U.S. patent application number 10/569311 was filed with the patent office on 2007-12-06 for live attenuated bacterial vaccine.
This patent application is currently assigned to The Board of Governors for Higher Education, State Of Rhode Island and Providence Plantations. Invention is credited to Paul S. Cohen.
Application Number | 20070280968 10/569311 |
Document ID | / |
Family ID | 34278638 |
Filed Date | 2007-12-06 |
United States Patent
Application |
20070280968 |
Kind Code |
A1 |
Cohen; Paul S. |
December 6, 2007 |
Live Attenuated Bacterial Vaccine
Abstract
A live, attenuated bacterium of the genus Escherichia, Yersinia
or Salmonella, that does not have a functional tRNA.sup.5.sub.leu,
and vaccines comprising the live, attenuated bacterium.
Inventors: |
Cohen; Paul S.;
(Narragansett, RI) |
Correspondence
Address: |
INTERVET INC.;PATENT DEPARTMENT
PO BOX 318
MILLSBORO
DE
19966-0318
US
|
Assignee: |
The Board of Governors for Higher
Education, State Of Rhode Island and Providence Plantations
Kingston
RI
02881-0811
|
Family ID: |
34278638 |
Appl. No.: |
10/569311 |
Filed: |
August 26, 2004 |
PCT Filed: |
August 26, 2004 |
PCT NO: |
PCT/US04/27896 |
371 Date: |
February 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60498988 |
Aug 29, 2003 |
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60498961 |
Aug 29, 2003 |
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Current U.S.
Class: |
424/257.1 ;
424/234.1; 424/258.1; 435/252.1; 435/252.8 |
Current CPC
Class: |
A61K 2039/523 20130101;
A61P 31/04 20180101; A61P 37/04 20180101; Y02A 50/407 20180101;
A61K 2039/522 20130101; Y02A 50/30 20180101; A61K 39/02 20130101;
A61K 39/0275 20130101; Y02A 50/482 20180101 |
Class at
Publication: |
424/257.1 ;
424/234.1; 424/258.1; 435/252.1; 435/252.8 |
International
Class: |
A61K 39/02 20060101
A61K039/02; A61K 39/108 20060101 A61K039/108; A61K 39/112 20060101
A61K039/112; A61P 31/04 20060101 A61P031/04; C12N 1/20 20060101
C12N001/20 |
Claims
1. A live, attenuated bacterium of the genus Escherichia, Yersinia
or Salmonella, said bacterium not having a functional
tRNA.sub.5.sup.leu.
2. The live, attenuated bacterium according to claim 1, said
bacterium not having a functional tRNA.sub.5.sup.leu as a result of
a mutation in the leux gene.
3. The live, attenuated bacterium according to claim 1, wherein
said bacterium is selected from the group consisting of E. coli, S.
enterica serotype typhimurium, enteritidis, choleraesuis, dublin,
typhi, gallinarum, abortusovi, abortus-equi and pullorum.
4. The live, attenuated bacterium according to claim 2, wherein the
mutation comprises an insertion and/or a deletion.
5. The live, attenuated bacterium according to claim 1, wherein
said bacterium carries a heterologous gene.
6. The live, attenuated bacterium according to claim 5, wherein the
heterologous gene is inserted in the leux gene.
7. A vaccine for the protection of animals and humans against
infection with a pathogenic bacterium or the pathogenic effects
thereof, comprising the bacterium of claim 1 and a pharmaceutically
acceptable carrier.
8. The vaccine according to claim 7, comprising an adjuvant.
9. The vaccine according to claim 7 in a freeze-dried form.
10-11. (canceled)
12. A method for protecting a susceptible human or animal from
infection with a pathogenic bacterium, comprising administering an
effective amount of vaccine according to claim 7.
Description
[0001] The present invention relates to live attenuated bacteria
for use in a medicament, to vaccines based upon such bacteria
useful for the prevention of microbial pathogenesis, to the use of
live attenuated bacteria for the manufacture of such vaccines and
to methods for the preparation of such vaccines.
[0002] Immunity to microbial pathogenesis is one means by which a
warm blooded animal avoids pathogenesis, or suffers a less intense
pathogenic state. Incomplete immunity to a given pathogen results
in morbidity and mortality in a population exposed to a pathogen.
