DD253182A1 - METHOD FOR PRODUCING STABLE SALMONELLA IMPF STEMS WITH A HUMAN ADAPTED OPTIMAL ATTENUATION HOUSE - Google Patents

Abstract

The invention relates to a process for the preparation of stable Salmonella typhi vaccines with a man-adapted, optimal attenuation, realized in the present case over three- and four-marker vaccine with graded virulence reduction - by using in vitro not (or 10 7) revertierenden aspartic acid - auxotrophic clones with - in the present case - moderate attenuation; in which two and three further low to moderately attenuating mutations are incorporated, specifically: a hst marker or colored marker (DD-WP 218 836 A 1 and DD-WP registered) and one or two metabolic drift markers (functionally modified RNA Polymerase, gyrase, etc.) for the purpose of increasing the stability (potentiation of individual stabilities) to levels 10 21 and 10 28reduction or loss of potency for vaccine strain infective chain formation as a result of impaired outer membrane permeability (according to WHO definition: attenuation marker) adjustment of attenuation level to the optimum value for humans.

Description

Characteristic of the known state of the art

In the patents NL-OS 7810480; DD-WP 155294; DD-WP 218836; DD-WP 235828A1; (Method for producing live vaccines with two or more independently virulence-reducing markers: pure and metabolic drift mutations) and DD-WP 218836A1 and DD-WP filed (method for producing stable and epidemiologically epidemiologically safe live vaccines) has been

- on the one hand, the state of the art set out in accordance with the latest WHO information (ICP / BVM008 / [S] 6144B, July 1981) and the recommendations of the symposium "Enteric infections in man and animals: Standardization of immunological procedures", Dublin 1982 ( in: Developments in Biological Standardization, Vol. 53, S. Karger, Basle) ζ.Zt. is still valid;

- On the other hand, proposed principle solutions for the production of vaccine strain mutants that meet the safety requirements of the WHO by coupling two or more markers and by using an anti-epidemic marker.

The high immunogenicity of such potential vaccine strains is due to their residual, below clinical threshold multiplication in the host and the associated sufficient but temporally limited persistence in the tissue. Their epidemiological-epizootological safety is based on mutation-induced permeability of the outer membrane, recognizable and unavoidable. a. a macrolide antibiotics and surfactant hypersensitivity, which causes the germs in the outside world to die progressively, so that infection chains usually can not be achieved.

Details of the prior art including dergal-E mutation (Germanier), the Sm-id mutants (Likhoded, Shuster), and the aro mutant (Stocker), including the recently reviewed S. typhi aro ~ / pur ~ / (his ~) mutants can be found in the patents cited above, the materials of the symposium "Enteric infections in man and animals: Standardization of immunological procedures", Dublin, 1982 (in: Development in Biological Standardization, Vol.53, S.Karger , Basel) and the following publications:

- Linde, K., ZbI. Bact. Hyg., I. Alb. Orig. A249, 203-214 (1981),

- Linde, K., ZbI. Bact. Hyg., I. Alb. Orig. A249,350-361 (1981),

Linde, K., Wonitzki, Ch., Randhagen, B., ZbI. Bact. Hyg. I.Abt. Orig. A250,478-489 (1981),

Linde, K., Arch, exper. Vet. Med., Leipzig 36, 657-772 (1982),

Linde, K., Wonitzki, Ch., Beer, J., Randhagen, B., Keller, H.-P., Z. ges. Hyg. 30, 141-147 (1984),

- Levine, Myron, M., WORLD HEALTH, Diarrheal Diseases, April 1986, p.24-26,

- WHO / CDD / 86.15 Diarrhoeal Disease Control Program p. 12-13

- WHO / CDD / 86.16 Control of Diarrheal Diseases p.71-73.

