Note: Descriptions are shown in the official language in which they were submitted.
WO 94/11024 214 8 8 2 9 PCI/US93/10600
- !-
COMPOSITION PROTECTIVE AGAINST
P. MULTOCIDA PASTEURELLOSIS INFECT:ltON
' .
~ackground of the Invention
Pasteurella multocida has been recognized as an
important veterinary pathogen in disease processes of a
variety of domestic and feral mammals and avian species.
For ~xample, ~. multocida is associated with atrop~
rhinitis and pneumonia of swine and with enzootic
pneumonia in cattle. P. multocida is also the
etiological agent of fowl cholera in avian species. P.
multocida associated diseases cause major economic
losses to the swine, cattle and avian industries. -~
Pasteurellosis or fowl cholera in turkeys is a ~-
highly contagious disease which occurs as a hyperacute,
acute or chronic form. Pasteurella multocida belonging
to different capsule types and somatic serotypes is the
e~iological agent of fowl cholera. The hyperacute and -~
acute forms of the disease are characterized by
septicemia, irreversible lesi~ns in the lung, liver and ~-
spleen, and eventual death caused by the endotoxin of `~
P. multocida. The chronic form is associat~d with high
morbidity and the development of a carrier state. ;
The turkey industry in the United States is
part of the $26 billion poultry industry and it produced
4.9 billion pounds of live weight in 1988 from 242.47
million turkeys, valued at $1.95 billion. The per
capita consumption of turkey meat increased from 6.1
pounds in 1960 to 17.9 pounds in 1990. Death due to
diseases has been a major cause of monetary loss to the
U.S. turkey industry. In 1988 (the latest year for
which statistics are a~ailable), disease cost the U.S.
turkey industry an estimated $222 million, of which 50%
was from respiratory diseases. The National Tur~ey
Federation and the American Association of Avian
Pathologlsts have recognized avian pasteurellosis, also
known as fowl cholera, as one of the three most
important diseases wreaking substantial economic losses
to the U.S. turkey industry through increased mortality,
WO94/11024 PCT/US93/i~60
2 ~ 4~ 8 2 9 2
^ condemnation and medication costs. Carpenter et al.,
Avian Dis., 31:16-2i (1988). 1 ~-
With the recognition of the involvement of
P. multocida in various animal diseases, efforts have
5 been made to prevent this disease by vaccination with an , ,-
array of commerc~ial bacterins and attenuated li-ve
vaccines. Bierer et al.l Poult. Sci., 5l:408-4l6
(lg72). However, efficacy and epidemiologic data
available for these vaccines indicate that they are not
~otally ef~ective in preventing the disease. It is now
established that bacterins prepared from strains grown
in vitro on artificial media, induce pr~tection only
. . .
a~ainst the somatic serotype from which the bacterin is
made, i.e., serotype-specific immunity. Heddleston,
Avian Dis., 6:315-321 (1962); Heddleston et al., A~ian
Dis., l~:626-635 (1970). The immunogen that is
responsible for serotype-specific immunity has been
identified as the lipopolysaccharide, while the
immunogen which induce cross-protective immunity are the ~;
membrane associated proteins, called cross-protection
factor (CPF) immunogens. Heddleston e~ al., Poult.
Sci., 54:217-221 (lg74). ~-
_. _ ,. .
Live vaccines induce cross-protecti~e immunity
against challenge exposure with multiple somatic -
serotypes of P. multocida. Bierer et al., cited supra.
A serious disadvantage often encount0red with the
available li~e Yaccines is that, in previously
compromised turkeys, they actually cause sys~emic
in ection and death. Hofacre et al., National Turkey
Federation Pasteurellosis SYmposium at pages 12-16
(l~89). Another important disadvantage has been the
short duxation of immunity induced by both bacterins and
live vaccines. The protection never lasts beyond four
weeks. In a recent symposium on fowl cholera disease
sponsored by the National Turkey Federation, ss~eral
speakers challenged the researchers to develop a new
generation of superior vaccines that are safe and yet
. .
-".`;
,: .'
W0~4/1l0~4 21 4 8829 PCT/US93/~0600
.". ;-.-
still provide a broad spectrum of protection against all j
16 somatic serotypes of P. multocida. ¦
Thus, there is also a need for a vaccine ¦
specific for pasteurellosis that is simple to
administer, yet provides long-lasting cross-protecti~
immunity without adversely affecting the host. There is
a need for a highly immunogenic avirulent live vaccine
for fowl cholera which can be administered orally.
- -.
Summary of the Inv~ntion
The invention provides vaccines and methods for
protecting an animal against P. mul~ocida associated
pasteurellosis. A ~accine of the invention can be :~
comprised of an ef~ective amount of a stable avirulent
immunogenic P. multocida mutant or a recombinantly
produced P. multocida ~irulence factor in a liquid non-
toxic carrier. The avirulent immunogenic mutants can be
a transposon-mediated mutant or a mutant having at leas~
one genetically modified virulence gene located on a
9.4 kb EcorV fragment of the P. multocida genome. The
methods of the invention include the steps of producing
an avirulent immunogenic mutant and administering an
effective amount of the mutant to protect an animal -~
against pasteurellosis.
