DE2530275A1 - Salmonella-coli hybrids useful for vaccination - produced by transferring 09.12 determinant salmonella chromosomes to a genetically related recipient strain - Google Patents

Abstract

Novel hybrid is characterised by having a surface antigen with a Kauffmann-White specificity of 09.12. New process for producing a stable microorganism utilisable as a live germ in a vaccine which is free from virulence and immunogenic comprises transferring the chromosome fragments of a Salmonella strain which are determined for 09.12 antigen synthesis onto a genetically related recipient strain and selecting the resulting hybrid with the desired metabolic efficiencies from the hybridisation mixt. Process for propagating the new hybrid by cultivating on a suitable medium is also claimed. The Salmonella-coli hybrid is useful for vaccination against Salmonellosis esp. Salmonella typhi infections. Results of tests described in the specification show that immunisation of mice with hybrid strain F 1193 (ATCC 31161) gives better protection against artificial infection than does vaccination with acetone-inactivated S. typhi Ty2. The pref. hybrid is Salmonella-E. coli hybrid ATCC 31161.

Description

Salmonella coli hybrids, process for their production and propagation and non-pathogenic live vaccine against salmonellosis. The invention relates to a reproducible hybrids or

Recombinants from Salmonella bacteria as donors of the antigen specificity the Salmonella serogroup D1 and Escherichia coli bacteria as recipients of these serological specificity, process for the production of the Salmonella coli hybrids, Method for propagating the hybrids and one to be administered orally and parenterally Vaccine against salmonellosis, especially against typhoid infections, which is considered essential immunizing component containing the new Salmonella coli hybrids.

It is known that the administration of killed typhoid bacteria leads to humoral immune reactions in test animals and humans, which is determined by determination are detectable by serum antibodies. However, it is also known that a there is no exact correlation between humoral immunity and protection against infection. Animal experiments have shown that there is essentially local and cellular immune mechanisms are important for active protection against infection and that these are preferably carried out by Live vaccine administration. The previously used or However, recommended live germs have a number of disadvantages. For example in streptomycin-dependent typhoid germs, a reversion to streptomycin independence and therefore pathogenicity cannot be ruled out. Also are in such a If long-term administration of streptomycin is necessary, which among other things causes a disadvantageous Change in the intestinal flora of the vaccinee occurs, the normalization of which occurs after the Vaccination can take a long time.

The system is also difficult to control and only in countries with feasible with a well-developed health service. In Canadian Patent No. 939608 is a Salmonella typhi mutant with a deletion defect in the Galactose operon described as a live typhoid vaccine.

It is a germ of weakened pathogenicity, however still with almost all the biochemical characteristics of S.typhi. It therefore arises still the task of finding or producing a stable microorganism, which is suitable as a living germ for the production of a particularly orally administrable Vaccine to be used and the essential property of which is to be to be free from virulence and have adequate immunogenic properties.

It has now been found that Salmonella coli hybrids which are suitable for Salmonella strains of serogroup D1 characteristic O-antigen combination 9 and 12 of the Kauffmann-White scheme, but otherwise behave like E. coli, are particularly suitable for the intended purpose.

The present invention accordingly relates to new Salmonella coli hybrids. They are characterized in that they are characteristic of the Salmonella serogroup D1 0 antigen combination 9 and 12 of the Kauffmann-White scheme, otherwise but behave like E. coli germs.

The invention relates in particular to the Salmonella coli hybrids, which has been deposited with the American Type Culture Collection under No. 31161.

The invention also relates to processes for producing such characterized Salmonella coli hybrids. The principle for making the hybrids consists in a genetic transfer of the chromosome fragments that determine O-antigen synthesis from a Salmonella donor possessing these fragments to an Escherichia coli recipient strain and the subsequent isolation of the hybrid or recombinant with the essential metabolic properties of Escherichia coli, however the antigen specificity of the donor. For one such genetic Transfer are basically 3 types of procedures, against the application of which there are no fundamental ones Concerns exist after having the fundamental possibility of having a successful performed conjugation procedure was demonstrated.

