CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of Ser. No. 151,002, filed May 19, 1980, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Vaccination with live attenuated strains is extensively and successfully used in the prevention of various viral diseases of man, such as polio, smallpox, and measles. By contrast, live vaccines are used against only a few bacterial diseases of man or domestic animals: BCG vaccine for prevention of tuberculosis, strain 19 vaccine against bovine brucellosis and Sterne's spore vaccine against anthrax in cattle. Yet in many investigations of experimental Salmonella infections live vaccines have shown advantages over killed vaccines: (i) They frequently prevent, rather than merely postpone, multiplication of Salmonella in liver and spleen, which multiplication may lead to death; (ii) they provide good protection against challenge by oral route, in situations where killed vaccines, given by injection or orally, are relatively ineffective; (iii) in some instances injections of live vaccine confer ability to rapidly eliminate challenge bacteria from liver, spleen, etc., presumably through cell-mediated immunity, in contrast to killed vaccines which evoke mainly humoral immunity, without much ability to eliminate virulent bacteria. Some strains considered for use as live vaccines retain an unacceptable degree of virulence, by reversion or otherwise: Other reported disadvantages of some live vaccines include short persistence of immunity attributed to early disappearance of the vaccine strain from host tissues; and in some instances incomplete immunity, so that some vaccinated animals die after a large challenge inoculum of a virulent strain.

The non-virulent strains used as vaccines have been obtained in various ways. The BCG strain was derived by empirical procedures during prolonged in vitro cultivation, and probably owes its non-virulence to multiple unidentified mutations. Sterne's Bacillus anthracis spore vaccine is a strain which has lost the ability to synthesize the polypeptide capsule, important as a determinant of virulence but not as a "protective" antigen. Some experimenters have used as live vaccine merely a sublethal dose of a "wild" strain of relatively low virulence in the sense that the LD50 was a large number of bacteria--a situation in which there is evident risk of severe or fatal infection developing in some vaccinated subjects and of transmission to other hosts.

Recently, strains have been developed for use as live vaccines which are streptomycin-dependent mutants of strains of several pathogenic bacterial species. Shigella flexneri and Shigella sonnei streptomycin-dependent mutants have been extensively used as live vaccines given by mouth for protection and have found to be both safe and efficient. In experimental Salmonella infections, however, streptomycin-dependent mutants seem to have been only moderately satisfactory. In general "rough" mutants in Gram-negative bacterial species, i.e., mutants unable to manufacture normal lipopolysaccharide are non-virulent but have proven unsatisfactory as live vaccines because of failure to cause protection. Two exceptions may be noted. (i) In Salmonella mutation of gene galE prevents normal lipopolysaccharide synthesis unless the bacteria are provided with preformed galactose. A galE mutant of S. typhimurium was virtually non-virulent in small laboratory animals but evoked good immunity. As anti-O antibodies were produced the galE bacteria must have obtained sufficient galactose within the host tissues for them to make at least some O-specific lipopolysaccharide. Recently a galE mutant of S. typhi, given by feeding to human volunteers, proved non-virulent and conferred reasonable protection against later oral challenge with a virulent strain of the same species. Furthermore, first reports of a field trial of this strain, given by oral route to school children in Alexandria, Egypt, indicate that it gave very good protection against the risk of contracting typhoid fever, which has a high incidence in such children. The non-virulence of galE strains seems to be conditional on the presence of normal host cellular defense mechanisms, since administration of the cytotoxic agent cyclophosphamide to mice previously injected with a galE mutant of S. typhimurium, non-pathogenic to untreated animals, precipitated fatal infections due to multiplication of the galE strain. (ii) A "rough" mutant of S. dublin is in routine use in Great Britain as a live vaccine, given by parenteral injection, for protection of newborn calves against the frequently fatal Salmonella infections which were formerly prevalent; as the strain used appears to lack the O-specific part of lipopolysaccharide, it presumably acts by invoking "non-specific immunity," perhaps by causing activation of macrophages.

Since live vaccines have substantially greater probability of success in providing for protection for the host against a subsequent invasion of a virulent wild strain, it is desirable to develop new live vaccines which avoid the shortcomings of vaccines prepared previously. Because the immune response of the vertebrate host to antigens, in particular surface antigens, of the pathogenic microorganism is the basic mechanism of protection by vaccination a live vaccine should retain the antigenic complement of the wild-type strain. The live vaccine should be non-virulent, substantially incapable of multiplication in the host, and should have substantially no probability for reverting to a virulent wild strain.

2. Brief Description of the Prior Art

Sandhu et al., Infection and Immunity (1976) 13, 527 describes loss of virulence of Asperigillus fumigatus in a mutant auxotroph for p-aminobenzoic acid. Morris et al. Brit. J. Exptl. Path. (1976) 57:354 describes the effect of T and B lymphocyte depletion on the protection of mice vaccinated with a galE mutant of Salmonella typhimurium. Lyman et al., Inf. Imm. (1977) 15:491 compared the virulence of 0:9,12 and 0:4,5,12 S. typhimurium his⁺ transductants for mice. Descriptions of use of translocatable elements for causing deletions or inversions may be found in Kleckner, et al., J. Mol. Biol. (1977) 116, 125; Kleckner et al., ibid 127, 89; and Kleckner et al., Genetics, 90:427-464 (1978).