The principle solutions cited above as the prior art for the production of live vaccine mutants have the following disadvantages and their causes:

1.) Pure mutations "(does not apply, for example, to humans in whom sufficient amounts of non-phosphorylated purines are present in the serum or arootrophy, whose degree of attenuation depends on in vivo metabolite deficiency, and gal-E mutants in which there are no In vivo, as a result of galactose UDP-galactose enrichment, only the production of highly attenuated, low-proliferating or insufficiently persistent host cells, and therefore cellular immunity, can be stimulated for a shorter time than one-marker strains. pure typhimurium "

Live vaccine mutants for use in calves where, due to complete absence of non-phosphorylated purines, such mutants are at the limit of overattenuation. The introduction of a second virulence-reducing marker to guarantee the WHO safety and stability orders usually results in over-attenuation.

2.) The use of the anti-epidemic hst marker mediates on the one hand an interruption of infection chains (according to WHO definition at the same time attenuation marker), the simultaneous bile sensitivity, however, on the other hand requires a higher dose in oral immunization, which at doses 2:10 ¹⁰ germs / Vaccination may be associated with the risk of endotoxic side effects and higher costs, so that u. It may be necessary, instead of the hst marker, to avoid the bile-tolerant rbt revertant (WP-pending) or to set a lower attenuation level overall.

3.) Using the principle of virulence reduction via metabolic drift mutations (mutations in essential enzymes and metabolic compartments: selection via resistance to antibiotics and other noxious agents), there are unlimited possibilities for the production of two- or three-marker vaccine strains in any desired d. H. optimal optimal overall attenuation level, and thus high immunogenicity, adapted to the respective host species. A conditional disadvantage of this solution, however, is that the stability test of such markers is possible only indirectly through the detection of the unchanged resistance and Attenuierungsparameter (after> 10 nutrient media and / or animal passages); (a drawback, however, which is only academic in the use of two or more drift mutations due to the potentiating single stabilities).

Object of the invention

The aim of the invention is to bring about the removal of the following disadvantages of the known technical solutions:

1. Prevention of over-attenuation in three- or four-marker vaccine strains by using aro "and gal-E mutations, which give the single-marker parent strain a high degree of attenuation, which, as a rule, precludes marker coupling.

2. Necessary abandonment of the use of the pure "marker, in which the virulence reduction

- questionable and only in low altitude as a result of disturbed feedback errors occurs, however

Ostensibly based on the host species specific concentration of non-phosphorylated purines, so that, for example

• in calves with complete absence of the now essential growth factor a high attenuation results, while

• in humans and pigs with sufficient amounts of this "growth factor" at best, a low on feedback disorder induced virulence reduction is to be expected, and

• Mice in whom this marker mediates moderate attenuation as a (in any case limited meaningful) attenuation test system for S. typhi strains with pure markers.

3. Avoidance of (at least theoretically) the exclusive indirect use of metabolic drift mutations, by the use of at least one (or additional use of anti-epidemic markers of two) markers that can be tested for stability by direct methods into the potential (two), three and four marker vaccine strains.

Explanation of the essence of the invention

The object of the invention is to provide a method for producing stable, immunogenic Salmonella typhi live vaccines with Vi antigen in a man-matched, optimal Attenuierungshöhe and (usually) with reduced potency for Impfstamm-Infektkettenbildung, which or in which

- avoids mutations that like

• aro "and gal-E mediate a pronounced reduction in virulence and thus leave no or only too little attenuation reserve for a second, third and fourth attenuation marker;

• at best cause only slight attenuation in humans, but interfere with the testing for attenuation in the mouse model as markers in S. typhi;

- using the additional hst or rbt marker, the vaccine strains are characterized by a factor of <10 ~ ⁷ potentiating increase in stability as well as an anti-epidemic potency;

- in the case of the use of low or moderate attenuation clones with or without additional anti-epidemic markers (two-marker vaccine strains) in these one or two (more) additional - only by indirect methods for stability testable - the total attenuation height to the optimal value-raising mutations be incorporated, so that stable optimally attenuated three- or four-marker vaccine strains arise.

This object is achieved in that the Salmonella typhi wild strain stable, in vitro undetectable or <10 " ⁷ reverting asp" clones are isolated with different Attenuierungshöhe and introduced into a clone with low or moderate attenuation two or three more markers which interfere with the infectious chain formation potency and optimize the attenuation level to produce stable immunogenic three- and four-marker vaccine strains that are used for live vaccine production using methods known per se. The following asp "markers (different genotypes with different attenuation levels) have proved to be advantageous (usually) in combination with other anti-epidemic and virulence-reducing metabolic drift mutations we have developed:

- Aspartic acid auxotrophy with (clone or genotype dependent) low, moderate or high attenuation. The literature contains only three references of a general character.

