DD253184A1 - METHOD FOR PRODUCING STABLE SALMONELLA VACCINE STEM WITH AN ATTENANCE TREATMENT ADJUSTED TO ANY OF THE INDUSTRY SPECIES - Google Patents

Abstract

The invention relates to a method for the production of live vaccines for farm animals from stable Salmonella typhimurium one-, two- and Dreimarker Impfstaemmen in any desired - on Maeusemodell between i. p. LD50102.5 to 10 7 to 107 (wild strain 101) germinating buds - attenuation levels adapted to the host species; by using in vitro non-responders or aspartic acid auxotrophy cloning different virulence reduction as a single-marker vaccine; in which a second and / or third attenuating mutation is incorporated when needed (and when asp-clones with reversion frequencies 10 7 are used), namely a first or colored marker (DD-WP 218 836 A 1 and DD-WP announced) -Stickwechselrift drift marker (eg, functionally modified RNA polymerase or gyrase DD-WP 155 294) for the purpose of increasing the stability (potentiation of individual Stabilitaeten) to values 10 14 or 10 21Verminderung or loss of potency for Impfstamm-Infektkettenbildung than Sequence of impaired outer membrane permeability (according to WHO definition: Attenuation marker) Sets the attenuation level to the respective optimal value.

Description

Field of application of the invention

The invention relates to a method for producing stable Salmonella vaccine strains with an attenuation level adapted to the respective host species for use as a live vaccine, the

- attenuation level of a host species matched Virulenzreduktion corresponds to - on the one hand the inapparent infection and on the other hand sufficient (but temporary) persistence of the vaccine strains in the host tissue as a condition for a stable immunity guaranteed and the mouse model IP LD ₅₀ values between> 10 ^{2 '5} until <10 ⁷ to> 10 ⁷ (wild strain <10 ¹ ) germs can be adjusted

- Stability (no reverse mutation to the wild strain under non-selective field conditions) is guaranteed by the use of in vitro irreversibly reverting single marker strains or at reversion frequencies <10 ~ ⁷ via the incorporation of further one or two independently mutagenic or virulence reducing mutations

and which are suitable as live-virus vaccines for the active immunization of livestock against salmonellosis.

Characteristic of the known state of the art

In the patents NL-OS 7810480; DD-WP 155294; DD-WP 218836A1; 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 forth in accordance with the latest WHO information (ICP / BVM 008 / (S) 6144 B, 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) is currently 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-altered permeability of the outer membrane, recognizable i.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 state of the art, including the gal-E mutation (Germanier), the Sm-id mutants (Likhoded, Shuster) and the aro ~ mutants (Stocker) can be found in the patents cited at the outset, the materials 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) and the following publications:

- Linde, K., ZbI. Bact. Hyg., I. Alb. Orig. A 249,203-214 (1981),

- Linde, K., ZbI. Bact. Hyg., I. Alb. Orig. A 249, 350-361 (1981),

Linde, K., Wonitzki, Ch., Randhagen, B., ZbI. Bact. Hyg., I. Alb. Orig. A 250, 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, April 1986, IX ISSN 0043-8502, 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 (not all host species) or arootrophy, whose degree of attenuation depends on in vivo metabolite deficiency, and gal-E mutants which undergo galactolysis in vivo as a result of UDP-galactose accumulation, allow only the production of highly attenuated, in the host only slightly proliferating or insufficiently persistent and therefore the cellular immunity temporally shorter than possible stimulating Einmarker strains. This applies in particular to S. typhimurium pur "Live vaccine mutants for use in calves where - due to the complete absence of non-phosphorylated purines - such mutants are near the limits of overattenuation The introduction of a second virulence-reducing marker to guarantee WHO Safety or stability orders, usually leads to Überattenuierung.

2. Although the use of the anti-epidemic hst marker mediates an interruption of infection chains (according to the WHO definition at the same attenuation marker), the simultaneous bile sensitivity requires on the other hand with oral immunization a ~ 10 times higher dosage, which at doses> 10 ¹⁰ germs / Vaccination may be associated with the risk of endotoxic side effects and higher costs, so that there may be a need, instead of the hst marker to avoid the bile-tolerant colored revertant (WP-registered) or set a lower attenuation altogether.