In a private communication, Dr. John Roth, Department of Biology, University of Utah, developed two strains of S. typhimurium LT2, in each of which the transposon Tn10, conferring resistance to tetracycline, had been inserted into a gene of the aromatic biosynthetic pathway, thereby causing inability to synthesize the common precursor of the aromatic amino acids and of two bacterial metabolites, p-aminobenzoate, (precursor of the essential metabolite folic acid) and dihydroxybenzoate (precursor of the iron-chelating compound enterochelin or enterobactin). R. J. Yancey (1979) Infection and Immunity, 24, 174 report that a mutation causing inability to synthesize enterochelin secured in a mouse-virulent strain of S. typhimurium caused a very considerable reduction in virulence. The metabolic block was between chorismic acid and enterobactin, so that the mutation did not cause the requirement for p-aminobenzoate. In May, 1979, a paper was presented by Stocker and Hoiseth, entitled Effect of Genetic Defects in Iron Assimilation on Aromatic Biosynthesis on Virulence of Salmonella typhimurium.

SUMMARY OF THE INVENTION

Live vaccines are provided for vaccinating a host against a pathogenic microorganism, particularly bacteria. The live vaccines are prepared by producing auxotrophic mutants of a pathogenic strain, wherein normally at least one, usually two, biosynthetic pathways are blocked, so that the bacterial mutant requires for growth at least one nutrient which is normally not available in the host in the amount required by the microorganism.

The auxotrophic mutation is a result of a genetic change in the gene, which cannot be repaired by any single step. Such genetic changes include deletion of a polynucleotide segment of a gene, and inversion of a polynucleotide sequence forming a part of a gene immediately adjacent to an insertion of a foreign polynucleotide sequence in the gene. For the purposes of the invention, these changes will be referred to as "non-reverting mutations." Normally, the non-reverting mutation will not affect the antigenic constitution of the pathogenic microorganism, and, in particular, will not alter its surface antigens, some of which may be important determinants of pathogenicity, or may function as protective antigens in that immune response to them protects the host against infection by that microorganism. The resulting auxotrophic mutants have substantially zero probability of reversion, while having substantially the same, or the same, immunogenic characteristics as the virulent strain, so as to produce an effective immunogenic response.

In particular, auxotrophic mutants are obtained by employing a virulent strain, desirably having a genetic marker allowing its recognition, and introducing into it a non-reverting mutation in a gene, so as to produce a complete block in biosynthesis of an essential metabolite which is not normally available in vertebrate tissues, then isolating the mutant and screening for inability to revert, lack of virulence and immunizing ability when used as live vaccine. A number of different techniques can be used for obtaining non-reverting mutations and for efficiently isolating the auxotrophic non-reverting mutants.

The species of Salmonella showing particularly desirable properties of non-revertancy and activity as vaccines are designated as S. typhimurium SL1479 and S. dublin SL1438.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Live vaccines are provided which are particularly useful in vaccinating vertebrate hosts susceptible to disease from pathogenic microorganisms i.e. bacteria. The microorganisms are non-virulent due to being auxotrophic in having a non-reverting non-leaky block to at least one, usually a plurality of biosynthetic pathways, where the required nutrient(s) due to the block are normally not available in the host to be vaccinated. By employing various techniques, a mutation is introduced into a gene, the gene determining an enzyme indispensably needed for a particular step in a biosynthetic pathway, with resulting block in biosynthesis of an essential metabolite not available in host tissues. The mutation is non-reverting because restoration of normal gene function can occur only by random coincidental occurrence of more than one event, each such event being itself very infrequent.

In the case of a deletion mutation restoration of generic information would require many coincidental random nucleotide insertions, in tandem, to restore the lost genetic information. In the case of an insertion plus inversion mutation of a gene no longer able to specify an enzymically active protein because of inversion of a nucleotide sequence making up a part of the gene immediately adjacent to an inserted nucleotide sequence, restoration of function would require coincidence of precise deletion of the inserted sequence and precise re-inversion of the adjacent inverted sequence, each of these events having at most an exceedingly minute, probably undetectably low, frequency of occurrence. Thus each of the two sorts of "non-reverting" mutant has a substantially zero probability of reverting to prototrophy. In addition, it is desirable that the microorganism for use as live vaccine have one or more genetic "marker characters" making it easily distinguishable from other microorganisms of the same species, either wild strains or other live vaccine strains.

After manipulating the microorganism so as to introduce one or more non-reverting mutations into some members of the population the microorganisms are grown under conditions facilitating isolation of the auxotrophic mutants, either under conditions under which such mutants have a selective advantage over bacteria of the original type, or under conditions allowing their easy recognition from unaltered bacteria or mutants of other types. The isolated auxotrophic mutants are cloned and screened for virulence, inability to revert and ability to protect the host from a virulent pathogenic strain.