• Baconetal .: Brit. J. exp. Path. 31, (1950), 714-724

Three asp 'mutants of Salmonella typhi are moderately attenuated for mice

Baconetal .: Brit J. exp. Path. 32, (1951), 85-96

Injection of aspartic acid lowers the lethal dose of S.typhi asp in mice

• Herzberg: J. Inf. Dis. 110, (1962), 192-203

Of 7 Salmonella typhimurium asp ~ mutants, 1 clone is virulent for mice, 3 (apparently) low and 3 moderately attenuated.

A use of this by the aspartic acid auxotrophy mediated attenuation has not been described, since the

• reported levels of attenuation probably mediated (mistaken) beliefs: asp ~ single-marker vaccine strains are too virulent

• Linking the markers for the purpose of increasing stability and increasing attenuation only for a few years now and for the first time for us to discuss in a practice-relevant way;

• high reversion rates of most mutants (despite - as our own results show - retention of attenuation = obvious suppressor mutants, comparable to the reversion of streptomycin dependency to independency) and the reported occurrence of virulent asp "clones

obviously prejudiced: use of this attenuation principle unsuitable for the production of live vaccines.

We found that asp ~ mutants of Salmonella typhimurium-tested nine clones with a reversion frequency of <10 ~ ⁸ - decay into a spectrum of strains with varying degrees of virulence reduction (DD-WP applied), these three groups inter-transducing are recombined using phage P ²² to prototrophy.

With conditional congruence, seven of our tested S.Ty 2 hst / asp "clones - with an in vitro undetectable or <10 ~ ⁸ amount of reversion to prototrophy - behave with regard to their persistence behavior in mice as well as the mean germ count / liver and splenic homogenate on the 5th day after infection with i · P-10 ⁶ nuclei (s. Table 1, page 14)

may include attenuation groups Number of tribes Persistence in days (± 1 day) mid-day5 (± 50%) wild strain ./. 19 10x10 ⁴ S.ty2hst I ./. 1 17 15 6x10 ⁴ 5x10 ⁴ Il - 2 14 3x10 ⁴ 3 12 2x10 ⁴ III 1 5 4X10 ¹

For the production of three- and four-marker vaccine strains with anti-epidemic potency and an optimal level of attenuation adapted for the human (via metabolic drift mutations), therefore, corresponding hst / asp ~ clones, e.g. hst / asp ~ 6 - (see Table 1, page 14).

The construction of (two-marker strains using asp ~ clones without hst-markers), described in Examples, three- and four-marker vaccine strains using the clone hst / asp ~ 6 applies mutatis mutandis to all hst / asp ~ clones. (Genotypes) with lower, equal or higher degree of attenuation.

- Additional markers:

• hst mutation (DD-WP 218836 A1), incorporated here as the first marker, or colored revertant of the hst marker (DD-WP registered).

The stability of the above-mentioned asp and anti-epidemic markers can be determined by direct methods.

Metabolic drift mutants * z. B. RNA polymerase or gyrase (DD-WP 155294), which clone (genotype) specifically increase the Attenuierungsgrad the asp "monkey marker or hst / asp" Zweimarker strains to the desired, man-adapted, optimal Attenuierungshöhe ( see under examples).

* Equally advantageous is the use of appropriate clones (genotypes) with mutated z. Ribosome proteins (including streptomycin "resistance") or other chromosome "noxious" resistance. Such marker combinations, usable e.g. instead of deposited strain 4062 / I - for ease of distinguishing the different vaccine strains - were not listed among examples and deposit strains to show beyond doubt that different levels of attenuation were established by using different clones of the same (eg, gyrase) "resistance" phenotype can be.

