DD155294A1 - METHOD FOR PRODUCING STABILIZED AND HIGH-IMMUNOGEN BACTERIAL VACCINATES - Google Patents

Abstract

Molecular biology suggests that resistance mutations to antibiotics and other drugs may be due to different mutations in the particular gene. Such different mutations lead e.g. in ribosomes, enzymes, Zelloberflaechenstrukturen to various conformational changes, which trigger more or less severe or specific metabolic disorders in such resistance mutants. The experimental findings indicate that the non-proliferation-limiting metabolic disorders in chromosomal resistance mutants (eg Salmonella, Shigella, Pasteurella multocida) cause the occurrence of a spectrum of strains with different degrees of virulence. In animal experiments, a large proportion of resistance mutants are both attenuated and highly immunogenic. Using the example o.g. Pathogens are presented with a principle solution for the production of balanced attenuated, highly immunogenic live vaccines. The introduction of a second or third independently attenuating marker results in stable potential vaccine strains under field conditions as a result of the increasing single frequencies of retum mutation rates. The use of chromosomal resistance mutants for the production of bacterial live vaccines has so far been overlooked, since under in vivo conditions attenuated resistance mutants are eliminated.

Description

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Title of the invention

Process for the preparation of stable and highly immunogenic live bacterial vaccines

Application; the invention;

The invention relates to a method of producing genetically stable, virulently-attenuated and highly immunogenic mutant bacteria useful as live vaccines for the active immunization of livestock in industrial animal production and of human against cyclic and other bacterial infections such as e.g. SaI-monellosen, Pasteurellosen and Shigellosen, suitable and therefore useful in veterinary and human medicine ·

Characteristic of the known technical solution;

The applicability of live vaccines in humans and livestock against viral diseases (DTPS 62648, 70344, 86905, 89207, 88169, 110516, 110517, 116474, 117250, 129805) and against bacterial diseases is known in principle.

Known methods for producing vaccines, especially against bacterial diseases, are based either on the inactivation (killing) of the antigen carriers by means of chemical and / or physical methods DTPS (I00739, 106660, 127010), on extraction of cell components or cell substances (enterotoxins ) (DTPS 83010, 93537, 93697, 109029, 111585); In contrast, the production of bacterial live vaccine substances is based on spontaneous or artificial attenuation of the virulence of pathogenic microorganisms while at the same time preserving the antigenic structures and thus the immunogenic effect in the macroorganism Disease pathogens from the group of Bnterobacteriaceae were attenuated by altering the erythrocyte structure by way of spontaneous mutation or mutagenesis in their virulence so that bacterial mutants with streptomycin dependency and defined auxotrophic phenotypes were obtained and described as potential live vaccines (DTPS 93618 , 115919, Linde et al., Acta microbiol.Acad.Sci.hung 21_. 11-27 (1974)) · A Lach part of the hitherto known production methods for bacterial live vaccines with a single attenuating Mark he exists in the ^ ^ ' lying stability of the virule nzattenuierten mutants that are used as live vaccines · In return mutation of the genetic marker, with which the Virulenzattenuierung correlated, therefore, the restoration of the original virulence and thus vaccine complications is possible · It has therefore been tried in various ways, genetically stable bacterial live vaccines against Enterobacteriaceae infections to obtain.

For example, antigenic structures (O antigens) are available from Salmonella {(Kiefer and Mitarb., Infect. Immun., JQ, 1517-1513 (1976), J. Gen. Microbiol. <J2, 311-324 (1976), J. Org. from

Shigellafji Levine & Liitarb., J. Infect. JJ36, 577-532 (1977j) for avirulent Escherichia coli recipient strains on the

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Pathways of conjugation and interspecific recombination have been transferred. Thus, a reverse mutation to the virulent origin is principally impossible. A disadvantage of these strains is that they give no protection by oral administration, since these hybrids lack the ability to penetrate into cells or "tissues and for intracellular multiplication * Recombinative cross-breeding of these functions and the additional uptake of virulence plasmids would de facto Salmonella or» It has already been proposed to produce mutants for live vaccines which simultaneously carry two or more independently attenuating markers. This guarantees stability of virulence attenuation under non-selective conditions of use, since the simultaneous spontaneous Reverse mutation in two attenuating markers due to the potentiating reversion mutations of the single marker

10 ^l xf ° »/ 10 = ^ 10 becomes extremely unlikely.

In the coupling of two or more mutually independently attenuating markers, however, there is the danger that instead of a balanced Attenuierung - which protects the Hutztier or · humans with a single oral or parenteral vaccine application against a lethal or · disease-causing infection- over-attenuation occurs, so that after a single vaccine application, adequate protection against infection is not stimulated ·

The following is known about the problem of attenuation and chromosomal resistance mutation:

Chroraosomal resistance mutants ζ · Β. to fight against antibiotics

.C -ίο

in the order of 1:10 to 1:10 ^ germs, i. in the natural mutation rate of bacterial populations, on (V / alter u · Heilmeyer: Antibiotics Fibula 4 · Edition, Thieme Stuttgart, 1975, p.24-) · Regarding the virulence behavior of such mutants can be found in the same standard work.

the summary notes that

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"There is no definite evidence that antibiotic-resistant bacteria generally show higher or lower virulence, pathogenicity, and contagiousness." Resistant germs are not more dangerous than sensitive ones of the same type, but they represent a difficult chemotherapy problem "(p.66).

- "The generation time of resistant germs to sensitive pathogens is usually prolonged" (2nd edition 1965 p.48 / 49).

Lebek (Internist, χβ, 416-427 (1975)): "In human pathogens, chromosome antibiotic resistance plays only a subordinate role." Apart from the rarity of such mutations, mutations are mutated In particular, multiple mutations, which are necessary, for example, for the tetracycline-resistance of β-hemolytic streptococci and methicillin resistance of staphylococci until a therapeutically relevant level of resistance is required, simultaneously lead to the loss of certain properties which are important for the survival of the pathogens.

Analogous conditions should also be present in the selection of adapted to unphysiological environmental conditions mutants where the strains as a result of mutative adaptation to the altered propagation conditions have lost their virulence for the macroorganism (Fechner, vaccinations in pets, Hirzel, Leipzig I964) ·

Further concrete indications concerning the occurrence of attenuated germs among the antibiotic resistance mutants concern

Streptomycin and Salmonella typhi-murium, where virulence reduction is concomitant with prolonged generation time (Hobson, J. Hyg ££, 322-333 (1957)). Likewise, the mutation or transduction of streptomycin resistance in Shigella can lead to virulence abatement (Iyeheva and Aleshkin, Zh.Mikrobiol. (Moscow) 1977/9, 114-118).

- Erythromycin and Salmonella typhi-murium, the attenuated strains having a prolonged generation time and altered morphology (Gamalaya and Levashov, Zhe Mikrobiol (Moscow) 1976/3, 96-99, 1978/3, 8-15.

Pusidic acid and Salmonella typhi-murium, with concomitant changes in morphology, antigenicity, biochemical activity and prolonged generation time (Gamalaya and Levashov, Zh. Mikrobiol. (Moscow) 1976/2, 78-81; 1977/2 , 117-120). "Noteworthy is the observation that virulent clones also occur among the resistance mutants and that attenuation with resistance is co-transduced.

- Rifampicin and Shigellen (Osada et al., Jap. J. Microblol., 17, 243-249 (1973)) and mycobacteria (Daddy and M. Carbol is S. et al., Milan., 5 / 353-361 (1971) )).

- Isonicotinsäurehydrozid and for guinea pigs virulence-reduced mycobacteria (Bärtmann in Meissner, Schmiedel, wedge: mycobacteria and mycobacterial diseases, Part III, Bacteriological foundations of chemotherapy of tuberculosis, Fischer-Jena 1975 p.37).