The subject method for preparing the vaccines and the vaccines have a large number of advantages over prior vaccines. As contrasted with other vaccines, the subject invention provides for the exact cause of the loss of virulence. Unlike other live vaccine strains which are non-virulent because of alteration of lipopolysaccharide character, the subject vaccines will be substantially unaltered in O antigenic character, as well as other surface antigens which may have relevance to virulence and immunity, such as the major outer membrane proteins. Thus, the subject vaccines would stimulate production of anti-O antibodies which are known to be important components in the immunity obtainable by vaccination. The subject strains should be able to persist in the host for extended periods of time, usually weeks, to enhance the effectiveness of the immunizing effect. The auxotrophic mutants having non-reverting non-leaky mutations will have substantially zero likelihood of reverting to virulence. In view of the fact that the non-virulence depends upon the absence of relevant metabolities in host tissues and not on any host cellular function, the subject strains will be non-virulent even in immunodeficient hosts. Because of the nature of the metabolic defect of the subject strains, the subject strains should be unable to multiply in the lumen of the gut of animals infected by feeding and provided only with diet not supplemented with the relevant metabolites required by the subject strains, while such feeding should be safe, and efficacious in inducing antibody production in the gut wall.

In particular, among bacteria, the subject invention is particularly applicable to Salmonella, more particularly of groups A, B, or D, which includes most species which are specific pathogens of particular vertebrate hosts.

The subject invention finds particular application to a wide variety of Salmonella strains which are pathogens. Illustrative of the Salmonella causing disease for which live vaccines can be produced are S. typhimurium; S. abortus-ovi; S. abortus-equi; S. dublin; S. gallinarum; S. pullorum; S. choleraesuis; S. typhi, as well as others which are known or may be discovered to cause infections in mammals.

Other organisms for which the subject invention may also be employed include Shigella, particularly S. flexneri and S. sonnei; Haemophilus, particularly H. influenzae, more particularly type b; Bordetella, particularly B. pertussis; Neisseria, particularly N. meningitidis and N. gonorrohoeae; and Pasteuralla, particularly P. pestis and P. multocida.

The vaccines can be used with a wide variety of domestic animals, as well as man. Included among domestic animals which are treated by vaccines today or could be treated, if susceptible to bacterial diseases, are chickens, cows, pigs, horses, goats, and sheep, to name the more important domestic animals.

In preparing the live vaccines, one chooses a strain of the pathogen which desirably has a marker for distinguishing the auxotrophic mutant to be produced from other members of the strain. Various markers can be employed, such as resistance to antibiotic or synthetic antibacterial drug, block in a biosynthetic pathway causing requirement for an amino acid, e.g., histidine, or the like. The limitation on the particular marker is that it should not affect the immunogenic character of the microorganism, nor should it interfere with the processing of the microorganism to produce the live vaccine. The marker will alter the phenotype, to allow for recognition of the subject microorganism.

The subject organism will then be processed to provide a non-reverting mutation, preferably involving a polynucleotide of greater than five nucleotides, more preferably a polynucleotide of at least ten nucleotides, which blocks at least one, preferably a plurality of biosynthetic pathways, normally two or more. The blocked biosynthetic pathways should not be involved in the production of the antigens involved with the microorganisms' virulence, nor the host's immune response to infection by the microorganism. Various techniques can be employed for introducing deletions or insertion inversions, so as to achieve a microorganism having the desired "non-leaky" non-reverting biosynthetic pathway blocks.

The choice of gene will be governed by the ability to mutate the gene without destroying the viability of the microorganism; the essential nature of the product expressed by the gene; and the unlikely presence of the product in the intended host. Genes of particular interest include one of the aro genes, dap which is involved in the production of diaminopimelic acid; and pab which is involved in the production of p-aminobenzoic acid.

One technique for producing a non-reverting biosynthetic pathway block is the employment of translocatable elements, in particular transposons. Transposons are segments of double-stranded DNA, made up of some thousands of nucleotides, and normally comprising a gene for resistance to an antibiotic or other antibacterial drug, together with genes which can effect the insertion of a copy of the transposon at any of very many different sites in the DNA of the bacterium housing the transposon. Insertion of a transposon into a gene which specifies the amino acid sequence of an enzymically active protein causes complete loss of ability to synthesize that protein in active form. However the whole transposon is, at a low frequency (for instance, 10^-8 /bacterium/generation) deleted or excised from the gene into which it was inserted, this gene in consequence being restored to its original state, so that it again specifies an enzymically active protein. Such precise excision of a transposon causes loss of the resistance to the antibiotic or other antibacterial agent, which resulted from the action of the resistance gene of the transposon.

In addition to precise excision, loss of resistance conferred by the transposon occurs also by other kinds of events, which are much more frequent than precise excision and do not result in reconstitution of the original form of the gene, thus not resulting in restoration of the lost gene function. Two kinds of such event result in production of a non-reverting non-functional form of the gene into which the transposon was inserted: one event is deletion of a segment of DNA comprising the whole or a part of the transposon, including its resistance gene, and a segment of DNA extending to one side of the site of insertion, thus extending into, sometimes entirely through, part of the gene into which the transposon was inserted, to one side of the site of insertion; the other event is deletion of a part of the transposon and simultaneous inversion of a DNA segment which includes genetic material extending to one side from the site of the original insertion, i.e., part or the whole of the portion of the affected gene to one side of the site of the original insertion. Restoration of the affected gene to its original state can then occur only by precise deletion of the remaining part of the transposon together with re-inversion of exactly the inverted DNA segment, i.e., by the coincidental occurrence of two events each of which is expected to be exceedingly infrequent, probably undetectably rare.