Using asp "clones as well as anti-epidemic and metabolic drift markers (see above) with an in vitro undetectable or <10 ~ ⁷ reversion frequency, vaccine strains are obtained for use as live vaccines

- are stable under non-selective field conditions, since their overall stability in (two), three and four marker vaccine strains is at least (<10 " ¹⁴ ), <10 ~ ²¹ and <10 ~ ²⁸ , respectively;

- are immunogenic due to an optimal attenuation level adapted to the host;

- when using an anti-epidemic marker are not or less capable of vaccine strain Infektkettenbildung; so that the vaccine strains prepared according to this principle fully meet the WHO requirement for stability, immunogenicity and reduced transmissibility.

The following - from the two-mark strain hst / asp " ⁶ developed - three vaccine strains with graduated Attenuierungshöhe, namely a Dreimarker vaccine strain and two four-marker vaccine strains - which exemplify all produced by the same principle two-, three- and Four-marker vaccine strains of any desired attenuation level -

S.Ty 2 strains and vaccine strains Lyo number ip10 ⁶ germs / mouse persistence in days average.KZ on the 5th day wild strain 1466 19 10x10 ⁴ hst S.Ty2 colored 3 605 3874 17 18 6x10 ⁴ 8x10 " Dreimarker hst / asp "/ rif 4019 9 2x10 ² Four-marker colored / asp ~ / rif / nal colored / asp ~ / rif / nal18 4062 / I 4139 6 3 10 ¹ <10 ¹

are suitable for the production of highly immunogenic live vaccines according to known methods.

embodiment

Used strains

- Salmonella typhi Ty 2 (WHO) as wild strain (S.Ty 2)

- Salmonella typhi gal-E 21 a (Germani) vaccine strain

 This comparative vaccine strain does not have Vi antigen

breeding grounds

Nutrient agar and nutrient broth (dry medium, SIFIN, Berlin-Weissensee)

- Minimal medium (mm)

Modified (Falkowetal., J. Bacteriol 84 (1962) 1303-1312) M9 saline (Davis et Minigioli, J. Bacteriol 60 [1950] 17-28) with 2.5% watered agar and 0.5% Dextrose and 20 μg cysteine (Ferak, Berlin) and thiamine (Serva, Heidelberg) / ml minimal medium

 This Mm is used to propagate the wild trunk.

- Substituted Mm

Mm plus 100 μg aspartic acid (Reanal, Budapest) / ml Mm

 Antibiotic sensitivity

and the Wild Tribe

Surfactants ^ g / ml medium

Nalidixic acid 6,2

(Nevigramon: Hungary)

Rifampicin 12.5

(UMB Bucharest).

Oleandomycin> 100

(Medexport, USSR)

Sodium dodecyl sulfate: SDS> 20,000

(Serva, Heidelberg)

Sodium desoxycholate: SDO> 10000

(VEB Chem. Werk, Berlin)

animal experiments

1. Mouse persistence model as an indirect measure of attenuation

- 20-25 gram NMRI culture mice

- Detection of the temporal persistence of wild strain and S.Ty 2-derived mutants in mice, and maximum number of germs / liver-spleen homogenate on the 5th day after infection:

The mice are infected with 10 ⁶ (in more attenuated strains in addition 10 ⁸ ) germs of a 20 hours 37 ° C culture on Nähragar ip. Starting on the day after infection, 2 mice were killed daily. Liver and Mils taken under sterile conditions, triturated with diatomaceous earth, then added 10ml of physiological saline and then determined by Ausspateln of 0.1 ml of this stock suspension and logarithmic dilutions to Nähragar the colony (germ) number. The detection limit of this method is about 12 germs per liver-spleen homogenate. By averaging from <5 separate batches, the persistence duration (persistence curve) and the average bacterial count on the 5th day are determined, which correlates well with the persistence duration. The determined duration of persistence in days is the time at which most of the organ homogenates are sterile (although individual mice may already be "sterile" before or can still harbor a few germs after this time).

2. Chick embryo Test for the orienting demonstration of attenuation (following: Ketyi, Ann. Immunol., Hung. 9 [1966], 81-86)

10-12-day-old chicken embryos are infected intravascularly (via the free-prepared chorioallantoic membrane) with geometrically or logarithmically graduated bacterial counts of wild strain and mutants in 0.1 ml of physiological saline. After incubation, the death rate 12-15stündiger and from the LD ₅₀ is determined. Testing the stability of the markers

- asp "marker:

10 ⁸ and 10 ⁹ seeds of the vaccine strains are spatulated on Mm and determined after 48 and 72 hours incubation at 37 ⁰ C, the number of grown colonies.