It is generally agreed that vaccines based on live but attenuated
micro-organisms (live attenuated vaccines) induce a highly
effective type of immune response. Such vaccines have the advantage
that, once the animal host has been vaccinated, entry of the
microbial pathogen into the host induces an accelerated recall of
earlier, cell-mediated or humoral immunity which is able to control
further growth of the organism before the infection can assume
clinically significant proportions. Vaccines based on a killed
pathogen (killed vaccine) are generally conceded to be unable to
achieve this type of response. However, contrary to killed
vaccines, vaccines that contain a live pathogen present, depending
on the level of attenuation, the danger that the vaccinated host
upon vaccination may contract the disease against which protection
is being sought.
[0003] Vaccines against bacteria belonging to the closely related
families of Escherichia and Salmonella follow the general rules
given above. Many members of these families of bacteria are
pathogenic due to the fact that they infect the digestive tract and
the bladder. The pathogenic effect of these bacteria is closely
related to their ability to colonise the mucosal layers of the
digestive tract and the bladder. It is the phenomenon of
colonisation that leads to the prolonged presence of the pathogen
in the digestive tract and/or the bladder and to a very close
contact of the pathogen to the mucosal layers, which can also lead
to invasion of other tissues. Thus, at the same time,
paradoxically, its due to the fact that these bacteria colonise the
digestive tract and the bladder, and thus at the same time cause
disease, that the immune system is triggered to develop a certain
level of immune response. This immune response is clearly developed
too late to suppress the pathogenic effect of the colonising
bacteria.
[0004] It would thus be desirable to have a vaccine that possesses
the immunising attributes of a live micro-organism but that is not
capable of causing undesirable side effects upon vaccination.
[0005] For live attenuated vaccines there is however the following
paradox: an approach for attenuating bacteria is the removal of one
or more virulence factors. In most cases however, virulence factors
also play a role in inducing immunity. In those cases, deletion of
virulence factors unavoidably impairs the immunogenic capacities of
the bacterium. This is of course an unwanted situation. A live
vaccine should preferably retain the antigenic complement of the
wild-type strain, without being viulent.
[0006] It is an objective of the present invention to provide live
attenuated vaccines that do not suffer from several of the
drawbacks mentioned above.
[0007] Bacteria of the families Escherichia and Salmonella have
several virulence factors. An example of a single gene involved in
the synthesis of many virulence factors, including those playing a
role in colonisation in both Escherichia and Salmonella, is the
gene encoding LeuX. This gene encodes a specific tRNA:
tRNA.sub.5.sup.leu. SEQ ID NO 1 provides the sequence of a
Salmonella typhimurium leuX gene.
[0008] LeuX and its role in colonisation and fimbriae synthesis has
been described i.a. by Newman et al., (FEMS Microbiology Letters
122: 281-287 (1994)) Newman et al. (Microbial Pathogenesis 17:
301-311 (1994)) and by Collighan, R. J. and Woodward M. J. (vet.
Microbiol. 80: 235-245 (2001))
[0009] Ritter et al., in Mol. Microbiol. 17:109-121 (1995) have
thoroughly analysed the various roles of the LeuX gene product in
virulence. They showed that the presence of LeuX is crucial for the
stimulation of the synthesis of Type 1 fimbriae and flagella, both
involved in motility and colonisation, and is crucial for the
synthesis of proteins involved in iron uptake, for the synthesis of
enterobactin and for in vitro virulence.
[0010] All these characteristics are known to strongly contribute
to virulence. Therefore, they are at the same time the most
important targets for an immune response. An immune response
against fimbriae and flagella would interfere with colonisation and
motility, whereas an immune response against enterobactin and
proteins involved in iron uptake would block toxic effects and
deprive the bacterium from the possibility to obtain essential iron
respectively.
[0011] Thus, from a vaccine point of view, the key virulence
factors of choice to be present in a vaccine would be the virulence
factors expressed in the presence of LeuX. The vaccine of choice
would therefore (preferably) be a subunit vaccine comprising type 1
fimbriae, flagella, enterobactin and proteins involved in iron
uptake. Such a vaccine would first of all be safe, and secondly
most likely induce immunity against these 4 virulence factors and
by doing so provide protection against infection.
[0012] Live attenuated strains from which the LeuX gene is deleted,
do not make the virulence factors mentioned above. If such
LeuX-negative deletion-mutants were to be used in a vaccine, they
would not induce protection against these most crucial virulence
factors: type 1 fimbriae, flagella, enterobactin and proteins
involved in iron uptake.