The consequence of either of these two kinds of event, deletion or deletion plus inversion, occurring in a bacterium with a transposon insertion in a gene specifying a biosynthetic enzyme is to change the transposon containing antibiotic-resistant auxotrophic bacterium with a genetic lesion causing an enzyme defect, which though complete is liable to rare reversion, to an antibiotic-sensitive bacterium with the same enzyme defect, as before, absent the transposon, and now no longer subject to correction by rare reversion events. Thus when the transposon is inserted into a gene specifying an enzyme used in a biosynthetic pathway leading to a metabolite or metabolites essential to the bacterium, but non-available in its vertebrate host, the final result is a bacterial strain with a complete and non-reverting mutation causing non-virulence.

Alternatively, bacteria can be treated with nitrous acid, which is known to result in production of deletion mutations, most of them consisting in loss of many adjacent nucleotides. One would treat the culture of the microorganism with nitrous acid under conditions expected to produce mutants with a wide variety of different auxotrophic characters and then isolate from the population microorganisms being mutants with the desired auxotrophic trait(s).

Isolation of auxotrophic mutants can be facilitated by use of penicillin, to kill non-auxotrophic bacteria and so increase the proportion of nutritionally exacting mutants in the population. Once a mutant with the desired auxotrophic character has been isolated a large population of the mutant can be screened for presence of any descendants able to grow without the relevant metabolite, thus testing the probability of reversion of the mutation; this test is made more rigorous if the population is first exposed to a mutagenic agent, or agents, capable of inducing a wide variety of mutational change, i.e., base substitutions, frameshifts, etc. Furthermore if a recombination system is available, a mutant with a deletion of a segment of a gene covering the sites of two or more point mutants can be recognized by the absence of wild-type recombinants, when the deletion mutant is crossed with each of the point mutants in question. In these ways one can assure that the auxotrophic mutant investigated is almost certainly a result of deletion, rather than of point mutation.

A general transducing phage, such as phage P1, able to adsorb to bacteria of a wide range of genera (if necessary after appropriate genetic modification of their lipopolysaccharide character, to provide the necessary ability to adsorb this phage) can be used to transduce a non-functional biosynthetic gene, such as an aro gene inactivated by insertion of a transposon, from its original host to a pathogenic bacterial strain of a different species or genus, wherein it will have some probability of incorporation into the chromosome, there replacing the homologous wild-type gene, to produce an auxotrophic transductant. However, the frequency of such replacement is likely to be much reduced by the incomplete base-sequence homology of corresponding genes in bacteria of different genera. DNA-mediated transformation or bacterial conjugation can similarly be used to transfer an aro or other biosynthetic gene inactivated by transposon insertion or otherwise into bacteria of species or genera different from that of the original strain, to yield auxotrophic bacteria now non-virulent because of requirement for a metabolite not available in vertebrate host, and unable to revert to prototrophy due to the presence of either a deletion or insertion-inversion.

Conjugation may also be employed involving conjugational crossing of a virulent strain with a mutated gene of a non-virulent but amenable strain having the desired non-reverting mutated gene. By employing an Hfr or F⁺ strain with an F^- virulent strain, transfer of the mutated gene to the virulent strain can occur with recombination resulting in the replacement of the wild gene by the mutated gene. One would then select for the auxotroph as described previously.

In accordance with the subject invention, the vaccines are produced by introducing a non-reverting mutation in a gene, where the mutation is of a sufficient number of bases in tandem to insure a substantially zero probability of reversion and assurance of the non-expression of the gene, in the sense of its total inability to determine production of an enzymically active protein. In addition, the gene chosen will be involved in at least one, and preferably at least two biosynthetic pathways to produce metabolites which are either infrequently present in the host or completely absent. Therefore, the likelihood that a host for the vaccine will provide the necessary nutrients for growth will have a probability approximating zero. These requirements have been shown to be fulfilled by the aroA gene of Salmonella typhimurium, so that this gene is preferred, although other genes, as previously indicated, may also serve as the site for the non-reverting mutation.

In the case of transduction, the transposon has a marker which allows for selection of the transduced microorganism. For example, if the transposon provides resistance to a biostat or biocide e.g. antibiotic, by growing the transduced microorganism in a nutrient medium containing the bioactive agent, one selects for the transductant strain. One can then employ other techniques, as will be described below, to select for members of the transductant strain which undergo excision of all or part of the transposon where the transposon carries with it a portion of the gene into which it had been integrated or results in an inversion of a portion of the gene.

