Collaborative study for the establishment of a European Pharmacopoeia Biological Reference Preparation for Clostridia antiserum for serological potency testing of veterinary clostridial vaccines.

The European Directorate for the Quality of Medicines (EDQM) has organised an international collaborative study, divided in two phases, aimed at producing and establishing a suitable reference serum for serological potency testing of clostridial vaccines for batch consistency demonstration. In phase 1 a series of pools produced from sera provided by each manufacturer and raised against the broadest range of antigens possible were blended to obtain TN titres which were representative of the range normally elicited by the vaccines under test. Detailed statistical analysis of the data was not possible since only a few laboratories were able to participate and because limited replication of the assays was possible. Nevertheless, sufficient data were collected to conclude that the blend of sera would provide a suitable reference material for use by all manufacturers and regulatory authorities in respect of in vitro assay methods for the five components for which it was primarily designed, i.e. Clostridium (C.) perfringens beta and epsilon, septicum, novy and tetani. On the basis of these results a production scale blend of the serum pools was prepared, filled and freeze-dried by EDQM as a single batch, referred to as candidate (c) Ph. Eur. Biological Reference Preparation (BRP). In phase 2 of the study a larger group of laboratories, including both manufacturers and official medicines control laboratories (OMCL), were invited to participate in a study comparing the activity of the proposed rabbit multicomponent reference serum with the existing equine monovalent World Health Organization (WHO) International Standard (IS). These studies necessarily were conducted according to the methods described in the currently applicable monographs i.e. by toxin neutralisation test in mice (TN) to provide a definitive value for the antitoxin activity of the reference preparation in respect of the five components studied.

Alternatives to in vivo potency and protection tests for veterinary vaccines.

I firmly believe that we should use animal-based studies only when there is no satisfactory alternative. However, the development of meaningful alternatives requires good science. Regrettably, all too often, we can see examples of potency tests which have not been properly developed and validated and are therefore incapable of providing us with the information that they should be designed to provide.

Veterinary vaccines: In-VITRO--International Veterinary Industry Test Replacement Organisation.

The International Veterinary Industry Test Replacement Organisation (In-VITRO) was established in 1995 with the aim of developing, validating and harmonising in vitro alternatives to replace in vivo methods for in-process and potency testing of veterinary clostridial vaccines. The emphasis has been on the reduction of animal usage in the Clostridium chauvoei potency assay and its eventual replacement by an in vitro assay. Replacement of the toxin neutralisation assay for Cl. tetani by an internationally validated indirect ELISA has already started. A validation programme involving a collaboration organised through EDQM which could ultimately lead to the standardisation of in vitro tests for all clostridial vaccines is in progress. In addition In-VITRO is now considering the setting up of a programme for Erysipelas vaccines. The collaboration between manufacturers of veterinary vaccines in the development and validation of in vitro tests is a major step towards the reduction and replacement of animal tests.

The five Rs: refinement, reduction, replacement. A regulatory revolution.

Regulatory requirements have frequently been cited as a major reason for the continued performance of batch control tests using animals. Although such claims have not always been justified regulatory authorities and associated organizations such as the pharmacopoeial committees have sometimes been slow to respond to emerging knowledge and the potential of novel techniques. The regulatory approach to quality control tests in the wider context of quality assurance and total quality management is discussed together with the role which the regulatory authorities can play in promoting reduction in the use of animal based tests and the opportunities which exist for them to encourage manufacturers to explore other approaches. The need for caution on the part of regulatory authorities in requesting additional data which would necessitate further animal testing is stressed and the concept is proposed of a formal review of the benefits of animal testing applied on a batch basis whenever a marketing authorization is issued or renewed. The types of animal test most amenable to deletion or modification are considered together with those tests which present particular problems. Specific proposals are made for an immediate reduction in the numbers of animals used.

Reducing the use of the target animal batch safety test for veterinary vaccines.

The need to submit each batch of every veterinary vaccine to a target animal safety test is questioned. It is proposed that a risk/benefit analysis should be conducted, on a product by product basis, to determine whether the continued application of this test to each batch of a product is beneficial and justified. For an established product, the analysis should consider: the number of batches manufactured; the length of time for which the product has been manufactured; the testing experience and the incidence of reported adverse reactions; the level of GMP compliance and the standard of QA practised by the manufacturer; the method and conditions of manufacture; the use of animals for other batch tests; the recommendations for the use of the product. For a newly developed product the analysis should take into account: the safety data generated during development; the inherent risks in the product and its manufacture; the use of the other animal tests. It is suggested that the continued use of the test could be reduced on a phased basis e.g. after 10 consecutive satisfactory tests the frequency could be reduced by limiting it to the first batch of each production campaign or to every n th batch. Furthermore, the possibility of discontinuing the test altogether, for the product in question, could be considered. A return to full testing frequency would be required in the event of: a test failure; a batch related adverse reaction; introduction of a new seed; a change of manufacturer; a process of modification; a specification amendment; or an interruption of batch continuity. When a manufacturer has established consistency of production and testing, an alternative approach to the need for batch testing, with a view to avoiding or minimising the use of animals, is both desirable and possible.

Smooth lipopolysaccharide is the major protective antigen for mice in the surface extract from IATS serotype 6 contributing to the polyvalent Pseudomonas aeruginosa vaccine PEV.