The epidemiological and clinical safety of the attenuated "resistance" mutants, or of those within

) of a bacterial population caused by mutation "resistance" -attached germs, is also from subsequent Lite-

^ 5 ratur hints can be seen:

The BCG vaccine strain is cycloserine resistant (Boisvert, Ann.Inst.Pasteur HI, 180-192 (1966); Rist et al., Rev. Tuberc. Pneumol. 21, 1060-1065 (1967)). Reports of complications with this vaccine due to this resistance have not been disclosed.

The mecillinam-resistant Bnterobacteriaceae which can be selected in vitro do not occur in the patient because these mutants succumb to host defense as a result of their extended generation time or are overgrown in the intestine by the normal flora (Anderson, Journal of Antimicrobiol Chemotherapy 2 (Suppl. B) 89 -96 (1977)).

Object of the invention

The aim of the invention is to eliminate the deficiency mentioned above by providing a method by which a live vaccine resistant to bacterial infections, stable under field conditions and highly immunogenic, can be used. Salmonellosis, Pasteurellosis and Shigellosis can be obtained, in which on the one hand the possibility of vaccination complication due to reversion to the Y / ildstamm by incorporation of two or more independently attenuating individual markers is switched off and on the other hand Überattenuierung is avoided ·

Explanation of the essence of the invention

The object is achieved in that from populations of highly virulent infectious agents spontaneously occurring or after mutagenesis resistance mutants are selected against drugs with different mode of action and with different sites in the microorganism, among which - due to the genetically different resistance phenotype leading mutations - in a high percentage virulence-attenuated clones in which the resistance marker simultaneously correlates with the specific conformational changes leading to attenuation. The sequential selection for genetically different drug resistance markers on the one hand represents the decisive prerequisite for the selection of mutants with two or more mutually independent, genetically different attenuation inhibitors. Markers and on the other hand offers the possibility of doubling by incorporating different strong attenuating markers or multi-marker mutants with balanced attenuation, which on the one hand exclude epidemiological risks or cause no vaccine complications and on the other hand already protect against the lethal or the disease-causing wild strain infection in a single oral or parenteral administration.

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, With this summarized attenuation principle, a balanced and stable attenuation of virulence is evidently to be achieved for all bacteria (and probably also for viruses).

Features of the invention

Ea has surprisingly been found that among those of extremely virulent microorganism species such as "B. Salmonella, Pasteurella or Shigella, by spontaneous mutation or mutagenesis-derived resistance mutants to antibiotics and other agents, in a high percentage of clones that were attenuated, elicited no pathogenic symptoms in the test animal, but in mice injected with these mutants. Guinea pigs, these mutants were administered locally to the eye) immunity against infections with the homologous highly virulent parent strain

This made it possible to obtain a live vaccine by means of the usual methods of breeding, which is characterized by stability, balanced attenuation and highly immunogenic action and does not lead to a vaccination complication.

According to the invention, it is the provision of a wide range of attenuation marker-carrying clones in which the mutation

- has led to a conformational change ζ · Β · of vital enzymes, ribosome proteins or other essential cell building blocks ·

- Phenotypically in the sense of a pleiotropic effect in a high percentage of isolated clones attenuation caused.

- Can be selected with little laboratory effort.

"easily" can be recognized, correlated with the attenuating measurable in animal experiments and the selection marker with attenuation by co-transduction or conjugative

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Gene transfer are transferable,

- as a result of remaining or sufficient residual functions as single-marker mutant or as double-marker (or multiple-marker) mutant, an increase in specific gravity below the clinical threshold value is permitted; so that by a single oral or parenteral immunization necessary for the stimulation of a stable immunity antigen levels in the / / Irtsgewebe and sufficient antigen persistence (as a prerequisite for the initiation of cellular immunity as the main factor of protection against cyclic infections: Collins, Bacteriol Rev., 38, 371-402 (1974)).

- after incorporation into a non-proliferative, attenuated, high immunogenic single marker (or double marker) mutant into double marker (multiple marker) mutants (with two (or more) independently attenuating markers), in which under non-selective conditions, a simultaneous spontaneous reverse mutation in both or several attenuating markers is eliminated as a result of the potentiating individual frequencies.

The vaccine strains which can be prepared by the process according to the invention have the following advantages:

- stability under non-selective practical conditions; Frequency of reverse mutation to wild strain (i.e., concurrent

Reversion in two or more markers) ^ 10 ·

- Non-proliferation-limited, balanced attenuation, which guarantees the reproduction below the clinical threshold value and sufficient tissue persistence in the experimental animal or human animal (or human) necessary for triggering a reliable immunity.

- Immunogenicity with usually single oral (local) or parenteral immunization against a lethal or disease-leading wild strain infection.

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- Recognizability and separation of the vaccine strain of homologous wild strains using the simplest laboratory methods by detection of the or the "chromosomal antibiotics or drug resistance markers", (and the marker of the parent strains).

 j

Thus, as the examples below show, virulence-reduced "resistance" mutants are primarily not resistant and thus dangerous infectious agents, but rather an infection triggering with harmless, attenuated mutants with functionally altered ribosome proteins, vital enzymes or other essential cell building blocks which according to the invention the resistance to antibiotics or other agents ₀ only serves as a marker for in vitro selection,

By the following examples with Salmonella, Pasteurella, and Shigella the universal usability of the ⁿ resistance is occupied amines ⁿ -Attenuierung for the generation of stable highly immunogenic Lebendimpfst, without limiting the invention so that "At the same time coated with the examples and examination findings, such as the prejudice has been overcome, that chromosomal resistance mutants pose general risks for therapy and epidemic spread.

The suitability of the resistance marker for the production of highly immunogenic live vaccines against salmonellosis, pasteurellosis and shigellosis is completely surprising

- since it is generally accepted that any chemobiotics resistance causes clinical problems, as virulent resistance mutants are selected under the selection pressure of the antibiotics in vivo and are responsible for refractory recurrence, the continuation of infection and epidemiological spread including hospitalism,

however, it is altogether unknown that certain resistance mutations lead to conformational changes to which attenuation is coupled and Cto-transduced with this

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- can be.

Exemplary embodiments:

material and methods

Wild strains used, antibiotics, susceptibility of wild strains, nutrient media:

Trunk parameter Salmonella dublin 81 (ITPJ) S.typhimurium 415 (Moscow) Past.multocida B 393 (ITFJ) Shigella flexn.2a (Gamalaya) IDocMS Eye ~ 1 ₀ 5 2 χ 10 ⁶ Resistance / ml erythromycin 50 50 1.6 lincomycin 400 200 12.5 oleandomycin 200 200 6.25 streptothricin 12.5 12.5 , 12.5 nalidixic 0.8 0.8 3.2 6.4 rifampicin 12.5 12.5 0.8 25

antibiotics

breeding grounds

Commercial preparations of SW and NSW streptothricin derivative 3890 B: ZIMET Jena Mhragar for pasteurelles with the addition of 10% mutton blood and Mhrbouillon (dry medium) SIFIN

example 1

· · The recovery of the mutants with

mutant ribosome protein, mutant DNA replication complex or

mutant DNA-dependent polymerase is done in the following way:

, Approximately one osseous bacterial material is cultured in several passages on nutrient media with increasing concentrations of the respective antibiotic, so that chroincsomele

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Select resistance mutants. The achieved resistance values in / uq / ml were added

Salmonella Pasteurelleri Shigella

erythromycin 4000 200 lincomycin 16000 100 oleandomycin 1600 50 streptothricin 100 100 Malidixinsäure M 600 200

400

Approximately 10 germs are rinsed on nutrient agar or "blood agar with rifampicin and after 48 hours in particular small resistant colonies are isolated.

The clones with chromosomal antibiotic resistance obtained as expression of conformational change in essential cell units were tested for their virulence behavior and immunogenicity after the passage of 10 Mhragar passages for the purpose of "checking the" resistance "stability in the animal experiment as described under 3.