In order to isolate the auxotrophic strain, whether produced by transduction, mutagenesis, transformation or other means, it is desirable to provide selective pressure to the treated virulent strain to enhance the proportion of the desired auxotrophic strain. One technique is to employ a drug, e.g. a pencillin, which is only lethal to bacteria which are multiplying. By employing a nutrient medium which does not provide one or more of the metabolic products required by the auxotrophic strain due to the mutation introduced above, the auxotrophic strain will be inhibited from multiplying, while the virulent parent strain will multiply and so be killed by the penicillin. Normally, at least about 99% of the bacteria are killed and less than 100%, so that the surviving about 0.05 to 1% of the original bacteria have a greatly enhanced concentration of the auxotrophic strain. One can then grow the surviving bacteria in a supplemented medium which will allow for multiplication of the auxotrophic strain and repeat the penicillin killing process until one can isolate the auxotroph from a single colony and establish the complete absence of any of the virulent parent strain.

The resulting auxotrophic strain will be a non-virulent live vaccine, having the desired immunogenicity, in that the deletion will not affect the production of the antigens which trigger the natural immune response of the host. At the same time, the deletion results in a non-virulent live vaccine, incapable of growing in the host and incapable of reverting to a virulent strain.

Two particularly valuable strains were prepared generally as follows. A characterized Salmonella strain was exposed to a transducing phage grown on a different Salmonella strain which was aroA554::Tn10 and selection made for increased resistance to tetracycline. Tetracycline resistant aromatic-dependent strains were selected which grew poorly on Bochner medium. Transductants were then selected for low revertancy to aromatic independence, indicating the loss of Tn10. The transductants may be screened employing mice and a strain which is non-virulent and provides protection against inoculation with a virulent strain selected. As a matter of convenience, one may introduce a marker, for example, auxotrophy, to provide a specific nutritional requirement, which then allows a rapid determination of whether there has been revertancy in the event a host contracts Salmonella at the time of a subsequent vaccination with the vaccine prepared in accordance with this invention.

The subject vaccines may be used in a wide variety of vertebrates. The subject vaccines will find particular use with mammals, man, and domestic animals. Domestic animals include bovine, ovine, porcine, equine, caprine, domestic fowl, Leporidate e.g. rabbits, or other animal which may be held in captivity or may be a vector for a disease affecting a domestic vertebrate.

The manner of application of the vaccine may be varied widely, any of the conventional methods for administering a live vaccine being applicable. These include oral application, on a solid physiologically acceptable base, or in a physiologically acceptable dispersion, parenterally, by injection, or the like. The dosage of the vaccine (number of bacteria, number of administrations) will depend on route of administration and will vary according to the species to be protected. For mice, of weight 15-20 g, a single dose of ca. 3×10⁵ live bacteria of a non-reverting aro^- vaccine strain of S. typhimurium, given in a volume of 0.2 ml in saline by intraperitoneal injection, appears both safe and effective, as judged by the result of later parenteral challenge with virulent S. typhimurium. Mice have also been given ca. 3×10⁷ live bacteria of the same strain, in bread cubes moistened with 0.1 ml of broth culture, provided after six hours deprivation of other food; this dose proved harmless but immunizing efficacy is not yet known. Observations on vaccinated calves suggest that administration of live bacteria of the same strain, by intramuscular injection or by feeding, in doses a thousandfold greater than used for mice are safe, and may be appropriate. One or more additional administrations may be provided as booster doses, usually at convenient intervals, such as two to three weeks.

The formulations for the live vaccines may be varied widely, desirably the formulation providing an enhanced immunogenic response. The live vaccine may be provided on a sugar cube, in buffered saline, in a physiologically acceptable oil vehicle, or the like.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

A live vaccine derived from a Salmonella typhimurium was prepared as follows. As the parent strain, a mouse-virulent S. typhimurium SL4522 was employed which is a descendant of a "wild" S. typhimurium strain M7471 of the S. typhimurium subgroup called FIRN (Morgenroth and Duguid, Gent. Res. (1968), 11:151-169). This subgroup is defined by its inability to ferment rhamnose or inositol and failure to product type 1 pili or fimbriae. Strain M7471 was given a colicin factor or plasmid, ColE1-K30, by conjugation, then made, by successive exposures to a mutagen, maltose-negative, leucine-requiring and cystine-requiring followed by being made histidine-requiring by transducing in mutation hisC527 (an amber mutation). The genetic constitution of the starting strain, SL4522, is thus S. typhimurium M7471 (ColE1-K30) malB479 leu-1051 cysI1173 hisC527 (MacPhee et al. J. Gen. Microbiol. (1975) 87:1-10). This strain which retains its mouse virulence was reisolated from a mouse which had died from a small inoculum and the reisolate was designated SL3201.

S. typhimurium containing a transposon imparting tetracycline resistance and being present in gene aroA, designated as strain TT1455 and having genetic constitution S. typhimurium LT2 aroA554::Tn10, was employed as a source of the transposon employed in the subject preparation. This strain has been developed and is available from Dr. John Roth, Department of Biology, University of Utah, Salt Lake City and has been deposited at the ATCC with Deposit No. 33275. The insertion site of the transposon Tn10 is in gene aroA, which is located at position 19 on the linkage map and specifies the enzyme 3-enolpyruvylshikimate-5-phosphate synthetase. Strain TT1455 cannot grow on chemically defined media unless provided with the essential metabolites derived from chorismic acid, i.e. the aromatic amino acids tyrosine, phenylalanine, and tryptophan and two minor aromatic metabolites, p-aminobenzoate, needed as a precursor of folic acid, and 2,3-dihydroxybenzoic acid, as a precursor of the iron-chelating compound enterobactin (enterochelin).

