roller bottle plant. Proc. First Gen. Meet. Eur. Soc. Anim. Cell Techn. (eds. Spier, R. and Van Wezel A.L.) RIVM, Bilthoven, The Netherlands, 1976, 4 Table 1: Inactivation rates (with BEI-FA) of five SAT vaccine strains. Strain SAT 1-SAR 9/81 SAT 1- KNP 91/1 SAT 2 ZIM 7/83 SAT 2 KNP 89/2 SAT 3 KNP /9-3 Inactivation rate 2. log/hr 2. log/hr 3.1 log/hr 3.3 log/hr 2.2 log/hr Table 2: Mean virus neutralisation titres at 2 weeks p.v and challenge results. The animals were vaccinated with ¼ of a dose containing.4µg of the SAT 1 and SAT 2 strains and.8µg of SAT 3. Strain Mean titre SAT 1 KNP 196/91/1 1.8 SAT 1 SAR 9/81/1 2.4 SAT 2 KNP 19/89/2 2. Sat 2 Zim 7/83/2 2.3 SAT 3 KNP /9/3 2.3 Challenge (at 3 w. p.v.) was carried out with SAT 1 KNP 196/91-1. Out of animals 2 were protected and 1 was partly protected (1 lesion).

6 27 Text fig 1 Inactivation plots obtained with BEI alone (a) for one of the SAT vaccine strains and with BEI-FA (b f) for all vaccine strains produced at OVI. a) SAT1-SAR 9/81 - BEI b) SAT1-SAR 9/81 -BEI-FA Titre log Titre log c) SAT 1 - KNP 196/91-1 BEI-FA d) SAT 2 - ZIM 7/83 BEI-FA Titre log Titre log e) SAT 2 - KNP 19/89-2 BEI-FA f) SAT 3 - KNP /9-3 BEI-FA Titre log Titre log