The experimental data of single-marker "resistance" mutants with regard to virulence behavior and in attenuated clones with respect to immunogenicity are summarized in the

- Tables 1-5 for Salmonella typhi-murium (and S.dublin) - Tables 6-10 for Pasteurella multocida Carter type B tables for Shigella flexneri 2 «

The extensive data presented in Tables 1-11 are without prejudice to the special features of Salmonella's Pasteurella or Shigella and to the mutated essential cell component

as Grundgemeinsamkeit _s recognize that the "resistance clones ¹¹ disintegrated into a spectrum of strains with differentem virulence Mt varying frequency. always occur

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  - Attenuated clones:

The quantitative measure of the attenuation of virulence in these strains shows - visibly an example of "rifampicin resistance" and Salmonella, or from the stiob sample. The data given in the other "resistance mutants" - obviously related to the extended generation time as an expression of profound metabolic disorders,

- Virulents or clones with little virulence:

Such strains are causally responsible for overlooking the potential possibilities of vaccine strain selection based on "resistance attenuation".

Example 2 ί

Preparation of Stable, Balanced Attenuated Double Marker Mutantea by Way of Stepwise Selection:

In weakly attenuated single-marker mutants, e.g. Auxotrophic phenotypes (attenuation by co-mutation), adenine-dependent. Strains, mutants with defective glycerol kinase or "resistance" mutants (as described in Example 1, described and read as described in Example 3 · in animal experiments).

or weakly balanced attenuated double-marker mutants, e.g. Suppressor mutants as revertants of streptomycin-dependent or temperature-sensitive strains, or mutants with two genetically independent attenuating "resistance" markers

a second or further genetically differently effective "resistance" - markers - as in Example 1, described - incorporated. ;

As an independent variant, the incorporation of the ade "and / or gly-kin" "markers in suppressor mutants or" resistance "mutants (see above) is also appropriate.

The double-marker (multi-marker) clones thus obtained?

- as described under 3 · tested in animal experiments on virulence and immunogenicity, double marker (More.

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The initial strain and high immunogenicity remaining are available as stable, well-balanced attenuated vaccine strains after renewed nutrient agar passages and repeated animal experiments. "

The experimentally derived material from selected, stable, highly immunogenic double-marker (multiple) mutants with two (or more) independently attenuating! Markers in which a chromosomal resistance mutation as a second (or further) trait in a weakly attenuated or balanced attenuated single marker (double marker) mutant such as z _e B,. Auxotrophy phenotype (attenuation by co-mutation).

♦ Adenine dependence (attenuation due to reduced metabolite supply in vivo and pleiotropic effect).

»Revertant of Temperature Sensitive Mutant (Suppressor Mutant: Attenuation by Pleiotropic Effect)«

_# Attenuated chromosomal "antibiotic resistance" clone (attenuation by pleiotropic effect as a consequence of conformational changes in essential cell building blocks) · has been incorporated via the pathway of stepwise selection is compiled in the, or

- Tables 12-16 for Salmonella typhi-murium

 ; - Tables 17-18 for Pas expensive 11a multocida Carter type B - Table 19 for Shigella flexneri 2a.

Example 3

Testing for attenuation and immunogenicity: - Salmonella and Pasteurella: 18-20 gram ICR mice of both sexes (VEB Experimental Animal Production, 1291 Schönwalde)

, Screening for attenuation: 3e 10 mice receive ip ⁷ germs or log graded germ counts of 10 to

g g

(10) and are observed for 14 days for dying animals.

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• Immunogenicity testing: Surviving mice are i.p. on the fifteenth day after immunization. loaded with 1Cr wild strain germs (^ / 10O LDcq) and determines the number of dying animals until the 28th day.

Orientative assessment of virulence behavior

- "Immunization" with Salmonella

• 8-1ψψ / 10 mice: virulent

• 3-7Φ / 10 mice: weakly attenuated. 0-2% "/ 10 mice: attenuated

~ "Immunization" with Pasteurella

_• extended dying time: weakly attenuated

• single or all animals survive: attenuates Orientative assessment of immunogenicity

- Exposure of surviving (immunized) animals. - 70% Φ: lack of immunogenicity in

₀ 30-7OfS ^: weaker I once

• - 30% ^: high) immunization ^;

- Shigella: 250-300 gram guinea pigs

♦ Screening for attenuation: 3 guinea pig eyes each are infected with 4 χ 10 germs via a dropper pipette and evaluated for clinical symptoms in a differentiated manner, especially in the following three days.

♦ Determination of the elimination time: By daily vaccination of the eyes with serological controls of the cultures the duration of the germ excretion is determined. , Examination of immunogenicityj Approximately three days after the last isolation of the attenuated and weakly attenuated mutants from the eye, the infection challenge is carried out with 4 χ 10 wild strain germs and a subsequent clinical assessment of the eyes, especially the first three days.

Orientative assessment of virulence behavior

1 day 2 days 3 days 5-7 days

Virulent 1 2 3 3 weak atten. O 0-1 0-1 2 attenuated O O O O

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Orientative assessment of immunogenicity

2 days 3

full of protection O O O relative protection O. 1 1 no protection 1 2 2

0 = no reaction

1 = conjunctivitis, eye open

2 = strong junctivitis, incipient keratitis,

lie open

3 = purulent keratoconjunctivitis, eye closed

An assessment of the "resistance attenuation" on your suitability for the selection of highly immunogenic mutants as potential vaccine strains is possible on the basis of the available animal experiments, provided the information limits of the infection model are known.

Highly immunogenic attenuated or weakly attenuated monkey clones:

• Salmonella: Clear statements can be derived from single-marker resistance clones. Among the ribosome mutants (selection: oleandomycin, erythromycin, lincomycin and streptotiiricin), clones with mutated DNA replication complex (selection: nalidixic acid resistance) and strains with mutated DNA-dependent RNA polymerase (selection: rifampicin resistance) occur fully attenuated clones, which immunize against a lethal infection. That Such mutants with high immunogenicity or half -attenuated clones are suitable for marker coupling or for incorporation as a second marker into other highly attenuated high immunogenic single marker. Clones on. Theoretically, even defined single-marker clones, e.g. Salmonella typhi-murium olea 9 and 17, stthr 2 and 6, nali 17 and 21, rif 18 and 33 etc. as potential vaccine strains, since these and other markers Kloneη using indirect methods (Nähragar passages, selection of mutants however, immunized

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intercurrent or "at the" Impfseuchentod "dying mice) no Rückmu ations to the wild trunk could be observed. (Table 1-5)

• Pasteurella: The interpretation of the animal findings must j, consider that mice obviously have no defense mechanism against these pathogens and respond as a rule, in an infection with acute Seuchentod within 24 hours or · the pathogen eliminate without any appreciable to antibody formation leading in vivo Propagation (Linde, Arch. Esper. Vet.med. In press). From this point of view, the highly virulence-reduced lincomycin and erythromycin "resistance mutants" have proved to be overattenuated.

The attenuation as a consequence of an oleandomycin, streptophosphorus-j-nalidixic acid and rifampicin resistance is only recognizable by the prolonged dying time compared to the wild strain. (Table 6-10) The immunity of surviving mice, which is usually absent in the present infection model, does not speak against the suitability of these markers, since

• may have had a sub-lethal infection, which prevents the development of immunity due to severe organ changes

· In the case of surviving animals, the infection with the Hutante need not have been addressed, analogous to the conditions of an "immunization" with small numbers of virulent Salmonella bacteria, where the surviving animals did not undergo infection and consequently did not build up any immunity (Nakano and Saito, Nature"

222 , 1085-1086 (1969); own observations) *

Consequently, their suitability for the selection of potential vaccine strains can only be demonstrated when these markers are inserted into mutants that have been partially mutated (see below).