For the transduction, a high-transducing mutant, HT105/1 (Schmiger, Mol. Gen. Genet. (1972) 119:75-88) of the general transducing phage P22 was employed having an additional mutation int, hindering lysogenization and facilitating preparation of high-titer phage stocks. In accordance with conventional techniques, this phage was grown on strain TT1455; the resulting lysate, after removal of all bacteria by centifugation was further treated by membrane filtration. A plate of nutrient agar (Oxoid blood agar base No. 2, code CM55) supplemented with tetracycline, (25 μg/ml) was flood-inoculated with a broth culture of the recipient strain SL3201; drops of the phage lysate, volume approximately 0.01 ml, were applied to the plate and the plates were incubated at 37° for about 18 hrs. After incubation, each drop-area showed many large (approximately 2 mm diameter) colonies of tetracycline-resistant "complete" transductants (also many very small colonies of abortive transductants), whereas the agar between the drops showed no growth. Two tetracycline-resistant transductant clones were obtained, re-isolated from single colonies and tested to confirm freedom from phage P22, and were then designated strains SL3217 and SL3218. They showed the expected phenotype in requiring for growth provision of tyrosine, phenylalanine, tryptophan, p-aminobenzoate and 2,3-dihydroxybenzoate. The strains SL3217 and SL3218 showed the expected loss of mouse-virulence, provided that they were grown in the presence of tetracycline, to prevent accumulation of aromatic-independent revertants.

The isolation of tetracycline-sensitive variants is facilitated by the fact that tetracycline, at appropriate concentrations, prevents multiplication of tetracycline-sensitive bacteria, but does not kill them, whereas penicillin kills multiplying bacteria but spares non-multiplying bacteria. The technique of penicillin-selection was used for isolation of tetracycline-sensitive variants from the aroA::Tn10 strain SL3218. The strain was first grown in broth, without tetracycline to a concentration of approximately 9×10⁸ pfu/ml; this culture was then diluted 1:10 into broth containing tetracycline, 5 μg/ml, and the diluted culture incubated at 37° with aeration for 75 min; ampicillin, 2.5 mg/ml, was added and incubation with shaking was continued for 290 min; the culture was then held at room temperature without shaking overnight. Surviving bacteria were washed on a membrane filter to remove ampicillin, and then plated on an indicator medium containing dyes and a very low concentration of tetracycline. On this medium, tetracycline-sensitive bacteria produce small, dark colonies, whereas tetracycline-resistant bacteria produce large pale colonies. The ampicillin treatment reduced the number of viable bacteria by about 10^-5. Six of 386 survivor colonies tested proved to be tetracycline-sensitive. Two such isolants, designated SL3235 and SL3236 were shown to resemble their parent strain SL3218 in nutritional character, but to differ from it not only by their tetracycline-sensitivity, but also by their failure to produce any aromatic independent revertants, in tests which would have detected reversion at a frequency of one in 10¹¹ /bacteria/generation. One of these strains, SL3235, when used as live vaccine in mice and calves, showed no reversion to aromatic-independence of virulence. Another non-reverting aro^- vaccine strain was prepared from S2357/65, a Salmonella typhimurium strain known from experiments elsewhere to be highly virulent for calves. Strain S2357/65 is prototrophic: to provide a marker character it was made by transduction first hisD8557::Tn10 (therefore phenotypically with a histidine requirement not satisfied by histidinol, and tetracycline-resistant), then by a second transduction made hisD⁺ hisG46 (thus phenotypically with requirement for histidine or histidinol, and tetracycline-sensitive). This derivative, SL1323, was shown to cause fatal infection when fed to calves. The calf-passage strain, labeled SL1344, was next made aroA544::Tn10 by transduction from TT1455, as described above; the hisG46 aroA544::Tn10 strain so obtained was labeled SL1346. A tetracycline-sensitive mutant, still aromatic-requiring but now unable to revert to aromatic independence, was next isolated from SL1346 by a new method, i.e., selection on nutrient agar containing chlortetracycline, 50 mg/ml, added before autoclaving, and fusaric acid, 12 mg/ml, added after autoclaving. This medium prevents or at least greatly retards growth of tetracycline-resistant bacteria but allows good growth of tetracycline-sensitive bacteria. Both this strain and two aroA::Tn10 strains, SL3217 and SL3218 grown with tetracycline, to prevent accumulation of tetracycline-sensitive aro⁺, therefore virulent, revertants have been shown to be effective as live vaccine when administered to mice by intraperitoneal route. (i) Experiments with strains SL3217 and SL3218: CF1 mice given ca. 2×10⁵ live vaccine-strain bacteria, i.p.: challenge two months later with 2×10⁶ bacteria (i.e. more than 20,000 LD50) of virulent S. typhimurium strain SL3201, i.p.: no deaths in two months' observation. (ii) CBA/N×DBA/LN F₁ female mice given 10⁶ or 10⁵ live-vaccine strain SL3235, i.p.: challenged five weeks later with 10⁶ bacteria (ca. 100 LD50) of virulent S. typhimurium strain TML, i.p.: no deaths in fifteen days' observation. In other experiments the stable aro^- vaccine strain, SL3235, has been shown not to cause death (nor any obvious ill effects) when injected intraperitoneally even into mice exceptionally susceptible to Salmonella typhimurium infection, either in consequence of prior intravenous injection of macroparticulate silica, so as to destroy phagocytic function of macrophages, or in mice exceptionally susceptible because of a genetic defect in ability to respond to lipopolysaccharide, i.e., strain C3H/Heg. A non-reverting aromatic-dependent derivative thus obtained, number SL3261, has been shown to be non-virulent for mice and calves, by parenteral or oral routes. In addition, a derivative of type aroA::Tn10, similarly derived by transduction from a calf-virulent strain of the bovine-pathogenic species, Salmonella dublin, has been shown to be non-virulent for mice; and aroA::Tn10 derivatives, presumably non-virulent, have been made by transduction from several recently isolated strains of the human pathogen, Salmonella typhi.