~ transposon-mediated mutant can be a
transposon insertion or deletion mutant. The mutant can
be produced by introducing a transposon into the genome
of a virulent strain of P. multocida under conditions
fa~oring integration of the transposon. Suitable
3~ tran~posons include Tnl, Tn3, Tn5, TnphoA, Tn7, Tn9,
TnlO, and functional fragments thereof. The transposon ~ -
insertion mutants are selected for avirulence and the
ability to provide immunity against pasteurellosis.
Especially pre~erred mutants are those that provide
long-lasting cross-protective immunity against
pasteurellosis.
" ~,
W094/llU~4 ~`` PCI/US~60ol~ k
8 ~ 9 ';: ~ :
~ 4 1 ~
An avirulent mutant having a genetically
modified virulence gene located on a 9.4 kb EcorV l,
fragment of the P. multocida genome can be produced by ~;
standard methods of~mutagenesis. The virulence gene can
be genetically modified by transposon insertion or
deleti.on mutagenesis, chemical mutagenesis, restriction ~-
endonuclease and exonuclease mutagenesis, and polymerase --
chain reaction mediated mutagenesis. The mutants so -
produced can then be selected for avirulence and ~-
protection against pas~eurellosis. The genetic
modification to a virulence gene located on a 9 kb EcorV
fragment in the selected mutants can be verified by
standard methods, such as restriction enzyme mapping. -
A gene encoding a virulence factor on a 9.4 kb
15 EcorV fragment can be subcloned and transformed into a -~
suitable host so that a recombinant virulence factor can
be produced. The ~irulence gene i5 subcloned under -
appropriate transcriptional and translational control
regions to provide a high level expression of the ,~-
20 virulence factor. The virulence factor can be -
identified and purified by standard methods. The ~-
virulence factor can then be used to immunize animals -~
and provide protection against pasteurellosis.
The method of the invention provides for
administering an effective amount of the avirulenk
immunogenic P. multocida mutant to an animal to provide
protection against pasteurellosis. The mutant can be
administered by several routes, including the parenteral
routej nasal drops, aerosol, and preferably in the
drinking watar. The effective amount is that amount of
the mutan~ that provides for protection against
pasteurellosis, and preferably is about 1Oa CFV/ml to
about 109 CFU/ml. A wide variety of animals can ~e
immunized in the method of the invention including
cattle, pigs, ducks, turkeys, and chic~ens. The
preferred animal is thç turkey.
,
,., ".
~ . .
~ ~ w094~0~4 21~8829 PCT/IlS93/l~600
I ;
Brief Description of the Fiqures ' -
I
FIGURE 1 shows a restriction enzyme map of
TnphoA. ~ ;
FIGURE 2 shows Southern blot analysis of DNA
digests of avirulent transposon mediated mutants of P.
multocida.
:`
Detailed Description_of the In~entio~
~he invention provides vaccines and methods for
protecting animals against pasteurellosis including fowl
cholera. A vaccine is comprised of avirulent
immunogenic mutants of P. multocida that can provide ;~
i~munity against P. multocida associated pasteurellosis.
The vaccine can also be comprised of a recombinantly
produced P. multocida factor, and preferably the
virulence factor is a gene product encoded on a 9.4 kb
EcorV fragment of the genome P. multocid~. Once the
avirulent mutant or recombinant virulenc~ factor is
produced, an effective amount of the vaccine is
administered to the anim~l to provide for immunity
against pasteurellosis including fowl cholera.
A. Vaccines
An immunogenic bacterium employed as the active
componeht of a vaccine is a sta~le live avirulent
immunogenic mutant of Pa~teurella_multocida that
provides immu~ity against P. multocida. The mutant can
be administered to an animal without causing disease or
death and preferably provides long-lasting cross- j
protective immunity. The immunogenic bacteria can be a
transposon mediated mutant or a mutant having at least
one genetically modified virulence gene located on a
9.4 kb EcorV fragment of the P. multocida genomeO An ~ -
effective amount of the immunogenic avirulent mutant i ~`
3S~ bacteria or the recombinant virulence factor of the
inve~tion is combined with a physiologically acceptable ;~
non-toxîc liquid vehicle to form the vaccine.
''~'
W094/~l0~4 rCT/US93/~0600 ~ ~
2~ 29 6 ! :::
~ As used herei~;''stable" means that the mutant ll : maintains the desired ~Aaracteristics for multiple
passages through an animal or for multiple generations :
of growth. Preferably, the mutant has a reversion
5 frequency of less than about 10-5 to about 10~l, and more ; .;
preferably less than about 10-6 to about 10-8.
As used herein, "cross-protective immunity"
refers to the capacity of the avirulent immunogenic
1.'~ .,
mutant to protect the immunized animal from infection by
10 multiple virulent serotypes of P. multocida, and :-
preferably the immunogenic mutant protects against all ::~
virulent serotypes.
As us~d herein, "long-lasting immunity" refers
to the capability of the immunogenic mutant to generate
15 an immune response, preferably that lasts from at least ;~
about 6 weeks to about 20 weeks, and more preferably for ~ : -
the lifetime of ~he animal.