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, in fact, unlimited possibilities for producing two- or three-marker vaccine strains in any desired - i. 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-saturation in two- or three-mark vaccine strains by using pure (not valid for all host species), aro ", gal-E mutations, which confer a high level of attenuation, usually excluding marker couplings, to the single-marker outbred strain.

2. Avoiding the (at least theoretically) disregarding the use of metabolic drift mutations (at least theoretically) disadvantages of only indirectly possible stability of the individual markers, by incorporating at least one (or with additional use of anti-epidemic markers of two) with direct methods for stability testable markers (n ) into the potential one, two or three 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 live vaccines in any desired, d. H. appropriate Attenuierungshöhe adapted to respective host species and (usually) with reduced potency for Impfstamm-Infektkettenbildung set out which or in which

- Prevents mutations that, like pure (not for all host species), aro ", gal-E mediate a pronounced virulence reduction and thus leave no or only too little attenuation reserve for a second and third attenuation marker

- When using the additional hst or colored markers, the vaccine strains are characterized by a factor increase of <10 ~ ⁷ which enhances the stability and an anti-epidemic potency

- in the case of an insufficient attenuation level of the potential S. tm single-marker vaccine strains without or with additional hst or colored markers (two-marker vaccine strains) in these an additional testable only for stability by indirect method, the total Attenuierungshöhe on the optimal value raising mutation is incorporated, so that stable balanced attenuated two- or Dreimarker vaccine strains arise.

This object is achieved according to the invention by isolating stable, in vitro undetectable or <10 ~ ⁷ reverting asp ~ clones with different attenuation heights from S. tm wild strains and, if necessary, introducing a second and possibly a third marker into these subsequently. which interferes with the potency of infectious chain formation, or optimizes the attenuation level, to produce stable, immunogenic one, two or three marker vaccine strains that are used for live vaccine production using per se known methods. The asp "markers presented below (different genotypes with different levels of attenuation) as such, or in combination with other anti-epidemic and metabolic drift mutations we have developed, have proven advantageous:

- aspartic acid auxotrophy with (clone- or genotype-dependent) weak, moderate or high attenuation Only three clues of a general character are found in the literature.

Bacon et al .: Brit. J. exp. Path. 31, (1950), 714-724 Three asp ~ mutants of Salmonella typhi are moderately attenuated for mice

Bacon et al .: 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 was 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 attenuation levels presumably (mistaken) notions: asp "single-marker vaccine strains 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 (see Table 1 page 12)

These nine tested asp ~ clones can be subdivided under reservation into at least three attenuation groups:

Group I: ip LD ₅₀ mouse ~ 10 ² ' ³ germs = 1 strain

Group: ip LD ₅₀ mouse-10 ⁵ germs = 4 strains

Group: ip LD ₅₀ mouse ~ 10 ⁷ germs = 4 strains

Wild strain: 1.P-LD ₆₀ MaUSSiO ¹ germs _t

these three groups being mutually combinable by means of transduction-the experiment was carried out with the clones asp "23, asp" 12 and asp "15 (see Table 1, page 12) -using the phage P ^{22 to the} prototrophs. stable asp "deletion mutants) or multi-marker vaccine strains (for the purpose of modifying the attenuation level and / or introducing an antiepidemic potency and increasing stability) with a level of attenuation adapted to the respective host species (ie the different susceptibility of the host species to Salmonella typhimurium or other pathogens) Therefore, it is possible to use corresponding asp "clones with different levels of virulence reduction-such as, for example, asp" 23, asp "10 or asp" 15 (see Table 1, page 12). The construction of two- and three-marker vaccine strains using the clone asp "10 described in addition to the isolation of stable asp" single-marker vaccine strains of different attenuation levels applies mutatis mutandis to all asp "clones (genotypes) with a lower, equal or higher degree of attenuation ,

- Additional markers:

• hst mutation (DD-WP 218836A1) or reversion bile tolerance Revertantedes hst marker for selective gall tolerance (patent DD-WP registered).

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

• metabolic drift mutant For example, the RNA polymerase or gyrase (DD-WP 155294), which clone (genotype) -spezifisch specifically the degree of attenuation of the asp "single-marker vaccine strains or the asp" hst or colored two-marker vaccine strains (see above) on desired, ie Host species adapted to increase total attenuation height (see under examples).