The aro^- live-vaccine S. typhimurium SL1479 was prepared as follows. The parent strain was S. typhimurium UCD108-11 (Dr. Bradford Smith, University of California at Davis). A calf-passaged re-isolate of strain UCD108-11 was assigned stock number SL1420 and characterized as being nutritionally non-exacting, resistant to various antibiotics including tetracycline and smooth but resistant to the O-specific general transducing phages, including P22. Strain SL1420 was exposed to a transducing phage grown on an aroA554::Tn10 strain of S. typhimurium and selection made for increased resistance to tetracycline, by plating on a nutrient agar medium with tetracycline (50 μg/ml). Representative transductants of increased tetracycline resistance, aromatic-dependent and unaltered in phage sensitivity pattern were selected and one chosen and designated as strain SL1421. The parent strain grew well on Bochner medium (Bochner et al. (1980) J. Bact. 143:926-933) suggesting that the tetracycline resistance was determined by a mechanism other than the resistance conferred by Tn10. Strain SL1421 grew poorly on Bochner medium allowing for selection of colonies developing on plates of Bochner medium supplemented with dihydroxybenzoic acid. One variant was found to be aromatic-dependent and unaltered in phage pattern and did not yield aromatic-independent revertants at a detectable frequency (zero yield of Aro⁺ in a final population of approximately 9×10¹⁰ bacteria on plates of medium with a growth-limiting content of tryptophan). This mutant designated SL1452 was tested for virulence. Five BALB/c mice (males, age approximately 18 weeks old) each received about 3.5×10⁶ live bacteria of strain SL1449 by intraperitoneal (i.p.) injection.

The mice used were from a colony of the inbred line BALB/c, Caesarian-derived and barrier-maintained with a defined gut flora, in the Department of Radiology, Stanford University School of Medicine. The mice of this line have known high susceptibility to S. typhimurium infection and known inability to be protected against such infection by immunization with heat-killed vaccine (Robson and Vas (1972) J. Infect. Dis. 126:378-386). All survived to day 50 and looked well throughout.

To test the immunizing ability of strain SL1452, the five survivors of i.p. injection were challenged seven weeks after vaccination by i.p. injection of about 5×10⁵ bacteria of strain SL1420 (virulent ancestor strain). Four vaccinated control mice died on days 4, 4, 4, and 5 after challenge. One of the five vaccinated mice died by S. typhimurium infection and the other four vaccinated mice survived and looked well to day 14 after challenge.

A genetic "marker" character was then introduced into the vaccine strain SL1452 to allow for identification. Strain SL1452 was treated with a transducing phage grown on an S. typhimurium strain carrying hisD8557::Tn10 and selection made for increased tetracycline resistance. Representative transductants were found to be of the expected nutritional character, Aro^- HisD^- (i.e. a requirement for histidine not satisfied by histidinol). The selected transductant was designated strain SL1474. This strain was exposed to a transducing phage grown on a hisC527 line of S. typhimurium and selection was made on a defined medium with aromatics and with histidinol as the histidine source. Some transductants were still aromatic-dependent and histidine-requiring, but able to use histidinol in place of histidine (i.e. of the nutritional character expected from replacement of hisD::Tn10 hisC⁺ of the recipient by hisD⁺ hisC527 of the donor). One such transductant numbered SL1479 of constitution UCD108-111 aroA554::Tn10/DI hisC527 was employed for tests in calves (DI-deletion-inversion event).

Ten calves, aged two weeks, were given S. typhimurium live vaccine strain SL1479 by intramuscular (i.m.) injection, usually about 1.5×10⁹ live bacteria as their first vaccination. None of them lost appetite or became seriously ill and only one developed diarrhea. None gave a positive stool culture for Salmonella. Three calves, aged two weeks, were given approximately 1.5×10¹¹ live bacteria of strain SL1479 by mouth as their first vaccination. None of them lost appetite but one developed diarrhea; all three gave positive stool cultures, as expected. Reactions to a second dose of live vaccine, by i.m. or oral route, were no more severe than to the first dose.

To test the protection conferred by vaccination, groups of calves aged five weeks, either non-vaccinated (controls) or vaccinated twice with live S. typhimurium SL1479 by i.m. or oral route, were challenged by oral administration of 1.5×10¹¹ live bacteria of a calf-virulent S. typhimurium strain, usually strain UCD108-111, but some calves were given strain SL1323.