[0013] Given this fact, LeuX is to be considered as a highly
unattractive candidate for deletion in a live attenuated vaccine
strain.
[0014] Moreover, bacteria that lack colonising abilities (due to
lack of type 1 fimbriae and flagella) as would be the case for
LeuX-negative deletion-mutants, will not be expected to come into
close contact with the host cells, and will consequently be assumed
to be washed out quickly. Therefore, they would even not be
expected to induce any substantial immunity against those virulence
factors that would still be present in the absence of the LeuX gene
product.
[0015] Surprisingly, however, it was found now that bacterial
strains, both from the family of Escherichia and Salmonella, not
having a functional tRNA.sub.5.sup.leu are very well capable of
inducing a protective immune response against virulent wild-type
bacteria in the host animal. This is indeed against all
expectations, since they do not induce any immunity against the key
virulence factors mentioned above.
[0016] Therefore, a first embodiment of the present invention
relates to live attenuated bacteria that have no functional
tRNA.sub.5.sup.leu, for use in a vaccine.
[0017] Dobrindt U., et al., (FEMS Microbiology letters 162:135-141
(1998)) have confirmed that it is indeed the LeuX-encoded
tRNA.sub.5.sup.leu that is responsible for e.g. survival of a
uropathogenic E. coli in mouse bladder mucus, and not the presence
of the pathogenicity islands as such. These pathogenicity islands
are the regions that encode e.g. haemolysins, fimbrial adhesins and
so on.
[0018] Due to its key position in the bacterial pathogenicity, the
tRNA.sub.5.sup.leu gene and its gene product tRNA.sub.5.sup.leu are
widespread in the bacterial realm. The tRNA.sub.5.sup.leu is highly
conserved. It can be found in e.g. Escherichia coli and in
Salmonella enterica species, such as serotype yphimurium,
enteritidis, galinarum and dublin and in Yersinia species such as
Y. pestis.
[0019] The gene itself and its complete nucleotide sequence in
Salmonella and Escherichia are discussed i.a. by Thorbjarnardottir,
S. et al. (J. Bacteriology 161: 219-222 (1985)), Yoshimura, M. et
al. (J. Mol. Biol. 177: 627-644 (1984)), Andersson, S. et al.
(Microbiol. Review 54: 198-210 (1990)), Komine, Y. et al., (J. Mol.
Biol. 212: 579-598 (1990)) and Blum, G. et al. (Infect. &
Immun. 62: 606-614 (1994)).
[0020] A functional tRNA.sub.5.sup.leu is understood to be a
tRNA.sub.5.sup.leu having the characteristics of the wild-type
tRNA.sub.5.sup.leu, i.e.: is capable of adding the amino acid
Leucine to a protein strand during its synthesis, if the codon
encoding leucine is UUG. Therefore, a tRNA.sub.5.sup.leu that is
defective in at least this function is considered to be a
non-functional tRNA.sub.5.sup.leu.
[0021] Live attenuated bacteria according to the invention can also
be obtained by introducing a mutation in the LeuX gene that
prevents the synthesis of functional tRNA.sub.5.sup.leu.
[0022] Therefore, a preferred embodiment of the present invention
relates to live attenuated bacteria not having a functional
tRNA.sub.5.sup.leu as a result of a mutation in the leux gene, for
use in a vaccine.
[0023] Such a mutation can be an insertion, a deletion, a
substitution or a combination thereof, provided that the mutation
leads to the failure to express a functional
tRNA.sub.5.sup.leu.
[0024] Live attenuated bacteria for use according to the invention
can be obtained in several ways. One possible way of obtaining such
bacteria is by means of classical methods such as the treatment of
wild-type bacteria having the tRNA.sub.5.sup.leu gene with
mutagenic agents such as base analogues, treatment with ultraviolet
light or temperature treatment.
[0025] Strains according to the invention can be easily selected on
the basis that they would lack at least the four virulence factors
mentioned above.
[0026] The nature of the mutation caused by classical mutation
techniques would however be unknown. This may be a point mutation
in the LeuX gene which may, although this is unlikely to happen,
eventually revert to wild-type.
[0027] In order to avoid this small risk, transposon mutagenesis
would be a good alternative. Mutagenesis by transposon mutagenesis,
is also a mutagenesis-technique well-known in the art. This is a
mutation accomplished at a localised site in the chromosome.