Use of asp "deletion mutants (theoretically possible repair of the defect in vivo but precluded under non-selective field conditions) as single-marker vaccine strains, as well as coupling of recurrent asp <10 ~ ⁷ with antiepidemic and / or metabolic drift -Markern (see above) obtained vaccine strains for use as live vaccines, the

- are stable as asp "deletion mutants under non-selective field conditions or have an overall stability of <10" ^u or <10 ~ ²¹ in the case of two or three-mark strains with reverting mutations,

- are immunogenic as a result of a host species-adapted balanced attenuation, and

- when using an antiepidemic 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 transmission capacity.

The listed below asp "single marker vaccine strains (reversion in vitro <10" ⁸ or not detectable), and two- and three-marker vaccine strains (using markers with a reversion frequency of <10 ~ ^7), such as. Salmonella typhimurium (S. tm)

-S.tmasp "23 ip LD ₅₀ mouse 10 ² ' ³ germs

- S.tmasp "10 ip LD ₅₀ mouse - 10 ⁴ · ³ germs

- S. tm asp "15 ip LD ₆₀ mouse - 10 ⁶⁷ germs

and multi-marker mutant constructed by asp "cloning at this attenuation level, such as" Stmasp "10 / h dysfunction ip LD ₅₀ mouse -10 ⁵ germs

- S.tmasp "10 / color 19 ip LD ₅₀ mouse ~ 10 ⁶ germs

- S.tmasp ~ 10 / hst / rif10 ip LD ₅₀ mouse-10 ⁶ · ⁵ germs

- S.tmasp "10 / hst / rif24 ip LD ₅₀ mouse-10 ⁷ germs

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

embodiments Used strain

Salmonella typhimurium (S. tm) wild strain 415 (Institute of Sera and Vaccines "Metschnikov", Moscow); i.p. LD ₅₀ mouse <10 ¹

breeding grounds

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

- Minimal medium (mm)

Modified (Falkow et al., J. Bacteriol 84.1303-1 312 11962]) M9 saline (Daviset Minigioli, J. Bacteriol. 60, 17-28 [195O]) with or without 2.5% watering Agar and 0.5% dextrose. The minimal medium is used as such for propagation of the S.tm wild strain

- Substituted minimal medium

Minimal medium plus 100 g aspartic acid (Reanal, Budapest) / ml minimal medium

antibiotics sensitivity and of the wild trunk surfactants jitg / ml medium nalidixic 6.2 (Nevigramon: Hungary rifampicin 12.5 (UMB Bucharest) oleandomycin > 100 (Medexport, USSR) Sodium dodecyl sulfate: SDS - 40,000 (Serva, Heidelberg Sodium Deoxycholate: SDO - 20,000 (VEB Chem. Werk, Berlin)

animal experiments

- used mice

T6-18 gram ICR mice (VEB experimental animal production Schönwalde)

- Comparative testing of wild strain and mutants for virulence and immunogenicity: i. p. Application logarithmically graduated bacterial counts in 0.5 ml physiological saline at 10 mice each

• Virulence behavior: Determination of the order of death within 2 weeks

· Immunogenicity: challenge the immunization-surviving or tolerant mice ip on day 15 with wild-type (> 100 LD ₅₀ ) in 0.5 ml saline and determine the number of dying mice within 2 weeks

Testing the stability of the markers

- asp markers:

10 ⁸ and 10 ⁹ germs of the vaccine strains are spatulated on minimal medium and determined after 48 and 72 hours incubation at 37 ⁰ C the number of grown colonies

Stability requirement:

• For single-marker vaccine strains: reversion frequency <10 ~ ⁸ or undetectable in vitro

• For multi-marker vaccine strains: reversion frequency <10 " ⁷

- hst and colored markers

see DD-WP 231491 and DD-WP registered

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

example 1

Preparation of Asp "Mutants and Selection of a Suitable Clone: The isolation of asparagine auxotrophic phenotypes is carried out by methods known per se (MNG mutagenesis, penicillin screening and replica plating, analogously for spontaneous mutants and transposon mutagenesis).