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Sbigellen: The occurrence of partially attenuated and fully attenuated nalidixic acid or rifampicin t resistance mutants with partial or full immunogenicity on the keratoconjunctivitis model suggests the suitability of these markers as a second characteristic in a double-label strain (Table 11).

Highly immunogenic stable dog-tag marker resistance mutants:

The material presented in extracts in Tables 12-19 demonstrates the suitability of attenuating chromosomal resistance mutations for the selection of stable highly immunogenic double-mutant mutants as potential vaccine strains which are usually subject to parenteral or oral (local) administration against a lethal or disease-borne infection challenge protect,

- Incorporation of an attenuating mutation in the 30 S ribosome protein (selection, streptothricin-dexviate 3890 B (ZIMET) resistance) as a second marker into the attenuated, highly immunogenic Salmonella typhi-murium single-marker mutants his ~ 155 (auxotropic phenotype, attenuation by co-mutation) or ade * 81 (adenine dependency, attenuation by reduced metabolite offer in vivo and pleiotropic effect) leads to stable highly immunogenic double-marker mutants,

• with one compared to the single-marker mutant by 1-3 log. Stages higher attenuation

• which the experimental animal mouse in immunization to the tested i.p. Germ number of 10 ^ - which is * 2 logs, levels below the fully tolerated dose -> 90% against lethal wild-strain infection,

whose additional attenuation is also suggested over a prolonged generation time compared to the single-marker mutant.

See Table 12

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- -. Incorporation of an attenuating chromosomal mutation in the DKA replication complex (selection: ITalidixic acid resistance) as a second marker into the attenuated, highly immunogenic single-marker mutant Salmonella typhi-murium olea 17 with mutated 50 S ribosomal protein leads to stable highly immunogenic double-marker mutants

• with a higher attenuation than the single-marker mutant umr-Ί log

, which the experimental animal mouse in immunization to the tested i.p. Germ number of ΊΟ - the ^ 2 log. Levels below the fully tolerated dose -; P 90% protect against lethal wild-strain infection See Table 13.

- The incorporation of an attenuating chromosomal mutation in the 50 S ribosome protein (selection: oleandomycin resistance) as a second marker into the attenuated high-immunogenic single-marker mutant Salmonella typhi-murium nali 21 with mutant DHA replication complex leads to stable highly immunogenic double-marker mutants

· With a higher attenuation than the single-marker mutant by / 1 log · stage

• which the "test animal mouse immunization up to the tested ip bacterial count of ΙΟ- ⁵ - the / ^ 2 log steps below the fully tolerated dose - protect against P 90% against the lethal wild-type strain infection

_• whose additional attenuation is also indicated by a slightly longer generation time compared to the single-marker mutant.

See Table 14.

- The incorporation of an attenuating chromosomal mutation in the 50 S ribosome protein (selection: oleandomycin resistance) or the DNA-dependent RNA polymerase (selection: rifampicin resistance) as the second marker into the weakly attenuated single-marker mutant Salmonella typhi-murium nali 2 with mutated DNA replication complex leads to stable highly immunogenic double-marker mutants

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with a higher attenuation than the single-marker mutant by ^ 3 log steps

which the experimental animal mouse in immunization to the tested i.p. Germ count of 10 - the / ^ i log - level below the fully tolerated dose -> 90% to protect against the lethal wild-type strain infection

• whose additional attenuation is also indicated over the extended generation time compared to the single-marker mutant »-

See Table 15.

- The incorporation of an attenuating chromosomal mutation of DNA-dependent RHA polymerase (selection: rifampicin resistance) as a second marker in the attenuated single-marker mutant Salmonella typhi-murium to "" i55 (auxotropic phenotype, attenuation by Eo mutation) leads to stable highly immunogenic double-marker mutants

• with one compared to the single-marker mutant by ^ 2 log ·. Stages higher attenuation

• the experimental animal mouse on immunization up to the tested ip · germ number of ΙΟ- ³ - the ^ 2 log steps

below the fully tolerated dose -> 90% to protect against the lethal wild-strain infection, as well as in oral immunization a measurable immunity to the; lethal infection burden

· Whose additional attenuation is also indicated over the extended generation time compared to the single-marker mutant. -

See Table 16.

- JSinbau an attenuating chromosomal mutation in the 50 S ribosome protein (selectionj Oleandomycin resistance) as a second marker in the attenuated single-marker mutant Pasteurella multocida B nali 6 (see Table 9) leads to stable highly immunogenic double-marker mutants

• with an additional significant attenuation compared to the single-marker mutant.

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which immunize the test animal mouse after a single ip immunization with a / ^ LD ^ j ^ ₅ to 95 ^ dose against the lethal challenge exposure

 See Table 17e

- The incorporation of an attenuating chromosomal mutation of DNA-dependent RNA polymerase (selection: rifampicin resistance) as a third marker in the attenuated double mutant Pasteurella multocida B 9b / 5 (revertant of a temperature-sensitive mutant = suppressor mutant) leads to stable highly immunogenic triple-marker mutants

• with an additional low-level attenuation $ compared to the suppressor mutant, especially when using i.p. 10 germs is recognizable (see Table 10)

"Immunize the experimental mouse with a single dose of i * p, immunization with a ^ LD _x .Q dose to ^ 90% against the lethal infection burden

 See Table 18.

- The incorporation of an attenuating chromosomal mutation of DNA-dependent RNA polymerase (selection: rifampicin resistance) as a second marker in the weakly attenuated single-marker muteins. Shigella flexneri 2a ade ~ 2 leads to stable highly immunogenic double-marker mutants. with one opposite to the single-marker mutant. Significant attenuation in the keratoconjunctivitis test with a single local inoculation ^ 80% of the guinea pig eyes against the wild-type infection causing a purulent keratoconjunctivitis.

See Table 19.

ίθ Example 4. -

Testing the frequency of reversion to the wild type

- Indirect stability test in random samples, which allows the statement: reversion frequency to virulence / * - * 10 '

Salmonella and Pasteuria:

\ 5> * 10 Nutrient agar passages with subsequent marker control are placed in front of the animal experiment. From individual

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immunized animals, which died intercurrent or on acute "Impfseuchentod", were isolated dip exciters from the liver and spleen and the markers and the attenuation checked, Eeversionen must not be observed in these samples.

• Shigella: pathogens isolated from the eye were checked for markers and attenuation * Eevisions should not be observed in these samples.

A highly selective laboratory test for the determination of the reverse mutation frequency to the wild strain, such as in the mutant types ade "" - ^ ade ⁺ or, gly-kin "* -> gly-kin ⁺ exists z _e Zt _# for the attenuated" resistance ^n- mutants Not. The indirect stability test is carried out on the basis of the experience with the marker adenine dependency. * In the case of this type of mutant and reversion frequency> 10 "* ', the isolation of virulent ade mutants succeeds only in a simplified manner.

5 7 rare cases from i.p. infected with 1Cr and 10 'vaccine strain germs and dying within 10 days of the "vaccine-death" mice. Mutagenisation of the potential vaccine strain immediately before the animal experiment and examination of the exciters isolated from the mice that died of "vaccine death" by means of "stamping technique" can indeed expect a high rate of reverse mutation, but likewise gives no exact data on stability under leaf conditions.

- Practice-relevant solution for the production of stable, well attenuated vaccine strains by a combination of markers, in which at least one attenuation feature can be determined exactly by means of highly selective laboratory methods on the frequency of reverse mutagenesisj

, Parent strain single-marker mutant with adenine dependency or the (weakly attenuated in Salmonella) feature gly-kin "*; Second- and / or third-party markers:" resistance "marker

♦. Starting strain: double-marker mutant with adenine dependency plus the (in Salmonella weak attenuation) feature gly-kin ""; Third-party markers: "resistance" markers.