As used herein, "an effecti~e amount" is the
amount of immunogenic avirulent mutant or virulence
factor that provides protection of the immunized animal
against P. mul~ocida associated pasteurellosis.
. ..
As used herein, a "transposon" refers to a DNA
sequence that can move from place to place in a genome
by processes which do not re~uire extensive DNA sequence
homology between the transposon and the site of
insertion nor the recombination enzymes need for
classical homologous crossing over.
An immunogenic avirulent mutant bacteria can ~e
a transposon-mediated mutant. The transposon-mediated
30 mutants are those mutants in which a transposon has been ~` ~
inserted or deleted from the genome of a virulent strain ~ ~:
of P. multocida. A transposon:insertion mutant is a ~-
mutant that has at least one transposon ox a functional
fragment thereof inserted in the genome at one or
35 multiple sites. Preferably, the transposon inserts . ~-
randomly in the genome. Transposon insertion mutants -
are then selected for the presence of transposon encoded
-.,.-'.'
,'',`.
21~8829
.. ~ W094/11024 PCT/~S93/10600 -:.
genes. The transposon insertion mutants can then be
further selec~ed for avirulence and for providing
immunity against P. multocida associated pasteurellosi~s
in animals. A transposon deletion mutant can be
produced from avirulent transposon insertion mutants by
selecting for mutants that ha~e lost the transposon
encoded genes but still maintain avirulence and the ~.
a~ility to protect animals against P. multocida .~.
associated pasteurellosis.
The txansposon-mediated mutants can be produced
by introduction of a transposon or functional fragment
thereof into P. multocida and selecting for avirulent
transposon insertion mutants. Suitable transposons are
those that encode a marker gene including Tnl, Tn3, Tn5,
TnphoA, Tn7, Tn~, and TnlO and functional fragments
thereof. The especially preferred transposon is TnphoA.
An avirulent immunogenic mutant can also be a
mutant having at least one genetically modified
virulence gene located on a 9.4 kb EcorV fragment of the ~
20 P. multocida genome. Virulence genes can be identified .~-
:
and mapped by transposon-mediated mutagenesis. A
virulence gene is one that is essentially non~unctional :~
or produces an essentially nonfunctional gene product in
an avirulent mutant but is functional in a virulent
P. multocida strain. An essentially nonfunctional gene
can be one that is not expressed at a level sufficient
.to provide the gene-associated function, including
virulence, to the mutant and/or one which is expressed ! -~
but produces a nonfunctional gene product. An
essentially nonfunctional virulence gene can be
identified by assaying for function, including virulence
of the gene product, and preferably a gene product ~.. --.`
having at least about 10- to about 1000-fold reduction r 5
in function is essentlally nonfunctional. .`
35 Alternatively, the gene pr~duct encoded by the :~:
essentially nonfunctional virulence gene can be . .
identified by a change in physical characteristics of -~;
'''..
, :.
"' `..
21~8829 PC~/US93/l~6olo
8 ~3 Rec'd ~ 1 9 O~T l99~ ,
the gene product including molecular weight, isoelectric
point, and amino acid composition. A preferred mutant
is one that has an essentially nonfunctional virulence
gene encoded on a 9 kb EcorV fragment of the P.
multocida genome. ~;
Once identified, ~irulence genes in virulent
strain of P. multocida can be rendered nonfunctional by
mutations or genetic modifications generated by standard ` :
methods known to those of skill in the art, including
transposon-mediated mutagenesis, chemical mutagenesis,
restriction enzyme andJor exonuclease-mediated
mutagenesis, and the llke. The P. multoclda mutants ~-
having at least one genetically modified virulence gene
are selected by screening for conversion of the virulent
strain of P. multocida into an avirulent strain and for
the ability to protect aga1nst pasteurellosis in -~
animals.
Specific examples of the avirulent mutants of ,-~
20 the invention include the avirulent transposon insertion ; -~
P. multocida mutants designated PmTn-294 and PmTn-396. -
Both mutants are characterized by expression of alkaline
phosphatase activity, loss of resistance to complement ~-
mediated killing, and loss of virulence in turkeys.
Preferred mutants of the invention include a mutant
having the characteristics of ATCC No. 55394, the mutant
is P. mul tocida PmTn 294 deposited with the American `
Type Culture Collection, Rockville, Maryland, on
February 17, 1993, and a mutant having the , -,
characteristics of ATCC No. 55395, the mutant is P. ~ -
mul tocida PmTn 396 deposited with the American Type , ~
Culture Collection, Rockville, Maryland, on February 17, ~ -
1993. . ~
A vaccine of the invention can also be -
comprised of an effective amount of at least one
recombinantly produced virulence factor from P. .
multoclda in a liquid non-toxic vehicle. A virulence - `
,'".~
, . ` ' .
. ~ .
' ,~
21~8829 ,-CI/US '~ 3 / 1 0 6 O O !~
8A $~ ~ec ~ Tf ~ OG I I~Y4 ~:
factor can be a gene product that is essentially '
nonfunctional in avirulent bacterial strain and
functional in a virulent P. multocida strain. The ~ I .