Stability requirement: not detectable in vitro up to <10 " ⁷

- hst and colored markers

see DD-WP 231 491 and DD-WP registered

- Rifampicin (RNA polymerase) and nalidixic acid resistance (gyrase) see DD-WP 155294

example 1

Preparation of asp ~ mutants - in this case starting from an hst mutant - and selection of a suitable clone:

The isolation of aspartic acid-auxotrophic phenotypes is carried out by methods known per se (MNG mutagenesis, penicillin screening and replica plating, analogously applies to spontaneous mutants and fransposone mutagenesis). The isolated in this procedure asp "clones were checked for their stability and all the tribes with a reversion to prototrophy of <10 ~ tested ^eight of their persistence in days and maximum bacterial counts in liver-spleen homogenate five days after infection on ip mouse model. (For orientation, the ip LD ₅₀ MaUS was also determined).

Table 1

S.Ty 2 hst mutant: influence of aspartic acid auxotrophy on the persistence behavior in days, the maximum germ count in the liver-spleen homogenate on the 5th day after the infection (and the ip LD ₅₀ after 24 hours) in seven asp ~ clones with a reversion frequency <10 ~ ⁸ on the ip mouse model.

S.Ty 2 Persistence in days av. Concentration camp on the 5th day ip LD ₅₀ germ count Wildstamm hst colored 19 17 18 10x10 "6x10" 8x10 " 5x10 ⁶ 1x10 ⁷ 1x10 ⁷ hst / asp "11 15 5x10 " 1x10 ⁸ hst / asp ~ 16 hst / asp ~ 10 14 14 3x10 "2x10 * 2x10 ⁸ 1x10 ⁸ hst / asp "14 hst / asp ~ 13 hst / asp" 6 12 12 12 2x10 ⁴ 2x10 ⁴ 1X10 " 1x10 ⁸ 2x10 ⁸ 2x10 ⁸ hst / asp ~ 15 5 4X10 ¹ 1x10 ⁹

For example, the preparation of the three- and four-marker vaccine strains described below under Examples 3 and 4 used the hst / asp ~ 6 binuclear mutant with moderately pronounced (additional) attenuation by the asp "mutation, analogously with asp" clone or higher attenuation to set the optimal human attenuation level.

Example 2

Production of the hst and colored mutants by incorporation of the anti-epidemic markers in the S.Ty 2 (or in other mutants already derived from this strain).

In S.Ty 2, as described in DD-WP 218836 A1 or DD-WP logged, a hst or. colored stains with a stability of <10 ~ ⁷ , whereby this strain receives a reduced or lacking potency for infection chain formation (according to WHO definition: attenuation marker) and its stability in the total reversion frequency of the vaccine strain with the potentiating factor < 10 ~ ⁷ inflows.

The hst 6 clone used for the further construction of the two-marker strains and three- and four-marker vaccine strains according to Examples 1, 3 and 4 gives this strain, as Table 1 indicates (see above), an additional (parenteral) low attenuation, Visible by shortening persistence duration from 19 to 17 days. Switching (reversion) to the colored marker, on the other hand, partially eliminates this low level of virulence reduction.

Example 3

Incorporation of a third mildly to moderately attenuating marker as a metabolic drift mutation, ζ. Β. a functionally altered RNA polymerase, in the S.Ty 2 hst / asp "6 Zweimarkerstamm, which - in the present case, the persistence in the ip mouse model of Dreimarker vaccine strain S.Ty 2 hst / asp" / rif 9-and proportional thereto the average germ counts on the 5th day after infection - (from 12 days of S.Ty 2 hst / asp "6) shortened to 9 days:

From the above-described S.Ty 2 hst / asp "6 two-marker strain, rifampicin resistance mutants were produced by spontaneous mutation as described in DD-WP 155294 and the clones were read and checked on the ip mouse persistence model, which was an additional Shorten the persistence period by several days (see above and Table 2, page 17)