In conjugation, the donor bacterium mediates an essential Part of his information laid down in deoxyribonucleic acid on the one hand genetically related recipients. The transmission of such information requires direct contact of the cells with one another, from the donor to the recipient the chromosome-like deoxyribonucleic acid part in which the information is deposited, is transmitted from one cell to the other. So-called Hfr strains (High frequency of recombination) are particularly suitable as chromosome donors. They transfer chromosome markers to the recipients with a particularly high frequency. For the mutants to be produced according to the invention, an S.typhi -Hfr- 5 .typhi-Hfr strain used. The chromosome parts responsible for O specificity the salmonellae are referred to as rfb genes that are close to the chromosome ring the histidine operon. The polymerization for the O specificity responsible polysaccharidic units is controlled by the rfc gene. The problem posed according to the invention is, genetically, to identify the rfb and rfc gene regions of the Salmonella donor to a suitable E. coli recipient. Thereafter the resulting hybrid is to be selected from the crossbreeding mixture.

The selection of the hybrids can be particularly advantageous if a S.typhi-Hfr strain is used for the crossing, which has a special metabolic property owns. This can be streptomycin sensitivity, for example. The choice of the E.coli recipients depend on the desired metabolic performance of the Hybrids and according to the intended use of the hybrids as a vaccine, i.e. according to the desired Properties of the germ effective as an antigen.

For oral administration, a human is preferably used Intestinal flora isolated as recipient and apathogenic E. coli strain as recipient used. Having a hybrid based on the named recipient is one significantly longer residence time in the intestinal canal of the vaccinee. The accordingly long-lasting antigen stimulation leads to a high and long-lasting protection against infection. When isolating germs suitable as recipients from stool samples E.coli only accepts gram-negative germs which, due to the biochemical Differential diagnosis showing characteristics typical of E. coli species. This one E.coli 08: K: H, (K and H antigens are missing), corresponds to criteria which does not form H2S, does not grow on ammonium citrate medium, forms indole and Lactose ferments and thus differs significantly from other Enterobacteriaceae.

The production of the hybrids by the Konjngationsverfahren is made in such a way that in the usual way increased germs of the S.typhi donor with those of the E. coli recipient, expediently with a numerical excess of the Recipients are mixed with one another in a suitable aqueous nutrient medium, then the mixture is incubated at least until the specificity is 09> 12 determining chromosome fragment is transferred from the donor to the recipient. The incubation depends on the ascertainable frequency of the transmission of the part of the gene to be transmitted. When using an Hfr strain of S.typhi the incubation should be carried out for approximately 2 hours and longer. The temperature, which must be observed during the incubation is that which is also usually observed during the Breeding of Salmonella or Coli germs is observed, namely about 37 ° C. the The concentration of the Salmonella Hfr donor is between 10 3 and 10 10 / ml, preferably 5 x 10 germs / ml and that of the E. coli recipient at 10 to 10 10 germs / ml, preferably 5 x 108 germs / ml. This incubation is preferably carried out for 2-4 hours at 370C, without the Mixture is specially treated.

Following the incubation period, the hybrids from the Po, pulation selected by the donor and recipient. This is preferably done in a special Selection of the culture media made in which the growth of the hybrid is preferred, that of the donor or recipient is prevented. Such a selection is on Easiest to make when the selection of the donor and recipient on the the required selection of the hybrid takes into account; for example by donor and recipients themselves in terms of their metabolic performance in terms of usability distinguish between certain amino acids or carbohydrates and also a different one Show sensitivity to antibiotics.

The selection of the hybrids can therefore often be selective on the corresponding the properties of the germs deposited solid selective media on which only the hybrid shows growth and reproduction and of which it is after a certain Incubation period can be isolated. Then the hybrid can be put on a solid nutrient medium or propagated in a liquid nutrient medium in suspension culture. The increase the hybrids, which are made by inoculating a suitable one according to fundamentally known methods Culture medium and maintaining favorable growth conditions for the hybrid takes place, is also claimed according to the invention. After achieving a sufficient Germ density is the hybrid from the culture medium e.g. by centrifugation or Separated by filtration and recovered.

The germs are usually propagated in small flasks on a laboratory scale or on an industrial scale in a fermenter.