~ ,.
....
J~;~
~''
'"'`'.
.., '
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, ' ;.'`,
- ',~'-., ',
~- .
'` '
. ', '`' :'
'~ME~
WO94/11024 214 8 8 2 9 PCr/U593/10600
g:
9 I .:
virulence factor can be identified as the gene p.roduct
of a virulence gene of P. multocida by methods known to
those of skill in the art including in vitro
transcription translation systems. Alternatively, the
5 virulence factor can be identified by a functional assay ~ ~-
including virulence or by the ability ~o produce ~ -
symptoms and lesions of the disease. A ~irulence factor
that i5 essentially nonfunctional in avirulent mutant
bacteria has at least about a 10- to 1000-fold reduction
in functional activity. Optionally, the virulence
factor can be identified by a change in its physical
characteristics when the same factor is compared between
virulent and avirulent mutants. To produce the
recombinant virulence factor, a virulence gene is cloned
15 from virulent P. multocida strains into an appropriate -
host organism by standard methods, such as described in
Sambrook et al., Nolecular Cloninq- A Laboratory Manual,
Cold Sp.ring Harbor Laboratory, Cold Spring, NY (1983), -~
and the recombinantly produced virulence factor is
expressed. The ~irulence factor is also preferably
selected for long-lasting cross-protective immunity
against pasteurellosis in an animal. An effecti~e
amount of the recombinant virulence factor is an amount
sufficient to provide for protection against
pasteurellosis, preferably about 5 mg/kg to about
10 mg/kg. A preferred recombinantly produced Yirulence
factor is a gene product encoded by a gene on a 9.4 kb
EcorY fragment of the P. multocida genome. `
To use the mutants of the present invention as ~ `
30 a vaccine, cells of the mutant are combined with a ---
suitable physiologically cceptable non-toxic liquid
vehicle. Specific examples of suitable liquid non-toxic
vehicles include buffered salt solutions, 0.85% saline ii
and, preferably, drinking water. The amount of cells i --
included in a given unit dosage form of the vaccine can
vary widely and depends upon factors such as age, weight
and physical condition of the animal. Such factors can
........ ..... . ..... . .... . .
~ ~ 4a~2 9 PCT/US93/10600 j,~ ~
be readily determined by the clinician or veterinarian
employing animal models or other test systems which are
well known to those of skill in the art. Preferably, an
effective amount of the mutant will range from about
5 1 X 106 to 1 x lO1l cfu/ml, and more preferably about
1 x 107 to 1 x 101 cfu!ml. A unit dose of the vaccine
can be administered parenterally, e.g., by subcutaneous
or intramuscular injection, however oral or aerosol ~-
delivery is preferred. The preferred vaccine can be
administered by mixing the mutant in the drinking water
and making the water available to the animals
~lternatively, the vaccine can be administered intra-
nasally by dropping into the nares or by aerosol. In a
preferred version, a vaccine comprised of 107 cfu/ml of a
mutant having the characteristics of ATCC No. 55394 or
ATCC No. 55395 is administered in the drinking water to ~-
turkeys.
The vaccines can be administered to a variety
of animals including cattle, pigs, ducks, turkeys, and
20 chickens to protect against pasteurellosis. The ; -
especially preferred animal is the turkey.
, . ....
B. Methods o Produci~g an Avirulent Tra~sposQn 7 ''
o f P Multocida a~d~Immunizi~g an Animal
2 5 ~ c~ l ~e ~
ThQ invention also provides a method of `-
immunizing an animal against pasteurellosis with a
stahle avirulent immunogenic transposon-mediated mutan~ , -
of P. multocida. The method involves the steps of
producing a stable avirulent immunogenic transposon-
mediated mu~ant and administering an effective amount of ~ ;
the mutant to the animal to provide immunity against P.
multocida associated pasteurellosis.
The preferred stable avirulent immunogenic
transposon-mediated mutants of P. multocida can be -~
produced by transposon-mediated mutagenesis. -
Transposon-mediated mutants include both those that have
a transposon inserted into the bacterial genome, known
',
':
,
WO94/110~4 2 1 ~ 8 8 2 9 PCT/Us93"0600 1~
J
as insertion mutants, and those wAere the transposon has
been inserted and then excised with a portion of the
bacterial gene, creating a nonreverting deletion mutant.
Transposon-mediated mutan~s are then selected for
S a~irulence and for the ability to protect against
pasteurellosis, and preferably for stability.
A transposon insertion mutant of P. multocida
can be produced by standard methods known to those of
skill in the art and as described by Taylor et al., J.
BacterioloqY, 171:1870 (1989). Briefly, a transposon in ~-
a suitable vector is introduced into P. multocida, ;
preferably a virulent strain, under conditions that
favor insertion of the transposon into the genome of the
bacteria~ For example, a transposon can be placed in a
15 suicide vector. A suicide vector is one that can be ~`
introduced into a wide variety of bacteria but is only ~`
capable of replicating in certain types of bacteria.