Example 4

Incorporation of a fourth mildly to moderately attenuating marker as a metabolic drift mutation, ζ. Β. a functionally altered gyrase in the S.Ty 2 hst / asp "/ rif 9 Dreimarker vaccine strain, which, as in the present case, the persistence in the ip mouse model of the four-marker vaccine strain S.Ty 2 hst / asp" / rif / nal - and proportional to this the mean germ counts on

5th day after infection - (from 9 days of S.Ty 2 hst / asp "/ rif 9) reduced to 3 days in clone nally and to 3 days in clone nal 18. From the S.Ty 2 hst / asp Three-mark vaccine strain were prepared as described in DD-WP 155294, by means of spontaneous mutation in two steps nalidixic acid resistance mutants with a resistance> 100jag nalidixic acid / ml nutrient agar and read those clones on the ip mouse persistence model and checked, which show an additional shortening of the persistence period of several days (see above and Table 2, page 1).

As Table 2 indicates, i.p. Mice model, the gradual increase in virulence reduction clearly demonstrated with the help of persistence behavior and maximum germ count 5 days after infection, while the chick embryo test merely provides guidance.

The listed four-marker vaccine strain S.Ty 2 hst / asp ~ / rif / nal 18 was deliberately selected in its attenuation level as slightly above the virulence reduction of S.typhi gal-E21 a (Germanier) (and may be used against clones with such Values are exchanged, which correspond exactly to those of the Germanier vaccine strain, or show a higher Attenuierung).

Table 2

S.Ty 2 wild strain with its directly derived single- and two-marker strains, as well as the three- and four-marker vaccine strains (with graded attenuation level) in comparison to the intemationl introduced vaccine strain S.typhi gal-E21a (Germanier): Persistence duration in days and mean germ count / liver-spleen homogenate on the 5th day after infection of the mice with ip 10 ⁶ and 10 ⁸ germs (as well as LD ₅₀ in the chick embryo test).

Salmonella typhi Ty 2 one-two-marker vaccine strains Three- Four- ip 10 ⁶ germs / mouse persist. KZ am in days 5th day 10x10 " ip 10 ⁸ germs / mouse persist. KZ am in days 5th day 100% * chick embryo germ count ip LD ₅₀ Wild Tribe Ty 2 19 6x10 "8x10 ⁴ 100% * 100% * 1-5 hst 6 colored 17 18 1x10 ⁴ 100% * 1-5 "1-5 hst asp "6 12 2x10 ² ~ 30% * 3x10 ⁴ 5-10 hst asp " rif 9 9 10 ¹ 12 2x10 ³ 100 colored - asp " rif NAL1 6 10 ¹ 10 3x10 ² not tested colored asp " rif 18 3 10 ¹ 8th 7x10 ¹ 10 ⁷ S.typhi gal-E 21 a 2 6 10 ⁴

Example 5

Separation of the S.Ty 2 three- and four-marker vaccine strains of wild strains:

The vaccine strains differ from wild strains as shown in Table 3.

Table 3

Separation of vaccine strains from wild strains by detectable or lacking growth on different nutrient media

S.typhi Ty 2 vaccine strains Nutrient agar 50, and so on Naïdixins. Nutrient agar with / ml 100 / ng rifampicin 60, ng oleandomyc. Mini times medium Dreimarker hst / asp "/ rif + 0 + © © ' Four-marker hst / asp ~ / rif / nal + + + 0 0 wild strain + 0 0 + +

Example 6

Mass culture, preparation and application of vaccine strains:

For the production of the live vaccine vaccine strains z. B. in a semi-synthetic nutrient medium at 37 ⁰ C until the end of the logarithmic phase in the fermentor bred. The bacterial suspensions are prepared as lyophilisate (or suitable coated form).

The resulting vaccine is usually orally administered as a single dose of ~ 10 ⁹ live germs.

Instead of two to three applications of a vaccine strain moving at the border to the overattenuation, the alternatives exist

- Use of a slightly lower attenuated vaccine strain, or

- Initial immunization with a vaccine strain and booster moving at the border of overattenuation after a reasonable time with a vaccine strain of lower attenuation level.