For example, if you have a streptomycin-sensitive S.typhi as Hfr donor strain used and a streptomycin-resistant E. coli recipient, who also has a defect in the histidine synthesis, a selective nutrient medium can be used which is designed as a minimal medium and contains streptomycin, the growth of both from donor colonies, because these are streptomycin-sensitive than also prevented by recipient colonies because they cannot produce histidine themselves will.

After incubation of the mixture of donor-recipient and hybrid on the streptomycin-containing agar plates for about 48 hours at 37 ° C the colonies then grown streptomycin-resistant recombinants, which the intact alleles of the donor for histidine synthesis that are isolated can.

The presence of Salmonella 09, 12 antigens in the recombinants is by a strong specific agglutination in a 09,12 antiserum as well indicated in a monospecific 09 or 012 factor serum. These recombinants are no longer agglutinable in an anti-E.coli-08 serum. That means that Donorgene, which are responsible for the 09, 12 antigen synthesis, not only in the recipient have been transferred, but that they also have that chromosome region in the recipient which contains the genes for the E. coli 08 antigen. They are accordingly haploid recombinants emerged, which are characterized by stability. Besides are reversions to pathogenic forms, as represented by the S.typhi donor germs, locked out.

In addition to the method described above for producing the Hybrids by conjugation can be used to transfer the corresponding chromosome fragments, namely the Salmonella rfb and rfc genes, into an E. coli recipient, also through a Carry out so-called transformation, with a corresponding fragment from the extracted isolated DNA from Salmonella on E. coli is transmitted.

Another type of procedure is called transduction, in which the transmission of the information responsible for the O-specificity and those who are considered important for the development of the corresponding antigens, done with the help of bacteriophages.

The hybrids obtainable by these processes become special advantageously used as an immunizing component for vaccines against salmonellosis. The vaccines are also the subject of the *) Both methods are described in "The Genetics of Bacteria and their Viruses" by William Hayes, Blackwell Publishing Scientific Publ., Oxford (1970) pp. 574-649.

Invention, in particular a vaccine against typhoid.

Particularly favorable properties in terms of usability the hybrids to be produced according to the invention as a vaccine, in particular as a vaccine, in which the living hybrid germs are to be applied orally, has the following combination, in which a S.typhi-Hfr strain with the designation WR4000 - deposited with the American Type Culture Collection under No. 31162 - and an E. coli strain as recipient the designation F 212 - deposited with the American Type Culture Collection under No. 31163 - with the characteristic features identified in more detail in the following table Features is used.

Table 1 Characteristics of donor and recipient strains serotype Auxotrophs sensitive.

Strain antigen loading marker carbohydrate against drawing required Growth fermentation Streptomyces factors donor: 5 O, .typhi WR4000 O> 9.12> Vi: d CysTrp RhaAraXyl 5 recipient: E. coli F212 08: K-: H- His Ara r Thr, threonine; Cys, Cysteine; Trp, tryptophan; His, histidine; Rha, rhamnose; Macaw, arabinose; Xyl, xylose; s, sensitive; r, resistant.

In general, (-) means that the amino acid is required, i. is not synthesized; or the sugar is not fermented.

The hybridization that takes place according to the method described takes place, for example, according to the following scheme: S.typhi Hfr WR4000 x E.coliF212 S (09, His str S (08) His rigid ½- His str selection Fell93 S (O9> 12) Table 2 below shows the biochemical changes which distinguish the hybrid of the designation F1193 to be produced according to the invention compared to the values characterizing the donor and the recipient.