The inability of the suicide ~ector to replicate favors
selection of bacteria having the transposon inserted "
into the genome. The vector is preferably introduced
into the P. multocida by transconjugation but can also ~`
be introduced by other methods known to those of skill 1--
in the art, such as electroporation or calcium phosphate ~-
precipitation. Once introduced into P. multocida,
transposon insertion mutants are selected by the
presence of transposon encoded marker genes and further l-
selected for avirulence for animals, for protection ~
against pasteurellosis ~nd stability. ; .
As used herein, a "transposon" is a DNA segment , ;
that can move to new locations in DNA molecules by
processes which do not require extensive DNA sequence i `~
homology or recombination~enzymes. Transposons can ~-
include marker genes encoding antibiotic resistance and
transposition enzymes, and are typically bounded by a
region of DNA sequences, known as insertion sequences,
that mediate insertion of the transposon into DNA. For
W0~4/11024 .~ PCT/US93/10600 -~ ~:
12 j
example, the DNA sequences at the termini of insertion
sequence 50 of t~ Tn5 transposon are~
~ ,
5' C~GACTCTTATACACAAGTAGCGTCCTGAACG. . . -
3' GACTGAGAATATGTGTTCATCGCAGGACTTGC. . . ~ .
: .
. . .GCGCAGGGGATCAAGATCTGATCAAGAGACAG tSEQ ID .. -
NO:l) .
. . .CGCGTCCCCTAGTTCTAGACTAGTTCTCTGTC .
' ' ''"'
as reported by Berg et al., Biotechnoloqy, l(5):4l7
(July l983). Transposons can be modified by metho~s
known to those of skill in the art, as long as they
retain the functional ability to insert into DNA. Some .
modified transposons are known as insertion sequences.
All or portions of ~ransposons can insert into one or . -.
more locations in a bacterial genome and, if they insert
into a ~ene, typically form a mutant no longer having
the function associated with that gene. Sui*able ,`-
transposons include Tnl, Tn3, Tn5, TnphoA, Tn7, Tn9,
TnlO, and functional fragments thereof. The especially `~.
preferred transposon is the TnphoA transposon, which
contains the lef~ insertion s~equence of Tn5 lin~ed to .
the gene encoding:a marker gene, such as the gene for
alkaline phosphatase, and an anti~iotic resistance gene,
such as kanamycin resistance and tetracycline .:.~
~esistance. `~.
Suit~ble vectors for introducing the transposon ~-
into P. multocida are vectors~that favor integration of
30 the transposon into the bacterial genome. Specific 7r "`',
examples include suicide plasmids that can coniugate .-~
with but cannot replicate in P. multocida.
~: Alternati~ely, P. multocida can be co-transfor~ed with a -~
plasmid containing the transpo~on and a plasmid of the '~
35 same incompatibility group so that ~he plasmids will not ..
be able to replicate in the cell. The preferred plasmid
is a suicide vector suc:h as pRT733 which can
~::
... ~.. . . . .. .. . .
2148829
: W094/11024 PCT/US93/10600
13
transCQnjUgate but not replicate in P. multocida and ~`
carries the TnphoA transposon.
P. multocida mutants with integrated
transposons are selected by identifying those bacteria ' ~ -
5 having at least one selectable marker gene encoded by ~`
t-he transposon. The selectab]e marker gene can include
antibiotic resistance ~enes, such as kanamycin ; `-~
resistance genes, tetracycline resistance genes, and the `~
like. Other marker genas can include reporter genes,
such as the chloramphenicol acetyltransferase gene t the
alkaline phosphatase gene, the ~-galactosidase gene, and ,~-
the like. The especially preferred marker gene is the
alkaline phosphatase gene because this marker gene
provides for selection of mutants having modified genes ~-
15 encoding membrane or secreted membranes. Alkaline !``
phosphatase is detected as a secreted enzyme and it is
believed that mutants secreting alkaline phosphatase
have the transposon inserted into a gene that encodes a --
membrane or secreted gene product.
The transposon insertion mutants are further
s~lected for avirulence. The avirulent mutants can be
identified by either in vitro or in vivo methods. -~-~
Avirulent mutants can be identified by in vitro assays
that correlate with the in vivo vixulence. A suitable
example includes a complement-mediated lysis assay;
virulent strains of P. multocida are resistant to
complement-mediated lysis, whereas avirulent strains are
susceptible to complement-mediated lysis.
Alternatively, a~irulent mutants can be identified and
30 selected by the inability to cause death or disease in '-
the animal by standard methods.
Avirulent mutants of the in~ention can be
further selected for the ability to protect animals ;
against pasteur~llosis. Different amounts of an
avirulent mutant can be administered to the animal.
~f~er about two weeks, the animals can be examined for
the presence of protective îmmunity specific for P. -
....;
.
WO94/1102~ PC~/US93/1~60
4~ 8 2 g 14
~ multocida by standard methods, including detecting
antibodies by the ELI~A;~`test. The animals can also be -~
challenged with at least one virulent strain of
P. multocida. The avirulent mutants that protect
against pasteurellosis caused by virulent P. multocida
can be identified as well as the effective amount of the
mutant providing protection against the diseas~.