Transposon-insertions can not be targeted to a specific gene. It is
however easy to pick up the LeuX mutants since they would lack at
least the four virulence factors mentioned above.
[0028] A much more elegant possibility to introduce a mutation, now
at a predetermined site, rather deliberately than randomly, is
offered by recombinant DNA-technology. Such a mutation may again be
an insertion, a deletion, a replacement of one nucleotide by
another one or a combination thereof, with the only proviso that
the mutated gene no longer encodes functional tRNA.sub.5.sup.leu.
Such a mutation can e.g. be made by deletion of a number of base
pairs. Even very small deletions such a stretches of 10 base pairs
can already render tRNA.sub.5.sup.leu non-functional. Even the
deletion of one single base pair may already lead to a
non-functional tRNA.sub.5.sup.leu. More preferably, a longer
stretch is removed, e.g. 50 or more base pairs. Even more
preferably, the whole tRNA.sub.5.sup.leu gene is deleted.
[0029] All techniques for the construction of
tRNA.sub.5.sup.leu-negative mutants are well-known standard
techniques. They relate to cloning of the tRNA.sub.5.sup.leu gene,
modification of the gene sequence by site-directed mutagenesis,
restriction enzyme digestion followed by re-ligation or
PCR-approaches and to subsequent replacement of the wild type
tRNA.sub.5.sup.leu gene with the mutant gene (alletic exchange or
allelic replacement). Standard recombinant DNA techniques such as
cloning the tRNA.sub.5.sup.leu gene in a plasmid, digestion of the
gene with a restriction enzyme, followed by endonuclease treatment,
re-ligation and homologous recombination in the host strain, are
all known in the art and described i.a. in Maniatis/Sambrook
(Sambrook, J. et al. Molecular cloning: a laboratory manual. ISBN
0-87969-309-6). Site-directed mutations can e.g. be made by means
of in vitro site directed mutagenesis using the Transformer.RTM.
kit sold by Clontech. PCR-techniques are extensively described in
(Dieffenbach & Dreksler; PCR primers, a laboratory manual. ISBN
0-87969-447-3 and ISBN 0-87969-447-5).
[0030] The tRNA.sub.5.sup.leu gene comprises not only the coding
sequence encoding tRNA.sub.5.sup.leu, but also regulatory sequences
such as the promoter. Therefore, not only mutations in the coding
regions but also mutations in those sequences essential for correct
transcription are considered to fall within the scope of the
invention.
[0031] In a more preferred embodiment, the invention relates to
live attenuated bacteria of the genera Escherichia and
Salmonella.
[0032] In an even more preferred form of the invention, the live
attenuated bacterium according to the invention is selected from
the group consisting of S. enterica serotype typhimurium,
enteritidis, choleraesuis, dublin, typhi, gailinarum, abortusovi,
abortus-equi, pullorum, E. coli or Y. pestis. These bacterial
genera comprise a large number of species that are pathogenic to
both humans and a variety of different animals.
[0033] In a still even more preferred form thereof, the live
attenuated bacterium according to the invention is S. enterica, E.
coli or Y. pestis.
[0034] In an even further preferred form, this embodiment relates
to live attenuated bacteria according to the invention in which the
mutation in the tRNA.sub.5.sup.leu gene has been made by
recombinant DNA technology.
[0035] Well-defined and deliberately made mutations involving the
deletion of fragments of the tRNA.sub.5.sup.leu gene or even the
whole gene or the insertion of heterologous DNA-fragments or both,
have the advantage, in comparison to classically induced mutations,
that they will not revert to the wild-type situation.
[0036] Thus, in a most preferred form, this embodiment of the
invention refers to live attenuated bacteria in which the
tRNA.sub.5.sup.leu gene comprises an insertion and/or a
deletion.