Example 5

Absorbance measurements of broth cultures as an indirect measure of the generation time and putative virulence: from a 20 hours Salmonella Nähragarkultur a suspension in physiological saline solution and by means Spekol (C _e Zeiss Jena) at 650 nm, the absorbance of

0.07: 10 microbes / ml. This suspension was diluted 1: 9 in calibrated tubes with nutrient broth, shaken in a water bath at 39 ⁰ O (mouse body temperature), after 2 and 3 hours, the absorbance read at 530 nm and the percentage is set compared to the wild strain (= 100 %) ,

As an example of the correlation of proliferation rate and virulence behavior, the data for mutants of the DNA-dependent RHÄ polymerase are listed in Table 5.

Example 6

Evidence of co-transduction of attenuation and rifampicin resistance

as proof that the present mutant types are primarily attenuated strains in which the resistance serves merely as a selection marker by comparative indirect determination of the generation time by means of extinction measurements (see above) of wild strain, attenuated rif-r donor, Transductants and Randomly Isolated rif-r Spontaneous Mutants: Transduction Approach:

Donor: S. typhi-murium rif-r 1; Phage: P22 temp.

Recipient: S, typhi-murium wild strain 415 (Moscow); Ratio phage: Recipient = 10: 1; Reaction time: 15 minutes; Expression time: 3 to 5 hours; Selection: Nutrient agar with lOOAAsRifampicin / ml,.

2 O "/ b 4 U

Co-transduction of rifampicin resistance and attenuation in S. typhi Murium, detected via the same as the donor restricted multiplication velocity (absorbance • in% after 2 hrs. 39 ⁰ C shaking) and the analog virulence on ip · mouse model of transductants in Comparison to the wild strain and randomly isolated spontaneous resistance mutants

transductants Yirul.- spontaneous mutants Virul.- Tribe- %-Value Ve rh * #-Value   Married. number Extinkt · ν ir Extinkt. Wildst. 100 I "" "" att donor 36 att (Att) 1 28 att 67 (Att) 2 31 att 79 vir 3 31 att 84 vir 4 31 att 85 vir 5 35 att 87 vir 6 36 att 88 vir 7 36 att 89 vir .8th 38 att 91 vir 9 40 att 102 vir 10 41 106

20 7 640

Example 7

Separation of vaccine strains from homologous wild strains: The separation of the vaccine strain from homologous wild strains, which is necessary when using a live vaccine and is easy to carry out in every laboratory, is carried out by the detection of

- Antibiotic or drug marker

additional recognition or / and attenuation features of single-marker (double-label) outbound mutants, e.g. Amino acid auxotrophy, adenine dependency, (glycerol kinase defect)

Detection: growth on minimal medium only when substituted with 20 / ug of the corresponding amino acid or »purine / ml medium (or addition of 0.3% glycerol phosphate (no growth in glycerol) as sole carbohydrate and energy source), and is outlined in the table below ,

Separation of vaccine strains from homologous wild strains by growth of strains to be tested on differentiation media -

Starting strain auxotrophic, ade

Nutrient Agar

I active ingredient

minimal medium

I active Contr. Ι material

" _A gly-kin"

substifc.Mm.

' .Work-

prototrophic

Nutrient Agar

I WiPk- Kontr, fabric

vaccine

wild strain

I 0

Example 8

Mass culture, preparation and application of vaccine strains according to Table 20 and 22:

For the preparation of the live vaccine, the vaccine strains are e.g. in a semi-synthetic nutrient medium

-25- 20 7 6 40

37 ⁰ C until the end of the logarithmic growth phase in the fermentor ängezüchtet ..

The bacterial suspensions are prepared as lyophilisate (or as liquid doses). The vaccine thus obtained

7 10

is usually administered as a single dose of 10 to 10 live germs to the respective farm animals or humans orally (or parenterally).

20 7 6

Table 1 Salmonella dublin

Attenuation and immunogenicity of clones with mutated ribosome protein (selection: lincomycin or erythromycin resistance) on the i.p. Mice Model, Immunization 10 'Mutan

tenkeimej load 15 Tagz-'IO 'wild strain bacteria (corresponding to _e ^ 100)

Linco- Immunis. Belastg. mutant Φ / Ν 1 ;O 5.10 5 0 3.10 10 0 8.10 7 1 5.9 3 1 7.9 4 1 8.9 8th 2 2.8 2 2 6.8 9 3 5.7 3 4 2.6 Wi, -1O ⁷ 10 10/10 St. 10 ⁵ , 6 10.7 NaOl- Contr, 0 0/10

erythema Immunis. Belastg. mutant '£ / 10 Φ / N 10 0 0/10 8th 0 2.10 3 O 6.10 2 1 0/9 6 1 1/9 * 7 1 2.9 5 2 1.8 9 3 0/7 3 1.7 1 4 1.6 Wi-10 ⁷ 10 10/10 St. 10 ⁵ 7 8.10 NaCl Contr. 0 0/10

LincomyclJn-Resist./Ug/ml - Erythromycia-Resist./Ug/ml

- Wild Tribe: 400 - Wild Tribe: 50

- mutants: ^ j 16000 - mutants: / ^ 4000

Table 2 Salmonella typhi-murium:

Attenuation and immunogenicity of clones with mutated eibosome protein (selection: oleandomycin resistance) on the i, p, mouse model} Immunization 1O ^ or, 10 mutant germs, load on 15 · day 10 ^ wild strain germs (^ .100

Ees, jug / ml oil ea Mu tante Immunis, # / 10 Belastg, olea courage. ί · ρ · 10 * Immunis, 4 * / ff Load ff / h 32OO 9,17,23 O 0 / 1Θ 9 8 7 6 5 13/20 0/20 0/20 0/20 D / 7 1/20 0/20 0/20 32OO 1,2,7 O 1-2 / 10 17 8 7 6 5 30/30 19/30 12/30 - 2 / 2O_ 1/11 i / 18 - 1/27. 3200 3,4, 5,10 O 3-5 / 10 20 7 6 5 8/10 7/10 4/10 0/2 0/3 0/6 1600 6,13,20 1-2 0-2 / 9-8 23 8 7 6 5_ _ 7 6 5 4 9/10 7/10 _ l / io_ 10/10 4/10 3/10 1 / 1O _- 1/1 0/3 _. 3/2. 0/6 2/7 .0 / 2. 1600 15.22 4-5 0 / 6-5 Wi-St, 5 3 10/10 10/10 10/10 10/10 1600 8,14,16, 18,21 6-8 0 / 4-2 NaOl Kon ti O 0/10 1600 12.19 10 · /. 200 Wi, -1O ⁵ St, 10 ³ 10 10 10/10 10/10 JTaOl Kontr. • 0 0 / 1Ö.

Table 3

Salmonella typhi-murium:

Attenuation and immunogenicity of clones with a mutated ribosome protein (selection: streptothricin derivative 3890 B (ZIMET) resistance) on the ip mouse model Immunization with 1Cr "or 10 ^x mutant germs Load on the 15th day 1Cr ⁵ virus strain germs (^ 100

Res · stthr. Immunis. Belastg / Ug / ml mutant , $ / 10 h £ / H 25 2 200 6 50 7 100 11 0 0/10 200 13 200 14 200 15 50 5 25 9 0 1.10 25 200 1 6 1.4 200 3 1 2.9 200 4 0 3.10 100 8th 1 0/9 50 12 6 2.4 200 16 0 6.10 Wi ₄ -IO ⁵ 10 10/10 St. 10 ³ 10 10/10 FaCl- Contr. 0 0/10

sttht: courage. i.p. Immunize φ / IT Belastg Ü 8 7 6 21/30 6/30 0/30 0/30 0/9 0/24 2/30 0/30 6 8 , ξ ·· 6 5 18/20 3/20 0/20 0/30 0/2 0/17 0/20 0/30 7 8 7 6 5 7/10 3 / .10 3/10 1/10 0/3 1/7 1/7 0/9 1 8 7 6 5 10/10 6/10 2/10 2/10 0/4 0/8 0/8 Wild, tribe 5 3 10/10 10/10 10/10 10/10 HaCl- Contr. « 0 0/10