Protection against pasteurellosis can be determined by
comparing the percentage of nonimmunized animals which
die or show symptoms of the disease after challenge with
those that were immunized with the avirulent mutant.
Symptoms of the diseases associated with P. multocida in
each species of animal are well known to those of skill
in the art. An avirulent mutant that protects about
90-lO0~ of animals from death or the symptoms of the
disease is preferred. :
~ An especially preferred avirulent mutant is one
- that provides long-lasting cross-protective immunity -
against pasteurellosis. An avirulent mutant can be
20 selected for provlding c~oss-protective immunity by 'i`
challenging ani~als immunized with the avirulent mutant -
with all of the virulent serotypes and identifying
avirulent mutants that provide protection against some
or all of the virulent serotypes of P. multocida. Long-
lasting immunity can be evaluated by challenging the
animals immunized with the avirulent mutants after
diferent time periods, from about 2 weeks to about
20 weeks. The preferred avirulent mutants can provide
immunity against pasteurellosis from at least about 6
30 weeks up to about 2~ weeks, and the especially preferred -~
mutants provide lifetime Lmmunity against pasteurellosis
for the animal. ~ ~
Preferably, the avirulent mutant of P. ~-
multocida is also a stable mutant. Stable mutants can -
35 be identified by growing the mutant for about lO to -~
50 generations without the loss of desireable
characteristics, such as avirulence and protection
'''.,'`
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~ WO94/l1024 2 1 ~ 8 8 2 ~ PCT~US93/10600 ~ ~
.... . .
. I , . ::
against pasteurellosis. Reversion frequ.ency can also
be measured to determine stability by standard methods
known to those of skill in the art. A stable avirulent ~-mutant of the invention preferably has a reversion
frequency of less than 10-5 to 10-1 and more pre~erably ~.-
of about than 10-6 to about 10-8. Alternatively, the
mutant can be passed through animals for about 10 to
20 passages and examined for the rate of the loss of the `;;
desired characteristics of avirulence and protection
. .
against pasteurellosis. A stable mutant is one that can
be passed through animals for about 10 to 20 passages -
and still maintain the desired characteristics.
... .
The transposon-mediated mutant of the invention -;
can also be aj transposon-mediated deletion mutant. A `
transposon-media~ed del~tion mutant can be selected and
isolated-from the transposon insertion mutants produc~d
and selected as described above. An avirulent
~;,
transposon insertion mutant can be grown under
conditions no longer selecting for the marker gene ~-
20 encoded by the transposon, such as a gene for antibiotic ;~
resistance or for alkaline phosphatase~ Bacteria which
have lost these marker g2nes can be further screened for
maintenance of the avirulence characteri~tic. It is
believed that, at a very low frequency of less than 10-8, `
the transposon can excise from the genome of the
bacteria and, if that excision is not perfect, can carry
some of the DNA sequence from the gene into which the
transposon initially inserted. When the transposon
excises in this manner, a tranæposon-mediated deletion
mutant is created. Transposon-deletion mutants can be
identified and isolated by screening for those mutants
that have lost the marker gene encoded by the transposon
while still maintaining the avirulence characteristic.
Once identified, the transposon deletion mutants can be
further selected for s~ability and for providing long-
lasting cross-protecti~e immunity against
pasteurellosis, as described above.
. z~48~9 PCT/US93/10600~ - r
16
In a pre~erred version, a transposon-mediated
mutant of P. multocida~~is produced. A suicide vector
encoding the TnphoA transposon is introduced into a
virulent strain of P. multocida by transconjugation.
... ..
5 Transconjugates with the TnphoA transposon inserted into
the genome are first selected by ~creening ~or
antibiotic resistance and for secretion of alkaline
phosphatase. ~utants that are resistant to antibiotics
and which secrete alkaline phosphatase are then screened
10 for avirulence in ~i~o and in vitro. An avirulent
mu~ant which secretes alkaline phosphatase, isolated as -
described herein, has been~deposited with the ATCC on -
February 17, 1993 and given Accession No. 55394.
Another avirulent mutant which secretes alkaline -~
15 phosphatase, isolated as described herein, has been -
deposited with the ATCC on February 17, 1993 and given --
Accession No. 55395. The mutant is then screened for
the ability to pro~ide protection against pasteurellosis
in an animal. The avirulent mutant also preferably is
20 stable and provides long-lasting cross-protective ~ -
immunity against pasteurellosis. `~
Once produced, the transposon-mediated mutants `~
of the invention are administered to the animal.
Administration can occur by any one of several routes
25 including parenteral,nasal drops, aerosol, and/or
through the drinking water. An effective dose of each
mutant can be determined as described above, but
preferably is about 108 to 109 CFUjml. The mutants can
be administered to a variety of animal species including
30 cattler pigs, ducks, chickens, and turkeys but is
preferably administered to turkeys.
~ ,~. . .
- .
,
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~;WO94/11024 2~4~829 PCr/US93/106~0
17
C. Method of Producing an Avirulent Immunoge~ic Mutant
Having at Least One Gen~tically Modif ied Virulence
Gene And Immurlizinq Animals A.qainst Pasteurellosis I .