Claims (9)

T .. Method for producing stable Salmonella typhi (S. Ty 2) vaccine strains with a human optimal level of attenuation - realized by providing vaccine strains with graded virulence reduction measured by persistence behavior in mice (and LD ₅₀ in the chick embryo test) - characterized in that, starting from an aspartic auxotrophy (asp ~) clone with moderate attenuation in conjunction with an anti-epidemic marker and further one or two mild to moderately virulence-reducing metabolic drift mutations, set up three-and four-marker vaccine strains and these using known processes are used for live vaccine production. 2. The method according to item!, Characterized in that as the first (or subsequent) marker in S. Ty2 the hst (high sensitivity to surfactants: DD-WP 218836A1) mutation with the potency for selective reversion to bile tolerance (colored : DD-WP logged in), which mediates this strain - a reduced potency for infection chain formation (according to WHO definition: attenuation marker) A stability which increases by a factor of <10 ~ ⁷ - And often an additional, parenterally coming to bear low virulence reduction. 3. The method according to item 1 and 2, characterized in that isolated from the S. Ty 2 / hst single-marker (or wild) strain using known methods asp ~ phenotypes and a in vitro not or <1CT ⁷ for prototrophy revertierenden clone with moderate (possibly low or high) attenuation for the further construction of the three- and four-marker vaccine strains. 4. The method according to item 1, 2 and 3, characterized in that in the S. Ty 2 hst / asp "two-marker strain as a third marker a low or moderately attenuating _t z., B. RNA polymerase (metabolic drift) mutation - according to DD-WP 155294, DD-WP 235828A1, DD-WP 218834 A1 - is incorporated, so that a Dreimarker Impfstamm arises whose persistence in mice from 19 days (see Ty 2) shortened to eg 9 days and which has an overall stability of <10 ~ ²¹ . 5. The method according to item 1,2,3 and 4, characterized in that in the S. Ty2 hst / asp ~ / rif Dreimarker vaccine strain, which may still have too high residual virulence, according to point 4 as the fourth marker another ^. B. gyrase (metabolic drift) mutation is incorporated, are read from which clones with little or moderate additional virulence reduction, so that four-mark vaccine strains arise, of which the one with the highest attenuation corresponds to S. typhi gal-E 21 (Germanier) vaccine strain and the second or the further four-marker vaccine strains in their attenuation level between the triple marker vaccine strain according to point 4 and are the S. typhi gal E21 a and which have a total stability of <10 ~ ^28th S. Method according to item 1 to 5, characterized in that - according to the desired application concept - the hst marker is transferred to a colored marker (DD-WP registered). 7. The method according to item 1 to 6, characterized in that the order of the stepwise marker installation is changed as desired. 8. The method according to item 1,4 and 5, characterized in that also other metabolic drift mutations are used as the specified. 9. The method according to item 1 to 7, characterized in that vaccine strains are made with more than four markers using only low virulence-reducing mutations. 10. The method according to item 1 to 8, characterized in that the following specified selection of three- and four-marker vaccine strains - which are exemplary of all produced according to the same principle and all desired Attenuierungshöhen vaccine strains - used for the production of stable, immunogenic live vaccines become. Field of application of the invention The invention relates to a method for the production of stable Salmonella typhi (S.Ty2) vaccine strains with a man adapted optimal attenuation level for use as a live vaccine, the - Attenuierungshöhe an optimally adjusted virulence reduction corresponds - which on the one hand, the inapparente infection and on the other hand, the sufficient (but temporary) persistence of vaccine strains in the host tissue as a prerequisite for a stable immunity guaranteed - and which on the mouse model to arbitrarily shortened persistence (as an indirect measure of Attenuierungshöhe ) can be adjusted. - Stability (no reverse mutation to the wild strain under non-selective field conditions) is guaranteed by the use of in vitro undetectable or <10 ~ ⁷ reverting single-marker strains and the incorporation of two to three (or more) independently of each other's transmissibility or virulence-reducing mutations and which are suitable as immunogenic vaccines from live pathogens for the active immunization of humans.