20 7 δ 40

Table 4 Salmonella typhi-murium:

Attenuation and immunogenicity of clones with mutated DNA replication complex (selection: nalidixic acid resistance) on the ip mouse model. Immunization 10 or 10 ^x mutant germs, load on 15 · day 10 ^ wild strain germs

Res. / J g / ml nali mutant Immunis. ψ / 10 Load f ^ / 10 i nali courage. i.p. - Immunis. ^ / U Belastg 1600 3200 6400 17 20 3,9,21 0 0/10 21 7 6 5 15/30 3/30 1/30 1/15 4/27 1/29 3200 10 5 2.5 17 7 6 5 20/20 6/20 1/20 -i 0/14 1/19 3200 2 7 3.1 9 8 7 6 5 7/10 5/10 0/10 0/10 0/3 0/5 0/10 4/10 3200 19 8th 0/2 10 6 5 10/10 6/10 0/4 6400 12800 18 5:14 8th 2.2 2 5 4 3 21/30 14/30 11/30 0/9 4/19 800, 1600 3200 6400 12800 4,6 7 11,15 16 8,12,13 10 Ji Wi-sn 5 3 30/30 30/30 30/30 30/30 Wi. -10 ^ St. 10 ³ 10 10 10/10 10/10 NaOl Kon. 0 0/10 NaCl Kontr. 0 0/10 •

Table 5 Salmonella typhi-murium:

Attenuation and immunogenicity of clones with imitated DNA-dependent RNA polymerase (selection: rifampicin resistance) on the ip mouse model. Immunization 10 ^ or 10 ^x mutant germs, load on 15 · day 1.Cr wild strain germs (^ -100

Percent value of the extinction of mutants compared to the wild strain after two hours 39 ⁰ C shake culture. Startkeim-

7 · number- '10' germs.

rif mutant Immune. •• £ / 20 Load ^ / N % Value Ext. rif- = ι ip courage.! iO ^x Immunis Φ / 20 Load $ / N rif 1 2 33 18 6 11 21 38 22 9 10 4 29 1 2 1 1 2 5 6 7 8 8 9 10 11 6/19 5/18 0/19 U / 19 0/18 0/15 0/14 1/13 1/12. 0/12 3/11 2/10 0/9 27 41 54 55 48 47 74 68 63 66 71 79 80 rif '8 ¹ 7 20 16 2 1 ./. 0/4 0/18 0/19 27 courage. 20 ./. > 81 rif I 8! 7 33! 6! 5 20 10 2 1 ./. 0/10 0/18 O / I9 Wi-10 ⁵ p. 10 ³ 20 20 20/20 20/20 100 wi.-! 5 con .; 3 20 20 20 20 NaCl Kontr. 0 0/20 NaCl ₁ ¹ Kon. 1. 0 0/20 Rifampicin-Resist./Ug/ml - wild strain: 12.5 - mutants:> 100

- 20 7 6 40

Table 6 and 7 Pasteurella multocida Carter type B: Attenuation and immunogenicity of clones with mutated ribosome protein (selection: lincomycin or erythromycin or oleandomycin resistance) on i.p. mouse model

mutant Immu 4v2 24 nisg. 0 M. 48 : 10 ^ 72 Germs St% 14d Belaj 24 3tg 15 mice 48 Day η η _β : 72 JiO ⁵ K. I Std. Σ * ™ Res. Μ g / ml line 1-7 line 8 8th 20 O 20 5-10 16-20 20 20/20 200 ery 1-10 O 6-14 20 20/20 200

olea 1 8th 19 20 7 14 19 9 1 1.1 - 100 olea 2 1 18 20 20 20 9 20/20 50 olea 3 7 17 16 20 20/20 100 olea 4 15 17 20 18 .18 0/1 100 olea 5 2 8th 20 50 olea 6 16 20 14 20 0/20 25 olea 7 7 Rough-form 20 100 olea 8 Robbery form 0 20 100 olea 9 0 20 100 olea 10 20 17 19 100 Wi.-1O ⁵ p. 10 ³ 20 20 12.5 1.6 6.2 NaCl Konti; 0

20 7 640

Table 8 and 9 Pasteurella multocida Carter type B: Attenuation and immunogenicity of clones with mutated ribosome protein (selection: Streptothricin derivative 3890 B (ZIMET) resistance) or with mutated DNA replication complex (selection: nalidixic acid resistance) on the ip _# mouse model

2 Immunisg. 0 M. * 10 germs Belastg.15. mice Day : 10 ⁵ K. Res " 3 48 η. Σ Hours. Φ / Ν 48 η. X hours / Ug / ml 4 24 18 72 14d 24 72 2 "$ / Ν 100 VJL 14 20 20 20 50 mutant 6 17 20 200 stthr 1 12 16 100 13 20 20 200 20 19 20 100 22 18 13 100 1 2 15 16 4 4.4 200 2 6 13 20 200 3 11 15 20 100 4 2 1 14 16 4 16 4.4 200 5 VJL 1 4 4 46 # 6 200 6 10 17 0/3 400 nali 7 2 4 16 1.4 200 nali 8th 5 17 18 3.1 200 nali "9 11 1 6 19 19/19 200- nali 10 4 6 15 20 200 nali Wi-1O ⁵ 1 17 20 20 200 nali St. 10 ³ 9 4 20 20 200 nali NAOL-K. 1 1 11 20 15 200 nali 1 VJI 3 15/15 12.5 nali 20 20 20 20 20 20/20 3.2 nali  18 20 16 20/20 0 0/20

2 0 7 6 4 0

Table 10 Pasteurella multocida Carter type B: attenuation of clones with mutated DNA-dependent RNA polymerase selection: rifampicin resistance) on i.p. mouse model

EZ 20 i £ / 10 mice to X £ 28 6 48 hours - 10 96 Res. mutant i.p. 6 24 72 9 10 ug / ml wild strain 10 4 8th 10 rif 1 2. 7 6 9 5 10 2 3 6 6 10 8th 6 10 3 2 3 4 10 4 2 3 1 10 10 IA VO 10 ⁵ 5 4 6 10 10 > 100 ί 7 2 1 5 7 10 8th 3 8th 7 10 10 9 3 2 8th 10 9 10 3 5 3 9 10 Sm-id 8/3 6 6 7 10 10 51 rif 1 1 2 7 10 2 2 2 2 10 9 10 10 3 4 10 ⁵ 4 5 1 ©. ^J 10 > 100 5 1 6 4 8th Sm-id 8/6 0 £ i rif 1 1 2 1 0 3 4 10 ⁵ 1 > 100 vn 0 6

Continued table concentration camp 20 10 ⁸ 7 s 10 Mice] after X - 4 φ / 20 mice 11 14 17 stood - 17 96 Res. i.p. 4 0 # / Io 28 y 48 6 6 3 6 7 72 8th 8th ug / ml mutant 10 ^ö 9 24 7 7 13 14 15 7 15 6 Tres 9! B / 1 1 0 6 3 2 2 3 4 3 6 rif 1 3 5 5 7 2 10 ⁸ 1 4 3 2 3 7 4 > 100 3 4 ro ro 4 8th 5 5 1 5 7 7 8th 6 Ί VO 8th 3 <1 Tres 9b / 5 1 7 2 4 rif 1 1 2 4 4 6 7 2 10 ⁸ 2 3 5 6 7 > 100 3 4 4 -3 7 3 5 6 1 2 6 1 17 9 <1 Tres 9b / 1 15 > 100 Tres 9b / 1 rif 3 5 <1 Tres 9b / 5 > 100 Tres 9b / 5. iif 1

- & - 20 7 6 40

Table 11 Shigella flexneri 2a

Attenuation and immunogenicity of clones with mutated DNA replication complex (selection: ITa lidixin acid e-resistance) or mutant DM-dependent RNA polymerase (selection: rifampicin resistance) in the keratoconjunctivitis test in guinea pig eye5 Exposure three days after the end of germination between the 1o. - 20th day.