The invention provides a method of immunizing , `
an animal against P. multocida associated pastel1rellosis
with a stable a~irulent immunogenic mutant of P.
multocida wherein the mutant has at least one
genetically modified virulence gene loca~ed in a 9.4 kb
10 EcorV fragment of the P. multocida genome. The method `
involves the steps of producing a stable a~irulent
immunogenic mutant having a genetically modified
~irulence gene and administering an effective amount to ~
an animal to provide immunlty against pasteurellosis. `
The avirulent immunogenic mutants can be
produced by first identifying virulence genes and then
genetically modifying the ~irulence genes located in a
9.4 kb EcorV fragment of the P. multocida genome. ~-~
Virulence genes of P. multocida can be identified by
20 using transposon insertion mutants to identify and to ;~
map the location of P. multocida virulence genes. It is
believed that insertion of a transposon into a gene can
result in inactivation of the gene. Transposon
insertion mutants ~howing a loss of virulence have
25 transposons inserted in genes required for virulence. - -~
The location of a transposon insertion in avirulent
mutants can be detected and mapped by standard methods
including Southern blot hybridi~ation and DNA ;-
sequencing.
Once identified and mapped, the virulence genes
can be genetically modified by standard methods known to ,- -
thosa of skill in the art. A virulence gene is
genetically modified resulting in the avirulence
phenotype of the mutant. The virulence gene can be ~7'- '
35 genetically modified so that ~ the gene is expressed at a
level below that required to produce virulence or it can ~ -
be modified to produce an essentially nonfunctional gene ;~
product.
.
WO94/11024 PCT/US93/10600,
2 ~ 4~ ~ 2 9 18
A genetically modified virulence gene can be
produced by standard methods of mutagenesis. Suitable
methods include transposon-mediated mutagenesis
~insertion or deletion), chemical mutagenesis,
restriction enzyme or exonuclease mutagenesis, and
polymerase chain reaction medlated mutagenesis. The
preferred method for generating the mutants is by
transposon-mediated mutagenesis. A preferred mutant of -~
the invention is a mutant having a genetically modified
10 virulence gene located in a 9.4 kb EcorV fragment of the `-
P. multocida genome.
A genetically modified virulence gene can be
detected by a variety of methods known to those of skill
in the art. The genetic modification can be detected by
a change in restriction enzyme mapping, ribosomal RNA
profile, or by a change detected by direct DNA
sequencing. Alternatively, the genetically modified
virulence gene can be detected by a functional assay for -~
the virulence gene product. A genetically modified
virulence gene preferably~expresses a gene product that
is essentially nonfunctional in the avirulent mutant. ~;
Essentially nonfunctional refers to at least about l0-
to l000-fold reduction of the functional activity of th~
gene product. Optionally, the genetically modified
virulence gene can be identified by a change in the
physico-chemical characteristics of the gene product, ~-~
such as a change in molecular weight, isoelectric point,
or amino acid composition or the like. ~
A mutant having a genetically modified ` ~-
30 virulence gene is also further selected for avirulence ~:
and for protection against pasteurellosis, as described
herein. In addition, the prefèrred mutant is selected
for long-lasting cross-protective immunity against ~ `;~;`
pasteurelloisis as~described~herein.
' `~
'' ~ : ' `,'-',"'
.
''':
~W094/1l024 21~8829 PCT/US93/10600
19 I '.. '
In a preferred version, an avirulent transposon
(TnphoA) insertion mutant, produced as described herein,
can be used to identify and locate a virulence gene of
P~ multocida. A virul~nce gene of P. multocida can be ~`
identified by Southern blot hybridi~ation with th~ pro~e
which hybridi~es to TnphoA sequences. A virulence gene
located on a 9.4 kb EcorV fragment of P. multocida can
be mapped by restriction enzymes and seguenced by direct
DNA sequencing methods. The virulence gene located on
the 9.4 kb EcorV fragment of a virulent P. multocida
strain can then be genetically modified by point `
mutation to generate an a~irulent mu~ant. The mutant is
then selected for a~irulencè and protection against -
pasteurellosis. The genetic modification of a virulence
gene located on the 9.4 kb EcorV fragment can be
verified by standard methods, including restriction
enzyme mapping or DNA sequencing.
Once produced, an avirulent mutant of
P. multocida having at least one genetically modified
virulence gene is administered to animals to provide for
protection against pasteureIlosis. The mutant can be
administered by parenteral route, nasal drops, aerosol,
and~preferably in the drinking water. An ef~ective
amount can be determined by injecting different amounts
of the mutant into~animals and determining the minimum
amount that p~otects against the disease. Preferably,
the effectiv2 dose is about 108 to 109 CFU/ml. The
mutants of P. multocida can be administered to animals
such as cattle, pigs~ ducks, turkeys, and chickens. ~he
preferred animal is the turkey.