All of 16 challenged control calves showed anorexia and depression; 15 had diarrhea and 14 died. Of seven calves which had had two doses of live vaccine by the i.m. route, three had diarrhea after challenge, two became anorectic and depressed and one died. The differences between control calves and those i.m. vaccinated in respect of death (14 or 16 versus 1 of 7) and of anorexia and depression (16 of 16 versus 2 of 7) are statistically significant (p[probability]--less than 0.001 for each comparison). Of three calves which had had two doses of SL1479 live vaccine by oral route, two had diarrhea after challenge and one was anorectic and depressed; none died. The differences between control and oral-vaccinated calves in respect of deaths (14 of 16 versus 0 of 3) and of anorexia and depression 16 of 16 versus 1 of 3) are statistically significant (p less than 0.01 for each comparison.)

The development of the S. dublin strain SL1438 substantially followed the procedure described for the S. typhimurium described above. The starting strain S. dublin S4454 (Dr. Clifford Wray, Central Veterinary Laboratory of the British Ministry of Agriculture, Weybridge, England) was found to be of the expected nutritional character (i.e. with an incomplete dependence on nicotinic acid, otherwise non-exacting), smooth, but resistant or only slightly sensitive to the general transducing O-specific phages P22, P22h, KB1 and A3, and sensitive to all tested antibiotics, including tetracycline. The strain was assigned number SL1363 and was shown to be virulent by intraperitoneal injection at 4×10⁴ live bacteria in BALB/c mice.

Strain SL1363 was exposed to transducing phage grown on S. typhimurium TT47 of geno type hisD8557::Tn10 and tetracycline-resistant transductants selected. A transductant of HisD^- nutritional phenotype and of unaltered phage sensitivity pattern was selected and designated SL1365 and exposed to phage grown on S. typhimurium strain of genotype hisG46 hisD⁺. Transductants were selected on medium with histidinol as the only histidine source and a transductant selected of unaltered phage pattern and requiring either histidine or histidinol (i.e. hisD⁺ hisG46) and assigned label SL1367. When three BALB/c mice previously deprived of food for several hours were given about 3×10⁷ bacteria of strain SL1367 on a cube of bread, all three died about seven days later. When the same strain was fed to a calf, it caused a rapidly fatal infection and a re-isolate was shown to be unaltered in nutritional phenotype. Strain SL1372 was then treated with phage grown on an aroA554::Tn10 strain of S. typhimurium and selection made for tetracycline resistance. A selected transductant which required aromatic supplements, as well as histidine and nicotinic acid, and had an unaltered phage pattern was designated SL1437. Tetracycline-sensitive mutants of strain SL1437 were selected by incubation on plates of Bochner medium, supplemented with 2,3-dihydroxybenzoic acid, so as to allow synthesis of enterobactin. A tetracycline-sensitive but still Aro^- variant was selected and assigned SL1438 and was shown not to produce aromatic-independent revertants at detectable frequency.

Strain SL1438 was given in different amounts to three groups of five mice i.p. from an overnight, 37° C., not-shaken broth culture. None of the vaccinated mice showed any apparent ill effects, even from 3×16⁶ bacteria, the largest dose given.

To test the immunizing ability of strain SL1438, the mice from the above experiment and also a control group of five non-vaccinated mice were challenged 30 days after vaccination by i.p. injection of 3×10⁵ bacteria of strain SL1372 (i.e. the virulent S. dublin strain made hisG46, re-isolated from a calf infected by feeding). The results compiled after 93 days of challenge, of the control, 5 of 5 died on days 4, 4, 5, 5 and 6. Of those vaccinated at a level of 3×10⁴, 3 of 5 died on days 5, 8 and 8, and 2 of 5 were sick but recovered. Of those vaccinated at a level of 3×10⁵, 2 of 5 died on days 7 and 13 and 3 of 5 looked sick, but recovered. Of those vaccinated at a level of 3×10⁶ live bacteria of strain SL1438, 0 of 5 died and 5 looked well throughout. At the level of 3×10⁶ live bacteria of strain SL1438, a single i.p. dose was found to protect the highly susceptible strain of BALB/c mice.

Five-week-old calves were then employed, either being vaccinated or non-vaccinated as controls, to test the adequacy of the S. dublin strain SL1438 as a live vaccine. Vaccination was by two i.m. doses of 1.5×10⁹ of the bacteria of the subject strain. All the calves were then challenged by administration of 1.5×10¹⁰ bacteria of a calf-virulent S. dublin strain SL1367. All three control calves died. None of the five vaccinated calves died.

The subject procedure can be used with a wide variety of pathogenic organisms by employing the appropriate transposon to achieve aro^- or other mutant derivatives. Other organisms of particular interest are Escherichia, particularly E. coli, Klebsiella particularly K. pneumoniae, or Shigella. By employing an appropriate transposon which is inserted into an appropriate aromatic-biosynthesis gene, techniques such as transduction with an appropriate general transducing phage e.g. P1, can be employed to provide for aro^- or other mutant non-virulent pathogenic organisms, which are non-revertant.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.