The isolated in this procedure asp ~ clones were checked for stability and all the tribes with a Reversiohshäufigkeitzur prototrophy of <10 ^-8 tested for their virulence on ip mouse model. Table 1 below shows the number of stable clones and their virulence behavior, which is subject to reservations about at least three groups.

Table 1

Sialmonella Typhimurium: Influence of aspartic acid auxotrophy on virulence behavior; tested in two separate experiments on nine clones (genotypes with a reversion frequency s 10 ~ ⁸ on the ip mouse model

asp clone number ip LD ₅₀ ICR-MaUS experiment A germ number 10 * Trial B 23 2.0 2.5 10 12 14 4.3 4.6 5.5 4.3 4.3 5.5

15 6.8 6.5 13 6.5 7.5

16 7.0 7.0 18 7.5 7.5 11 8.0 7.0

Wild strain <1 <1

Aspartic acid auxotrophy with in vitro undetectable or <10 ~ ⁸ reversion to prototrophy such as'

- S. tm asp ~ 23 ip LD ₅₀ mouse -10 ² ' ³ germs

- S. tm asp "10i.p. LD ₅₀ mouse ~ 10 ⁴ ' ³ germs

- S. tm asp "15 ip LD ₅₀ mouse-10 ⁶ · ⁷ germs

are suitable as single-marker vaccine strains for the production of live vaccines.

For the production of the two- or three-marker vaccine strains described below under Examples 2, 3 and 4, for example, the clone asp ~ 10 was used from the group II-asp ~ mutant with a moderately pronounced attenuation height. Analogously, the same with asp Perform cloning of lower or higher attenuation to set the attenuation level matched to the particular host species.

Example 2

Preparation of the two-marker vaccine strains asp ~ / hst and asp ~ / colored by incorporation of the anti-epidemic markers in the asp "10

Single marker mutant:

In the S. tmasp "10 clone described above, as described in DD-WP 218836A1 or DD-WP, a hst or colored marker with a stability <10" ^{7 was} incorporated, whereby stable asp ~ / hst resp colored two-mark vaccine strains arise as

eg asp "10 / hst or asp" 10 colored with an overall stability of <10 " ¹⁴

Example 3

Incorporation of the third attenuating metabolic drift marker, a rifampicin resistance (RNA polymerase) mutation or nalidixic acid resistance (gyrase) mutation with additional attenuation, which on the mouse model shows the ip LD ₅₀ values of the S.

tm two-mark stem asp "10hst or colored by ~ 10 ⁵ germs on 10 ⁶ to 10 ⁷ germs raises:

As described in DD-WP 155294, rifampicin-resistant mutenants or nalidixic acid mutants were prepared from the above-described S. tm asp "10hst or colored groups, and the clones were read and checked on the ip mouse model which contained the ip LD ₅₀ Value of S. tm asp "lOhstbzw. colored twins of

~ 10 ⁵ germs around 1-2 log. Raised levels.

About the i. p. Virulence behavior and the immunogenicity of the Dreimarker vaccine strains prepared in this way and the precursor vaccine strains asp "10 and asp" 10 / asp ~ 10 / hst or asp "10 / colored oriented Table 2

Table 2

Comparative level of attenuation and immunogenicity of single-marker vaccine strain S. tm asp "10, two-marker vaccine strains S. tm asp" 10 / hst, and the three-marker vaccine strains S. tm asp "10 / color 19; asp" 10 / hst / rif 10 and asp "/ hst / rif 24 on the ip mouse model

S. typhimurium Three- ip LD ₅₀ i.p. Immunis. i.p. Belastg. on the 15th day On Two mouse With with> 100 LD ₅₀ marker tribe -10 * germs 10 * germination conservation value

asp "10 asp" 10 asp "10 asp" 10 asp "10 asp" 10

Wild strain Contr.

hst colored beer

nal19 rif 10 rif 24

4.3 5.0 5.0 6.0 6.0 7.0

-90% -95% -95% -95% -95% -95%

0%

The two- and three-marker vaccine strains listed in Table 2 are suitable for the production of live vaccines, as examples of further multi-marker vaccine strains constructed in the same way with other asp) starting clones (equal, lower or higher attenuation).

