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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.

A wide variety of bacterial vaccines is in various stages of preclinical and clinical development. These products range from whole killed or live attenuated bacterial organisms to purified proteins, peptides and plasmid DNA. Although preclinical strategies may be directed by a set of common guidelines focused on demonstrating safety and biological activity, the exact developmental scheme will depend on product-specific characteristics. In general, preclinical data should support the proposed clinical formulation and include detailed information on the source and quality of starting materials, manufacturing processes, characterization of bacterial seed stocks, potency, general safety, purity, and identity. Data describing product validation and testing may be appropriate depending on the type of product, e.g., genetic stability for recombinant constructs, details on inactivation or attenuation methods for organisms or toxins, demonstration of potency of combination products, and safety and toxicology studies of plasmid DNA vaccines or vaccines with novel adjuvants. The choice of dose, route, and formulation to be used clinically may be greatly affected by rigorous preclinical developmental strategies.

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.

A block in the common aromatic biosynthesis (aro) pathway makes E. coli, Salmonella, etc., exacting for aromatic metabolites; two of these, paraaminobenzoic acid and dihydroxybenzoic acid, are not mammalian metabolites. Bacteria needing substances not available in host tissues should be unable to grow there and so be non-virulent. Transposon-generated deletion and deletion-inversion mutations at aroA caused loss of mouse virulence in Salmonella typhimurium; e.g., LD50, intraperitoneal route, increased from less than 10 to greater than 10(6), and LD50 by feeding increased from ca. 10(5) to greater than 10(8). Furthermore, a single injection of 10(5) live aro- bacteria protected mice of a Salmonella-susceptible line, BALB/c, against challenge, 4 weeks later, with 5 X 10(5) virulent S. typhimurium (i.e., greater than 10(4) LD50); a single dose of the live-vaccine strain given i.p. also protected against challenge one month later by feeding 2 X 10(7) of a virulent strain. Mice vaccinated by feeding 2 X 10(8) live aro- bacteria were unaffected by feeding, one month later, 2 X 10(7) of a virulent S. typhimurium strain ( = ca 100 LD50). Intravenous injection of microparticulate silica did not reduce the mouse i.p. LD50 of an aro- strain, and 3 i.p. injections of cyclophosphamide, 4 mgm, caused only a small reduction. Thus the reduced virulence of the aro- strain does much depend on the integrity of host cellular defense mechanisms. Non-reverting aro- derivatives made from proven calf-virulent S. typhimurium and S. dublin strains are being tested at the School of Veterinary Medicine, University of California at Davis. None of 26 calves given aro- live-vaccine bacteria intramuscularly (usually 10(9)) and none of 8 calves fed ca. 10(11) live aro- bacteria, died or became seriously ill. Vaccinated and control calves were challenged by feeding 10(11) bacteria of a virulent strain, S. typhimurium or S. dublin. This challenge caused death of 14 of 16 non-vaccinated calves. The results, to date, in vaccinated animals indicate that one aro- strain of each of the two Salmonella species is effective if given as a live vaccine in two intramuscular doses.

For the preparation of meningococcal group C polysaccharide-tetanus toxoid conjugate the reactive reagent N-ethyl-N'-(dimethylaminopropyl) carbodiimide is used. The application of this reagent results in a number of stable linkages (viz. "peptide" linkages between the polysaccharide and tetanus toxoid, intrachain ester linkages in the polysaccharide component and binding of the N-acylurea derivative of the reagent) and less stable ones (viz. anhydride linkages). As a consequence of the reaction, the reagent is converted to a non-reactive urea derivative. The toxic properties of the reagent and of the converted reagent were studied. These properties do not contraindicate the use of the coupling reagent for the preparation of vaccines for human use. In addition analytical methods have been developed for the quantitative evaluation of the coupling reagent, the reaction products and for the N-acylurea derivative of the reagent and of the residual reactivity of conjugates for primary aminogroups. Although no test was performed for the assay of ester linkages in the polysaccharide component of the conjugate, evidence is presented that such linkages may be present. The results of the test for residual reactivity indicated a spontaneous rearrangement of linkages after the preparation of the conjugate. In addition the influence of the ratio of coupling reagent-to-polysaccharide and tetanus toxoid on antigenic and immunogenic activities of the conjugate was studied. An increase of the ratio resulted in a decrease of the antigenic activity of the polysaccharide component but in an increase of its immunogenic activity as to the induction of IgG antibodies to the polysaccharide. The immunogenic activity of the polysaccharide component correlated rather well with the antigenic activity measured in heterologous enzyme-linked immunosorbent assay using antibodies to both components.