[0037] Given the large amount of vaccines given nowadays to both
pets and farm animals, it is clear that combined administration of
several vaccines would be desirable, if only for reasons of
decreased vaccination costs. It is therefore very attractive to use
live attenuated bacteria as a recombinant carrier for heterologous
genes, encoding antigens selected from other pathogenic
micro-organisms or viruses. Administration of such a recombinant
carrier has the advantage that immunity is induced against two or
more diseases at the same time. The live attenuated bacteria for
use in a vaccine, according to the present invention provide very
suitable carriers for heterologous genes, since the gene encoding
tRNA.sub.5.sup.leu can be used as an insertion site for such
heterologous genes. The use of the tRNA.sub.5.sup.leu gene as an
insertion site has the advantage that at the same time the
tRNA.sub.5.sup.leu gene is inactivated and the newly introduced
heterologous gene can be expressed (in concert with the homologous
bacterial genes). The construction of such recombinant carriers can
be done routinely, using standard molecular biology techniques such
as allelic exchange. Therefore, another embodiment of the invention
relates to live attenuated recombinant carrier bacteria, preferably
of the genera Escherichia, Salmonella and Yersinia that do not
produce a functional tRNA.sub.5.sup.leu, and in which a
heterologous gene is inserted.
[0038] Such a heterologous gene can, as mentioned above, e.g. be a
gene encoding an antigen selected from other pathogenic
micro-organisms or viruses. Such genes can e.g. be derived from
pathogenic herpesviruses (e.g. the genes encoding the structural
proteins of herpesviruses), retroviruses (e.g. the gp160 envelope
protein), adenoviruses and the like.
[0039] Also a heterologous gene can be obtained from pathogenic
bacteria. As an example, genes encoding protective antigens such as
bacterial toxins like Actinobacillus pleuropneumoniae toxins,
Clostridium toxins, outer membrane proteins and the like are very
suitable bacterial heterologous genes.
[0040] Another possibility is to insert a gene encoding a protein
involved in triggering the immune system, such as a cytokine, an
interleukin or an interferon, or another gene involved in
immune-regulation.
[0041] Insertion of the heterologous gene in the tRNA.sub.5.sup.leu
gene is advantageous, since in that case there is no need to find a
new suitable insertion site for the heterologous gene, and at the
same time the tRNA.sub.5.sup.leu gene is knocked out.
[0042] Thus, in a preferred form of this embodiment the
heterologous gene is inserted in the tRNA.sub.5.sup.leu gene. The
heterologous gene can be inserted somewhere in the
tRNA.sub.5.sup.leu gene or it can be inserted at the site of the
tRNA.sub.5.sup.leu gene while this gene has been partially or
completely deleted.
[0043] Because of their unexpected attenuated but immunogenic
character in vivo, the bacteria for use in a vaccine, according to
the invention are very suitable as a basis for live attenuated
vaccines. Thus, still another embodiment of the invention relates
to such live attenuated vaccines for the protection of animals and
humans against Escherichia, Yersinia or Salmonella infection or the
pathogenic effects thereof, that comprise a bacterium of which the
wild type form comprises a tRNA.sub.5.sup.leu gene.
[0044] Such vaccines comprise an immunogenically effective amount
of a live attenuated bacterium according to the invention or a live
recombinant carrier bacterium according to the invention, and a
pharmaceutically acceptable carrier.
[0045] Preferably, the vaccine comprises a live attenuated
bacterium according to the invention, selected from the group of
Escherichia, Salmonella and Yersinia.
[0046] Immunogenically effective means that the amount of live
attenuated bacteria administered at vaccination is sufficient to
induce in the host an effective immune response against virulent
forms of the bacterium.
[0047] In addition to an immunogenically effective amount of the
live attenuated bacterium described above, a vaccine according to
the present invention also contains a pharmaceutically acceptable
carrier. Such a carrier may be as simple as water, but it may e.g.
also comprise culture fluid in which the bacteria were cultured.
Another suitable carrier is e.g. a solution of physiological salt
concentration.
[0048] The useful dosage to be administered will vary depending on
the age, weight and animal vaccinated, the mode of administration
and the type of pathogen against which vaccination is sought.
[0049] The vaccine may comprise any dose of bacteria, sufficient to
evoke an immune response.
[0050] Doses ranging between 10.sup.3 and 10.sup.10 bacteria are
e.g. very suitable doses.
[0051] Optionally, one or more compounds having adjuvant activity
may be added to the vaccine. Adjuvants are non-specific stimulators
of the immune system. They enhance the immune response of the host
to the vaccine. Examples of adjuvants known in the art are Freunds
Complete and Incomplete adjuvant, vitamin E, non-ionic block
polymers, muramyldipeptides, ISCOMs (immune stimulating complexes,
cf. for instance European Patent EP 109942), Saponins, mineral oil,
vegetable oil, and Carbopol.
