[Preparation of stable Salmonella vaccine strains through combination of 2 independently attenuating markers with no limitation on growth].

Attenuation by only one single marker with no limitation on propagation (frequency of backmutation being less than 10(-7) will not provide sufficient safety against complications along with vaccination. (This notion, derived from both theoretical considerations and practical experience, does not rule out good stability of certain one-marker mutants under practice conditions.) Two independently attenuating markers with no limitation on propagation, however, do ensure full stability on account of potentiating single frequencies of backmutation (less than 10(-14)). The second attenuating marker must be measurable, and it must not be allowed to bring about substantive reduction in the one-marker mutant's immunogenicity. The following principle was conceptualised for the purpose of resolving the problem: A pool of attenuated highly immunogenic mutants with one single marker without limitation on propagation was rendered available, such as an S-form auxotrophic set of phenotypes (attenuation by co-mutation) or R-form mutants with the potential of tissue persistence. An attenuated second marker then was rendered available which was highly immunogenic as a "one-marker mutant", for example, mutants with adenine (purine) dependence with attenuation a pleiotropic effect and limited availability of metabolities. The second marker was introduced in a one-maker mutant, with verification of additional attenuation along with remaining immunogenicity. The following results were obtained: --Experimental parameters for attenuation and immunogenicity in mice: one single intraperitoneal immunisation, using 10(5), 10(6), 10(7), and 10(8) mutant germs; challenge on 20th day from intraperitoneal immunsation with about 100 LD50; lethality of controls 95 per cent or more; --S. typhimurium his-155/ade-4 and S. dublin met-91/ade-23: LD50: 10(8) germs; Immunogenicity: 90 per cent or more survived exposure to wild strain; --S. cholerae-suis R Dessau/ade-4: LD50: about 10(9) germs; immunogenicity: about 50 per cent only within endotoxic limits, about 10(8) germs or more; --S. typhimurium his-155/marker 2 (enzyme mutation): LD50 about 10(8) germs; immunogenicity: 90 per cent or more survived exposure to wild strain.