N , The body &. Right eye 2 3 lay I, burden with ι ,-Germinate + XiO ⁶ 3 12 5 With 4x10 germs 1-2 0-1 2-ß 0-1 5-7 \ Wildest, to X ' 2XID ₉₅ 0 Mu Findings after X I 3 1 I findings 2 protrude 1 tant © 3 8 1 2 0 2-3 0-1 - Ul 1 0 nal r 1 O 2-3 3 3 2 I 2 I 0 0-1 j 3 Y ¹ o rif-r Ι Ο I i Wild trunk f 1 I I I

Findings: 0 1 2

= no reaction

= Conjunctivitis} eye open

s = severe conjunctivitis, incipient keratitis; Eye open '

= purulent keratoconjunctivitis; Eye closed

20 7 6 40

Table 12a Salmonella typhi-murium

Attenuation and immunogenicity of monolayer clone his ™ "155 (aux" ~~ phenotype, attenuation by co-mutation) and the double-marker clone his * "i55 / sfcthr 3 (selection: streptothricin derivative 389OB '(ZIMET) resistance) as a second attenuating marker on the i _e p · mouse model, immunization 1O ^X mutant germs on 15 days 10 strain of viral strain, (corresponding to ¹ ^ 100 LD ^ q) · percent of extinction of mutants compared to wild strain after 2 hours 39 ⁰ C Shake culture, starting germ count / ~ Ί Ο ⁷ germs

mutants i «p · 10 ^ Immunization / 30 Load factor / ir % 7 / ert Exfcinkt. Sese ug / ml his ~ 155 8 7 6 5 30 27 12 3 · / · 2/3 3/18 0/27 85 3.2 his "" i55 stthr 3 8 7 6 5 18 4 0 0 1/12 3/20 0/30 0/30 55 100 Wildest, control 5 3 30 30 30/30 30/30 100 12.5 UACL-Contr · 0 0/30

20 7 640

Table 12b Salmonella typhi-murium:

Attenuation and immunogenicity of the single-marker clone ade "" 81 (adenine dependency: attenuation by limited metabolite offer in vivo and pleiotropic effect) and the double-marker clone ade ~ "81 / stthr 3 with mutated em bibosome protein (selection: streptothricin derivative 3890 (ZIMET ) Resistance as a second attenuating marker on the ip mouse model Immunization 10 ^x mutant germs, load on 15 days 10 wild-type (? Ti00 LD ₅₀ ).

. Percent of the extinction of mutants in comparison, to the wild strain after 2 hours 39 ⁰ C Schiittelkultur, starting germ count n / 10 'germs

mutants i.p * 10 ^ Immunis. / 30 Load · S % Value Sinktin · E, μg / ml ade "81 8 7 6 5 19 3 0 O 4/11 2/27 1/30 2/30 88 3.2 good bye 81 8 7 6 5 1 O 0 O 2/29 0/30 2/30 4/30 • 60 100 Wildest, control 5 3 30 30 30/30 30/30 100 12.5 NaCl contr, 0 0/30

-33-2 0 7 64

Table 13 Salmonella typhi-murium:

Attenuation and immunogenicity of single-marker clone olea 17 with mutated ribosome protein (selection: oleandomycin resistance) and the DoppeImarker clone olea 17 / nali 2 with mutated DNA replication complex (selection: nalidixic acid resistance) as a second attenuating marker on i »p, Mausemodell; Immunization 10 mutant germs, load on the 15th day 10 ^ wild strain germs (^ 100 LD ₅₀ ) * Percentage of the extinction of mutants in comparison to the wild strain after 2 hours 39 ⁰ O culture of skins, starting bacterial count ΛΊ 0? Germs.

olea / nali mutant ! • Ρα 10 ^x Immune's $ 40 Load, φ / Ν % Value Sinkt. Balidix. Res »g / ml olea 17 8 7 6 4 40 27 18 3 O 1/3 1/22 2/37 1/40 65 3200 25 olea 17 nali 2 8 7 6 5 4 33 14 1 0 O 5/7 3/26 1/30 0/40 3/40 45 3200 1600 WiId, - tribe 5 3 40 40 40/40 40/40 100 200 0.8 NaCl control, O 0/40

-W- 2 0 7 6

Table 14 Salmonella typhi-murium:

Attenuation and immunogenicity of single-marker clone nali 21 with mutated DNA replication complex (selection: nalidixic acid resistance) and double-marker clone nali 21 / olea with mutated eibosome protein (selection: oleandomycin resistance) as a second attenuating marker on the ip · mouse model} Immunization 1O ^X Mutant germs, loading on 15. ^ ag 1O ^ wild strain germs (^, 100 LDc ₀ ^ Percent value of the extinction of mutants in comparison to the wild strain after 3 hours 39 ⁰ G shake culture · start germ count 'ViCr germs.

nali / olea mutant ip · 1O ^X Immunis. φ / 20 Belastg. φ / Ν % Value Extinct * Nalidix. Oleand. Res.ug/ml 21 8 7 6 5 18 7 1 O 2/2 1/13 1/19 0/20 68 6400 200 nali 21 olea 5 8 7 6 5 6 1 0 0 0/14 1/19 1/20 1/20 44 6400 1600 WiId.- tribe · 5 3 20 19 20/20 20/20 100 0.8 200 HaOl contr. Ό 0/20

20 7 6 40

Table 15 Salmonella typhi-muriumi

Attenuation and immunogenicity of monolayer clone nali 2 with mutated DNA replication complex (selection: Na lidixic acid resistance) and the double-marker clones nali 2 olea 11 or nali 2 / rif 10 with mutated ribosome proteia (selection: oleandomycin resistance) or mutated JDNA-dependent RNA polymerase (selection: rifampicin resistance) as a second attenuating marker on the i, p. Mouse model; Immunization 10 ^x mutant germs, loading on the 15th day 1O ^ monocytes (™ 100 UDc ₀ ) · percent d. Extinction of mutants compared to the wild strain after 2 h. 39 ⁰ C shaking culture; Start ^ K)? seed

ip · 10 ^x mutant Immunization »Φ / 30 Belastg. • S / ff % Value extinct nali rif olea Res.ug/ml 4 nali 2 3 2 14 11, 7 1/16 4/19 10/23 82 3200 12.5 -t © O 7 nali 2 6 olea 11 5 4 17 8 1 0 0/13 0/22 1/29 1/29 41 3200 400 7 nali 2 6 rif 10 5 4 22 9 1 0 1/8 1/21 0/29 0/30 40 3200> 100 Wildstu 3 contr. 2 30 25 ./. 5.5 100 0.8 NaCl control, 0 0/30

Air 2 O 7.6 4 0

Table 16 Salmonella typhi-murium:

Attenuation and immunogenicity of single-marker Klor} his ~ 155 (auxotrophic phenotype, attenuation by co-mutation) and double-marker clone his ~ 155 / rif 2 with mutant DHA-dependent RHA polymerase (selection: rifampicin resistance) as the second attenuating Marker on the ip and oral mouse model. Immunization 1O ^X Mutant germs , exercise on day 15 ip ΛΟτ or oral 10? Mutant germs (^ 100 LD ₅₀ ).