[0052] Adjuvants, specially suitable for mucosal application are
e.g. the E. coli heat-labile toxin (LT) or Cholera toxin (CT).
[0053] Other suitable adjuvants are for example aluminium
hydroxide, aluminium phosphate or aluminium oxide, oil-emulsions
(e.g. of Bayol F.sup.(R) or Marcol 52.sup.(R)), saponins or
vitamin-E solubilisate.
[0054] Therefore, in a preferred form, the vaccines according to
the present invention comprise an adjuvant.
[0055] Other examples of pharmaceutically acceptable carriers or
diluents useful in the present invention include stabilisers such
as SPGA, carbohydrates (e.g. sorbitol, mannitol, starch, sucrose,
glucose, dextran), proteins such as albumin or casein, protein
containing agents such as bovine serum or skimmed milk and buffers
(e.g. phosphate buffer). Especially when such stabilisers are added
to the vaccine, the vaccine is very suitable for freeze-drying.
Therefore, in a more preferred form, the vaccine is in a
freeze-dried form.
[0056] Still another embodiment relates to the use of a bacterium
according to the invention for the manufacture of a vaccine for the
protection of animals and humans against infection with a wild type
bacterium or the pathogenic effects of infection.
[0057] For administration to animals or humans, the vaccine
according to the present invention can be given inter alia
intranasally, intradermally, subcutaneously, orally, by aerosol or
intramuscularly. For application to poultry, wing web and eye-drop
administration are very suitable.
[0058] The skilled person would know how to administer a vaccine
according to the invention, because the method would most likely
not differ from the methods followed for vaccination with presently
existing bacterial vaccines. A vaccine according to the invention,
especially when it comprises bacteria belonging to the family of E.
coli, Salmonella or Yersinia would preferably be given orally.
[0059] Still another embodiment of the invention relates to methods
for the preparation of a vaccine according to the invention. Such
methods comprise the admixing of a live attenuated bacterium
according to the invention or a live recombinant carrier bacterium
according to the invention, and a pharmaceutically acceptable
carrier.
EXAMPLES
Example 1
Construction of leuX Mutants
[0060] The Wanner system (Proc. Natl. Acad. Sci. U.S.A. Jun. 6,
2000. 97(12):6640-45.) was used to replace the S. typhimurium SR-11
leuX gene with a kanamycin resistance gene. A wild-type S.
typhimurium; SR-11 was used in this example, but the principle
described is equally applicable to all bacteria carrying the leuX
gene.
[0061] Sequences homologous to S. typhimurium SR-11 leuX flanking
sequences were added to the 5' end of primers to a kanamycin
cassette. Forward Primer: 5' GGT ATA ATC CAC MC GTT TTCCGC ATA CCT
CTT CAG gtgtaggctggagctgcttcg and Reverse Primer: 5' MA AAG CCA CGA
TTA GGC AGC TAA TTA TTG CAT CAC catatgaatatcctccttag (upper case
letters are leuX sequences and lower case letters are the kanamycin
primers) were used to PCR the kanamycin cassette from the plasmid
pKD4. To amplify the kanamycin (.about.1700 bp) cassette from the
plasmid pKD4, the primer set containing the priming sites were used
in a standard PCR reaction with Fisher Taq DNA polymerase (1.5 mM
MgCl.sub.2). Cycling conditions were 1.times.94.degree. C. 4 min;
35.times.94.degree. C. 30 sec, 55.degree. C. 15 sec, 72.degree. C.
75-105 sec; 1.times.72.degree. C. 7 min. Eight 100 .mu.l reactions
were pooled, 5 .mu.l was checked on a gel, and the linear PCR
product was ethanol precipitated and resuspended in 2-4 .mu.l of
water.
[0062] S. typhimurium SR-11 cells were electroporated with the
temperature sensitive plasmid pKD46. When these cells are grown at
30.degree. C. in the presence of arabinose the plasmid expresses
the lambda Red recombinase. The cells (A.sub.600=0.6) were made
competent for electroporation by centrifugation and washing
3-4.times. with cold 10% glycerol. The linear PCR product was then
electroporated into the competent cells. The recombinase acts to
replace the wild type leuX gene with the deleted leuX gene
containing the kanamycin cassette. S. typhimurium SR-11 clones
containing the deleted leuX gene were selected by overnight growth
at 37.degree. C. on Luria agar plates containing kanamycin (40
.mu.g/ml), which also resulted in loss of the temperature sensitive
pKD46 plasmid.