Taxonomic property part of donor, recipient and hybrid donor Recipient hybrid S.typhi WR4000 E. coli 08 F1193 F212 H2S formation + + dextrose-V evaluation + + + gas formation + + NH4 citrate growth + indole formation + + lactose utilization + + + Adonit recovery Inositol recovery Sucrose recovery Mannitol recovery + + + Dulcit utilization Salicin utilization Rhamnose utilization + + Urease formation Lysine decarboxylase ornithine decarboxylase - + + motility + colony morphology flat raised raised grayish whitish whitish matt glossy moist glossy moist glossy smooth-edged smooth-edged smooth-edged 7 If mice intraperitoneally 10 live bacteria, which were obtained according to Example 1, applied, forms as follows shown a vaccination protection. For comparison, 107 bacteria of the Salmonella strain are used typhi Ty2> which were killed by acetone, also intraperitoneally on mice applied. The immunization is repeated in both groups 10 days later. 4 weeks after the last vaccination, the mice become 5 × 10 5 cells of the virulent S.typhi Ty2 applied intraperitoneally. A non-immunized one serves as a control Group of mice infected in the same way with 5 x 10 living bacteria of S.typhi Ty2 became. The course of the infection is followed by looking into the following Given in the table kills 5 mice at intervals of time and the number of infectious germs present in the spleen and liver is determined. For one Typical results of such an experiment are shown in Table 3 below. The results show that those with the living cells of the hybrids according to Example 1 immunized mice eliminate the infectious germs more effectively than those with the Acetone-inactivated cells from Ty2 vaccinated mice.

Table 3 days after the number of infectious germs vaccinated in the liver and spleen and infection of non-vaccinated mice vaccinated with: Recombinant F1193 Ty 2 (inact.) No vaccine 2 2 5 1 2 x 10 4 x 10 5.5 x 10 2 2.5 s 103 8 x 103 4.5 x 105 3 5.5 x 102 7.5 x 103 8.5 x 105 4 2 x 10 8.5 x 103 2.5 x 105 6 1 x 10 5 x 103 2 x 10 8 60 2 x 103 4 x 104 10 <25 1 x 103 1.5 x 104 In experiments on chimpanzees and pigs, in which similar physiological conditions exist as in humans, can after single oral administration of 1011 germs according to the present invention, their excretion be detected in the faeces for a period of at least 2 months.

The detection of the Salmonella-E.coli hybrids according to the invention can particularly easy and diagnostic of the excretion of S.typhi infectious germs be differentiated. It shows the O-specific serological behavior of S.typhi and the metabolic behavior of E. coli. This can be used for specific evidence the hybrid can be used.

Consisting of the effectiveness of the vaccines to be produced according to the invention from living germs of the hybrids according to example 1 can also be carried out using the following model prove.

The model consists in the infection of mice with a virulent mouse Salmonella typhimurium hybrids with Salmonella typhi-O specificity. This hybrid In contrast to S. typhi (mouse avirulent), it creates a real infection in the mouse and can therefore be used to test typhoid vaccines such as those of the present invention non-pathogenic E. coli hybrids.

For this purpose, mice are brought to life in the manner described above Cells of the hybrid F1193 according to Example 1 and, as a comparison, with acetone inactivated Ty2 germs immunized and 4 weeks later with a mouse virulent S.typhimurium recombinant infected with 09, 12 specificity. As a control, non-vaccinated mice are infected. The animals are observed for 30 days and those killed by the infection Animals are registered. Typical results for such an experiment are listed in Table 4. The results show that the vaccination with living S, typhi-E.coli recombinants according to Example 1 causes significant protection.

Table 4 Immunization with post-infection survivors survivors / total Recombinant F1193 20/20 100 S.typhiTy2 (inact.) 13/20 65 non-vaccinated 0/20 The example given below is intended to explain the invention in more detail.

Example 1 x 108 S.typhi WR4000 germs and 5 x 10 E. coli F212 germs per ml in 10 ml of a standard I nutrient broth from E. Merck, Darmstadt, cat. no. 7882/1974 to be designated nutrient solution are mixed together and the mixture for 2 hours leave at 37 C. The germ suspension is then centrifuged at 6000 g, the Sediment suspended in 1 ml of sterile water. Aliquots of approximately 0.1 ml are made plated on a minimal agar of the following composition: 22g Difco Bakto-Agar and 0.75g sodium citrate x 5 H2 0 are mixed in one liter of distilled water and sterilized. Separately, a saline solution is sterilized, which is per liter of distilled water. the following Salt contains: 20 g NH4C1 4 g NH4N03 8 g Na2SO4 12 g K2HP04 4 g KH2 PO4 0.4 gMgS04 Then 900 ml of the agar solution and 300 ml of the saline solution are mixed together and a glucose solution sterilized separately from the solutions by filtration added so that the final concentration of glucose is 0.3%. Finally will Streptomycin added to the mixture so that the resulting minimal agar has a concentration of 400 pg of streptomycin per ml contains. The agar poured into Petri dishes becomes with the germ suspension, the donor as well as recipient - as well as hybrid germs contains, inoculated, incubated for 48 hours at 37 ° C. The colonies that then grew are streptomycin-resistant recombinants, which also contain the intact allele of the donor for histidine synthesis. They are therefore called His recombinants designated.