Percent d. Extinction of mutants in comparison to the wild strain after 2 h. 39 ⁰ C shaking culture, start ^ iO 'germs.

mutants i "p. 1Ο ^Χ Immunis. Φ / 30 • 00000 Belastg. Ψ / Ν % Value Extinct. Rifamp * Res.ug/ml his ~ 155 8 7 6 5 26 17 7 1 29 1/4 2/13 1/23 Ο / 29 85 12.5 his ~ 155 rif 2 8 7 6 5 12 1 O O O 3/13 I / 29 2/30 1/30 > 100 Wildst. EC control 5 3 30 30 3Ο / 3Ο 29 / 3Ο .. - 12.5 orally orally his ~ 155 rif 2 10 9 8 7 6 3/30 5/30 8/30 10/30 15/30 wild strain 7 30/30 Nagl-Kont, U / 30

2 0 7 6 4 0

Table 17 Pasteurella multocida Carter type Βϊ attenuation and inuminogeneity of the DoppeI marker clone nali 6 / olea 16 with mutant DUA replication complex (selection: uralidixic acid resistance) and mutated ribosome protein (selection: oleandomycin resistance) as second attenuating marker at i _# p * mouse model ·

mutant Immu with. i "p. 1O ^X germination Φ / Ή mice Exposure 15 · day ip 10 ⁵ WiIdst, -K * 5VlT immune _e mouse Res. / ug / ^m l nali 6 / +) olea 16 1O ⁵ 10 ⁴ 1O ³ 6/40 7/40 1/40 0/34 2/33 1/30 200 50 WiIdst. Contr. 1O ⁵ 1O ³ 30/30 30/30 30/30 30/30 3.2 6.25 NaCl control 0/30 0/30

+) single-marker mutant: see Table 9

20 7 640

Table 18 Pasteure 11a multocida Carter type B: attenuation and immunogenicity of the triple-marker clone tres 9b / 5 / rif 1, consisting of the double-marker mutant Tres 9b / 5 (revertant of a temperature-sensitive mutant = suppressor mutant) with mutated DNA-dependent RNA Polymerase (selection: rifampicin resistance) as the third attenuating and at the same time recognition marker on the ip mouse model

mutants Imm Φ / 1 unis, 00 ηε : 10 ^ ϊ me X Hours 14d 2 Mice 48 in n, - 72 SC Std. Res., Μ g / ml Tres 9b / 5 1 3 4 9 5 6 6/91 Tres 9b / 5 rif 1 1 3 4 19 17 2 4/96 > 100 φ Wi.-10 ⁵ J St. 10 ³ 18 20 20 20 20 20 20 20/20 20/20 _o NaCl- eontr. 1 1 1 1.19

see also Table 10: Absterbeordnung bei i.p. 10

mutant germs

20 7 6 40

Table 19 Shigella flexneri 2a,

Attenuation and immunogenicity of single-marker clone ade ~ 2 (adenine dependency: attenuation due to limited metabolite supply in vivo and pleiotropic effect) and double «

P marker clone ade 2 / rif 12 with mutated DNA-dependent ΉΈΑ- polymerase (selection: rifampicin resistance) as second marker in the keratoconjunctivitis test on guinea pig eye ©

Immune. : right eye "^ 2 3 0 = no reaction Load on Day 15 With 2 ) Days besinn 3 5 with 4x6 germs   1-3 L ² "" ³ 1 = conjunctivis, 4x1 O ^b WiId.- K. (2xID Ö 0 0 Findings after X days O 0 = 84% 1 = 16% 2 = strong Kc Findings after X days 0 = 84% 1 = 14% Eye open 0 = 86% 2 = 14% 0 = 86% 3 = 14% mutant 1 3 3 ι η .i nkt 5 1 3 -Vltis- 3  3 ade * ~ 2 O +) Germination: 10 - 2C 0 goodbye 2 rif 12 O findings: 0 WxId stem 1 1 end of Ke ratitis

3 =

Eye open

purulent keratoconjunctivitis, eye closed

sen

20 7 6 40

Table 20 Salmonella typhi-murium (S.tm)

Attenuated highly immunogenic single-marker mutants

Amp. Laboratory inspection 9 Client number 22 10 56 ZIMET 2253 S * tm olea 17 AF 22 10 57 B 445 2261 S.tm olea 2 AF 22 10 60 B 446 2269 "Tm stthr 6 AF 22 10 61 B 447 2273 S.tm stthr 17 AF 22 10 62 B 448 2245 S.tm nali 21 AF 22 10 63 B 449 2249 S.tm nali 18 AF 22 10 58 B 450 2519 S.tm rif 33 AF 22 10 59 B 451 2520 S.tm rif AF B 452

Table 21 Salmonella typhirnmrium (S _e tm)

.. ^: . Pasteurella multocida Carter. Type B (Past) j _: \ -. "'. ... -":. '': - ''} '_ Shigella flexaeri 2a (Shigemasa) Attenuated highly immunogenic or "weakly attenuated single marker 4iutanten (as well as suppressor = ditag mutants) as starting strains for ditag vaccine strains with two independent attenuating markers (or · as starting strains for the installation of a third attenuating marker, which also serves as a recognition marker)

Amp. Laboratory certification 155 81 Tribe Hummer 22 10 53 22 10 54 ZIMET 1770 1244 S.tm his "S.tm ade" * 17 21 2 6 AF AF 22 10 37 22 10 63 22 10 64 62 45 14 B 453 so. so. 2231 1830 S.tm olea Setm nali S.tm nali Past nali 9V5 2 AF AF AF AF 62 45 16 53 40 19 B 457 1533 65 Past Tres Shig bye "* AF AF B 459 B 460

2 O 7 6 Λ O

Table 22 Salmonella typhi-murium (S, tm)

Pasteurella multocida Carter Type B (Past)

Shigella flexneri 2a (Shig)

Stable, attenuated, highly immunogenic double-marker mutants (or suppressor mutants with a third attenuation marker and at the same time

Amp * Laborbeζeiclmung his ~ 155 81 sttfrr rif 3 Tribe Uummer 22 10 66 ZIMET 461 2546 S.tm ade "" 17 stthr 3 AF 22 10 65 B 462 2562 S.tm olea 21 nali 2 AF 22 10 67 B 463 2485 S.tm nali 2 olea 5 AF 22 10 68 B 464 2497 S.tm nali 155 olea 11 AF 22 10 69 B 465 2494 S.tm his " ' 2 rif 2 AF 22 10 55 B 466 1884 S.tm nali 6 rif 10 AF 22 10 70 B 467 2479 S.tm nali olea 16 AF 62 45 17 B 468 2316 Past Tr.es 9V5 rif 1 AF 62 45 14 B 469 1662 Past ade " 2 12 AF 53 40 20 B 470 70 Shigenobu AF B

Claims (5)

- 26 - 2 0 7 6 Invention claims: 1. A process for the preparation of stable and 'highly immunogenic bacterial live vaccines, in particular against SaI-monellosen, Pasteurellosen and Shigellosen, characterized in that a) chromosomal resistance mutants to antibiotics and other active substances are isolated from virulent starting populations of the infectious agents by spontaneous mutation or by known methods of mutagenesis be liert and t>) from the mutants identified under 1) on the appropriate animal or "infection model" weak to balanced attenuated high immunogenic single marker clone and either used as such for vaccine strains or c) in analogue or other cause weakly balanced to balanced attenuated one-marker or double-marker Liutanten be incorporated as starting strains. Method according to item 1, characterized in that single-label mutants, e.g. with chromosomal "resistance" attenuation, with purine dependency or (weakly attenuated for Salmonella) feature glycerol kinase defect can be used. Method according to item 1, characterized in that as starting strains double-marker mutants e.g. with two sub-type "resistance" attenuation markers, with purine dependence and glycerol kinase defect, or suppressor mutants. 4. Method according to points 1-3, characterized in that the vaccine strains obtained are multiplied by the usual methods of fermentative fermentation in mass cultivars and the resulting bacterial suspension is prepared as lyophilizate or as liquid doses for oral or parenteral application. 5. Use of vaccine strains obtained according to points 1-3 for the production of highly immunogenic live vaccines.