Example 4

Preparation of S. tm asp "/ Metabolism Drone Two-marker vaccine strains for use as a live vaccine A suitable metabolic drift mutation is incorporated in a suitable asp" clone (without anti-epidemic markers) analogously to Example 3.

(It is also possible, although purely academic, to obtain such two-marker vaccine strains by experimentally reverting the antiepidemic marker to wild-type tolerance from a Three Marker vaccine strain described in Example 3.)

Example 5

Separation of the single-marker vaccine strains S. tm asp ", the two-marker vaccine strains S. tm asp" 10 / hst or colored and the three-marker vaccine strains S. tm asp "10 / hst or rbt / rif or nal 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

Salmonella typhimurium vaccine strains Nutrient agar (NA) NA with 50 ng nalidixins. or 100 g of Rifampic./ml Na with 60 μg oleandome / ml Mini times medium One-marker asp " + 0 + 0 Two-markers asp "/ hst or colored + 0 • 0 0 asp "/ rif or nal + + + 0 Three-markers asp "/ hst or rbt / rif or nal + + 0 0 wild strain + 0 + +

Examples

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 as liquid doses). The vaccine thus obtained is usually administered as a single dose of 10 ⁷ to 10 ¹⁰ live germs to farm animals orally (possibly parenterally).

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

- Use of a slightly lower attenuating vaccine strain, or

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

Claims (6)

1. A process for the preparation of stable Salmonella vaccine strains with an appropriate host species adapted Attenuierungshöhe, characterized in that one uses the virulence-reducing marker aspartic acid auxotrophy (asp ~) as such, or in conjunction with anti-epidemic and / or metabolic drift mutations, and the thus using immunogenic vaccine strains prepared using methods known per se for the production of live vaccines. 2. The method according to item 1, characterized in that are isolated by per se known methods asp ~ phenotypes and stable in vitro not or <10 ~ ⁸ reverting to prototrophy clones of different Attenuierungshöhe such. Clones (genotypes) with an ip LD ₅₀ mouse ~ 10 ² ' ³ or ~ 10 ⁵ or ~ 10 ⁷ (wild strain <10 ¹ ) germs, used as single-marker vaccine strains. 3. The method according to item 1 and 2, characterized in that indicated in item 2 asp "clones (and those with a reversion frequency to <10 ~ ⁷ ) a hst- (DD-WP 218836A1) or colored (DD-WP registered ) Marker is built so that two markers vaccine strains arise with a - anti-epidemic (no or reduced potency for vaccine strain infection chain formation) potency - ip LD ₅₀ mouse of -1O ³ , ~ 10 ⁵ and ~ 10 ⁷ and • - by the factor <IfJ "" ⁷ increasing potency. 4. The method according to item 1,2 and 3, characterized in that in Zweimarker vaccine strains prepared according to point 3 -according to DD-WP 155294, DD-WP 235828A1, DD-WP 218834A1 - Metabolic drift mutations with little or moderate (additional) Attenuierung be incorporated so that Dreimarker vaccine strains with an anti-epidemic potency arise, which On the mouse model all attenuation levels between ip LD ₅₀ > 10 ⁴ to <10 ⁷ to> 10 ⁷ include germs and - have an overall stability of at least <10 ~ ²¹ . 5. The method according to item 1, 2 and 4, characterized in that in asp 2 clones (and those with a reversion frequency up to <1CT ⁷ ) according to point 2 in accordance with item 4, an additional, the Attenuierungshöhe modifying, metabolic drift mutation is incorporated, so that , "Zweimarker vaccine strains without anti-epidemic markers arise, which On the mouse model all attenuation levels between ip LD ₅₀ > 10 ⁴ to <10 ⁷ to> 10 ⁷ include germs and - have a stability of at least <10 ~ ¹⁴ . 6. The method according to item 1, 2, 3, 4 and 5, characterized in that the following specified selection of one-, two- and three-mark vaccine strains, which - as an example, all for all produced by the same principle and all desired Attenuierungshöhen comprehensive Vaccine strains with asp ~ markers alone or in combination with anti-epidemic and / or metabolic drift markers - used for the production of stable, immunogenic live vaccines.