Those hybrid germs which have the O specificities 9 and 12, the are detectable with appropriate antisera, represent the invention product represent.

A recombinant produced by the method given by way of example with the designation F1193 is in 100 ml standard I nutrient broth from E. Merck, Darmstadt, cultivated with shaking at 37 ° C. The bacteria will be after 4 hours Incubation precipitated by centrifugation at 6000 g. The sediment is then suspended in 100 ml of an aqueous protective medium. The protective medium contains im present case 5% skimmed milk powder and 1% gelatin. Portions of 1 ml each lyophilized in 6 ml ampoules and stored at 4 ° C.

The propagation of the new hybrid is made by the content one ampoule suspended in 700 ml nutrient broth in a 1 liter glass flask and incubated with shaking for 12 hours at 37 ° C. This is called the preculture Germ suspension is added to 14 l standard I nutrient broth from E. Merck, Darmstadt, transferred and placed in a fermenter under constant pH and aeration conditions (pH 7.2 and 1.5 1 air per minute per liter of medium) cultivated for 6 hours at 37 ° C. The purity of the fermenter culture is determined by taking samples at regular intervals controlled. At the end of cultivation, the bacteria are placed in a refrigerated centrifuge sedimented at 6000 g and suspended in 500 ml of the protective medium described above. Portions of 1 ml each are lyophilized in 6 ml ampoules. The contents of one ampoule contains 10 -10 bacterial cells. Administered e.g. in 5 ml of warm milk, is enough Ampoule contents to protect an adult chimpanzee against an acute S.typhi infection and protect against typhoid fever. It can also be done in this way immunized chimpanzees as well as pigs treated in the same way be shown 11 that a single oral application of 10 germs of the invention Hybrids that are excreted in the faeces over a period of at least 2 months can be proven.

Claims (11)

PATENT CLAIMS Salmonella coli hybrids, characterized in that they contain the surface antigen with the specificity 09.12 according to Kauffmann-White. 2) Salmonella E. coli hybrids according to paragraph 1, characterized in that that the E. coli is a non-pathogenic intestinal inhabitant. 3) Salmonella coli hybrids ATCC 31161. 4) Method of producing a stable, as a living germ in a Vaccine usable microorganism that is free of virulence and immunogenic, thereby characterized in that the 09,12-antigen synthesis determining chromosome fragments of a Salmonella strain transferred to a genetically related recipient strain and the resulting hybrid with the desired metabolic performance from the Intersection mix is selected. 5) Method according to claim 4, characterized in that as recipient an E. coli strain is used. 6) A method for producing a hybrid according to claim 5, characterized characterized in that the E. coli recipient is a strain E. coli 08: K: H, which may be streptomycin-resistant is used. 7) A method for producing a hybrid according to claim 3, characterized characterized in that a non-pathogenic E. coli intestinal inhabitant is used as the recipient will. 8) A method for producing a hybrid according to claim 4, characterized characterized 'that as a Salmonella strain a S.typhi-Hfr strain, which may have a special Metabolic property such as streptomycin sensitivity is used. 9) Process according to claim 4 to 8, characterized in that one a live germ suspension of a Salmonella typhi donor with that of an E. coli recipient mixes, incubated at least until the O-antigens 09,12 determining Chromosome fragments are transferred from the donor to the recipient and subsequently isolates the resulting hybrid. 10) method for propagating a hybrid according to claim 1 to 3, characterized in that a suitable nutrient medium is inoculated with the hybrid, maintains favorable growing conditions for the hybrid and eventually them isolated. 11) vaccines against salmonellosis, characterized in that they have a Hybrid according to claim 1 in one suitable for oral or parenteral administration Form of preparation if necessary Contains with pharmaceutically customary excipients.