The nature of the protective antigen in one of the sixteen monovalent extracts (viz., extract-6) contributing to the pseudomonas polyvalent extract vaccine (PEV) was studied in a mouse challenge assay. Selective removal, by filtration through Sep-Pak C18 cartridges, of two major protein antigens with molecular weights of 16,200 and 21,000 had no effect on the protection afforded by extract-6. When analyzed on the basis of 2-keto-3-deoxyoctonate, lipopolysaccharide (LPS) purified by hot phenol extraction (LPS-A) from Pseudomonas aeruginosa (International Antigenic Typing System serotype 6) could account in full for the protective capacity of extract-6. Comparative analysis of LPS heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining indicated that both extract-6 and LPS-A possessed similar spectra of smooth LPS molecules, containing between 10 and approximately equal to 50 O-antigen repeating units. Differences in the profiles of heterogeneity displayed by LPS in LPS-A and extract-6 were restricted to molecular species with short O-antigen chains. Subfractionation of LPS molecules on the basis of number of O-antigen repeating units was achieved by gel filtration in the presence of deoxycholate. Protection experiments performed on the subfractionated species of LPS-A revealed a relationship between O-antigen chain length and protective capacity; molecules with over 18 O-antigen repeating units being 50 to 100 times more protective than those with zero-two repeating units. The results indicate that most of the protection afforded by LPS-A and extract-6 can be accounted for by LPS molecules possessing extended (10 or more) O-antigen repeating units.

Effect of toxic shock syndrome toxin 1 on chicken embryos.

Staphylococcus aureus strains associated with toxic shock syndrome produce toxic shock syndrome toxin 1 (TSST1). This toxin has a variety of biological effects, including enhanced lethality in rabbits in the presence of sublethal amounts of lipopolysaccharide (LPS). Because chicken embryos are highly susceptible to LPS, the synergistic effect of TSST1 and LPS was examined in this system. Although TSST1 per se had no effect on chicken embryos, it potentiated the lethal effect of LPS. The 50% lethal dose of LPS was greatly reduced in the presence of up to 10 micrograms of TSST1 per ml. However, at high doses of TSST1 (greater than 100 micrograms/ml), no enhanced lethality was observed. The lowest dose of TSST1 tested which potentiated lethality was 10 ng/ml.

The effects of laboratory animal diets on the potency tests of bacterial vaccines.

Comparative studies of the responses elicited by mice fed on PCD and FFG diets to a number of bacterial vaccines have shown a significant reduction in the immune response to tetanus toxoid but not to Clostridium septicum toxoid and increased resistance to challenge with E. coli and syngeneic tumour cells but not to Pasteurella multocida. These differences cannot readily be explained in terms of differences between the identifiable constituents of the diets and illustrte the dangers of vaccine potency tests that require an absolute level of response to the material under test.

Development, preparation and safety testing of a Clostridium welchii type C toxoid. II. Laboratory evaluation of potency.

The results obtained with four laboratory tests on four candidate formulations of Clostridium welchii type C vaccine for use in man have been compared with clinical responses to the same vaccines. Quantal response assays in mice appeared to reflect the ranking of the four vaccines in human subjects better than did the guinea pig tests. They also enabled the potency of the vaccine preparations to be related to an existing International Reference Preparation. Mouse assays in which the animals received two spaced doses of vaccine prior to challenge yielded marginally more satisfactory results in terms of precision and reflection of human responses than did assays involving a single dose of vaccine.

Experience with the quality control testing of C. parvum suspension for clinical trial.

The control of C. parvum suspension presents special problems. The intended application of this preparation is in a therapeutic area where clinicians are accustomed to using defined chemical entities the biological activity of which are usually predictable according to weight of active principle administered, the route and number of doses. But the essential character of C. parvum is that of a vaccine in which the character of the active principle or principles is not clearly identified and can vary according to certain conditions. The control of the preparation therefore depends on the nature and relevance of tests for biological activity. These problems are exacerbated by uncertainty as to which of several possible mechanisms of antitumour activity might prove relevant in clinical practice. Experience with these problems as they have affected the quality control of large numbers of batches of C. parvum suspension prepared for clinical trial, will be discussed.

Studies on the toxicity of a variety of human vaccines as measured by laboratory tests.

The results obtained from our laboratory tests for pyrogenic activity on 8 bacterial vaccines are compared with the clinical reactogenicity of the same 8 vaccines in man. It is concluded that, although none of the tests correlate exactly with clinical reactogenicity, the rabbit pyrogenicity test provides the best indication of such activity. The limulus test, although valuable as a means of detecting contaminant endotoxin in pyrogen free preparations appears to be of little value as a gauge of pyrogenicity in bacterial vaccines.

A comparison of pyrogenicity and related properties seen in a suspension of Corynebacterium parvum and a gramnegative organism.

When the pyrogenic properties of C. parvum and a gramnegative organism, S. typhimurium, were compared it was found that the onset of the pyrogenic response to C. parvum was delayed relative to that of S. typhimurium and that secondary responses rarely occurred. In further experiments the interaction of C. parvum and S. typhimurium was studied. Although synergism was not demonstrated, the kinetics of the response to mixtures of the two vaccines was anomalous while pre-treatment of rabbits with C. parvum resulted in the elimination of the secondary pyrogenic response to S. typhimurium suspension. The implications of these results are discussed in terms of the known characteristics of endogenous pyrogen, lipopolysaccharide and peptidoglycan.

The assay of clostridial vaccines in mice.

The proposed establishment of a multicomponent clostridial standard vaccine inevitably raises the question of how such a preparation might be used in the standardization of batches of vaccine. Traditional multipoint parallel line assays based on the antitoxin responses of rabbits and guinea pigs are likely to prove prohibitively costly, whilst simpler methods based on single dose levels of either vaccine fail to quantitate the relationship between test vaccine and standard.