[0063] Primers 5' and 3' of the leuX deletion/antibiotic cassette
insertion, i.e. upstream and downstream, were used to verify the
mutants. For the S. typhimurium SR-11 leuX mutant, primers
containing a PstI site and homologous to regions 5' and 3' of the
leuX gene (the primers are called Pst Leux 5': ctagctgcag
gcgttaatctgctggagaaggc; and Pst Leux 3': ctagctgcag
acgaccaacacggaaagaccac) were used to amplify the expected
approximate 1700 bp band (versus a 400 bp wildtype band). 2.5 mM
MgCl.sub.2, with Finnzyme DyNAzymelI polymerase was used in the
reaction. Cycling conditions were 1.times.94.degree. C. 4 min;
30.times.94.degree. C. 30 sec, 55.degree. C. 30 sec, 72.degree. C.
105 sec; 1.times.72.degree. C. 7 min.
Example 2
Animal Tests
Experimental Design
[0064] To test both safety and efficacy, broilers were inoculated
by spray on day of hatch and inoculated orally at 15 days of age
with approximately 10.sup.7 CFU of SR11-LeuX.sup.(-) (The LeuX-
minus mutant).
[0065] Safety was assessed by clinical observation after
vaccination. Also, cloacal swabs were taken at days 8, 15, 22 and
30 to determine the presence of the vaccine strain in the
intestinal tract. Swabs were used to inoculate Brilliant Green
Agars (BGA) directly and after enrichment in Rappaport Vassiliades
Broth. At 30 days of age, 5 vaccinated animals were necropsied and
their livers and spleens were cultured to determine if the vaccine
strain had invaded from the intestinal track.
[0066] To test for efficacy, the animals received an oral challenge
infection with 1.4.times.10.sup.6 CFU of a tetracycline resistant
wild-type S.t. strain at 30 days of age. Two weeks after challenge
infection, the animals were euthanized and the livers, spleens,
cloacal swabs and swabs of the cecum contents were cultured for the
challenge strain. Organs and swabs were inoculated on BGA
containing tetracycline (BGAtet) directly and also after incubation
in an enrichment medium (buffered peptone water containing
tetracycline).
Animals
[0067] Hatching eggs were obtained from a Salmonella free broiler
breeder flock.
Results
[0068] No clinical abnormalities were observed after the spray and
oral vaccinations. The SR11-LeuX.sup.(-) strain was cultured from
cloacal swabs of some of the vaccinated animals on days 8, 15, 22
and 30. At 30 days of age, the vaccine strain was still shed by 53%
of the vaccinated animals, but it was not reisolated from the
livers and spleens of the 5 animals that were necropsied.
[0069] As shown in Table 1, the challenge strain was isolated from
the liver and spleen of non-vaccinated animals. In addition, the
challenge strain was reisolated from the cloaca and caecum of
practically all non-vaccinated control animals.
[0070] Vaccination with SR11-LeuX.sup.(-) resulted in complete
clearance of the challenge strain 14 days post infection. In
addition, no challenge strain was found in the liver and spleen of
vaccinated animals.
[0071] The results show, that LeuX.sup.(-) bacteria, even if the
LeuX.sup.(-) mutation is the only attenuating mutation, are safe
and efficacious in live vaccines. TABLE-US-00001 TABLE 1 S.t.
(tet.sup.r) positive Group SR11-LeuX.sup.(-) Control Spleen
0/14.sup.a 10/12 Liver 0/14.sup.a 10/12 Cloaca 0/14.sup.a 8/12
Caecum 0/14.sup.a 11/12 .sup.asignificantly different from control
(p < 0.05, Fisher exact test)
[0072]
Sequence CWU 1
1
1 1 210 DNA Salmonella typhimurium 1 acttagcgat acaggtgtat
gctcgtgctt gcatggtggc gtatgacagg tataatccac 60 aacgttttcc
gcatacctct tcagtgccga agtggcgaaa tcggtagacg cagttgattc 120
aaaatcaacc gtagaaatac gtgccggttc gagtccggcc ttcggcacca agtgatgcaa
180 taattagctg cctaatggtg gctttttttg 210
* * * * *