D. Method for Clon~ng a P. multoc~-da V~rulsnc~ Ge~e and ~-
Purifvi~q Rec~o~binantly Produced Virulence_Factor
The invention provides a vaccine comprised of a
recombinant P. multocida virulence factor encoded in a
9.4 kb EcorY fragment of P. multocida. The recombinant
~irulence factor can be produced by cloning a gene
:~-
L ~
WO94~ 4 PCT/US93/10600 ~
~,~.488~9
encoding a virulence gene into a suitable host by , -.
standard methodsr as described in Sambrook et al., cited ... -
supra. The recombinantly produced virulence factor can . ~
then be identified and purified from the host cell.
For example, a virulence gene located on a .. : `
9.4 ~b EcorV fragment,.isolated.as described herein, can
be subcloned into a vector such as the plasmid pBR322. .
The virul~nce gene is preferably subcloned at a location ~
in the pBR322 such ~hat it i5 under the control of the
lO appropriate transcriptional and translational control :.
regions to provide for a high level of gene expression .. ~:~
in ~he host cell. The subcloned virulence gene can be .~
introduced into a suitable host, such as E. coli.and -~.
expression of the subcloned virulence gene can be
monitored by standard methods, including Western blot
using an antibody such as pasteurellosis convalescent
serum. The recombinant virulence factor can be isolated ~.
and purifîed from E coli cell lysates by standard
methods, including affinity, size exclusion, and/or HPLC
chromatography.
The virulence factor can then be tested for the
ability to protect against pasteurellosis by immunizing
an animal with different amounts of the purified .
xecombinant virulence factor. The immunized animals can .:~
25 be analyzed for the developme~t of protective antibody .~.
response by standard methods, including E~ISA. The
immunized animals are also challenged with at least on
.virulent serotype of P. multocida to validate whether
the virulence factor provides protective immunity . ~.
against pasteurellosis. The virulence fartor of the
invention provides for protection against pasteurellosis ~ .
and preferably long-1asting cross protec~ive immunity.
., ~ ..
'--~,:
., ,-.
,. W094/11024 2148829 PCI/US93/10600 i~
21 1,
EXAMPLE 1 ! -
Ganerat~ on of TnphoA Muta~ts of Pasteurella multocida ¦ :
Nutants of Pasteurella multocida were generated
~y transposon mutagenesis. The transposon utilized was
a modified Tn5 (TnphoA) carrying the left insertion
sequence of Tn5 linked to the gene for alkaline
phosphatase without the natural promoter or signal
sequences for the alkaline phosphatase gene. The
transposon is present in a plasmid pRT733 which is a
pGM703.1 derivativa carrying the TnphoA and kanamycin
resistance gene and is available from J. Mekalanos,
Department of Microbiology and Molecular Genetics, -
Harvard Medical School. The plasmid pRT733 is a broad
host range suicide vector. The plasmid can conjugate
with a wide variety of bacteria but is only capable of
replicating in those bacterial strains carrying the
A-pir transducing phage. The plasmid cannot replicate
without a protein encoded by the A-pir transducing
phage. The alkaline phosphatase gene, when inserted
into the bacterial genome along with the transposon,
serves as a marker for genes that encode secreted,
excreted and membrane bound proteins. The alkaline
phosphatase is only active when excreted and has shown '`~
to be active as a fusion protein. -
The pRT733 plasmid was introduced into a -~
virulent complement resistance streptomycin resistant
recipient strain of P. multocida designated Pm-P1059tSmR) ` -~
and mutants containing transpositions were selected in a ' ~;~
single step. E. coIi R12SMlO lysogenized with A-pir
carrying pRT733 were mated with Pm-P1059(SmR) overnight
at 37C on an LB plateO Pm-P1059(Sm~) insertion mutants ,
were selected on LB plates containing streptomycin
(100 ~g/ml) and kanamycin (225 ~g/ml)~ Selected
colonies were then incubated on LB plates containing the s
antibiotics and 5-bromo-4-chloro-3-
indolyl-phospha~e-P-toluidine-(XP) (20 ~g/ml for 18-24
hours. The XP is a chromogenic substrate for the
~4~a~9 2~ PC~/US93/]060~
; j .
alkaline phosphatase e~zyme and indicates the presence
~.... I ,.
of secreted alkaline phosphatase by the mutant. Blue j
colonies were indicative of insertion mutants secretinq
alkaline phosphatase.
Forty-two TnphoA insertion mutants were
isolated. The ~nphoA mutants were screened for alkaline
phosphatase activity, expression of fusion proteins, `;
expression of iron-regulated outer mem~rane proteins, i~;~
loss of complement resistance, and loss of virulence for
turkeys. Alkaline phosphatase activity as a fusion
protein was measured with the chromogenic substrate XP
or P-nitrophenol as described in Taylor et al.,
Bacteriol., 171:1870 ~1989). The iron-regulated
outer membrane proteins having molecular weights of 94
kDa, 84 kDa, and 76 kDa were de~ected by standard
Western blot methods using antisera specific for these
iron-regulated membrane proteins. `~
Two mutants, designated PmTn-294 and PmTn-396,
were positive for alkaline phosphatase activity,
20 expression of fusion proteins and iron-regulated outer `~
membrane proteins. These two mutants were further
characterized for virulence in turkeys.
E:XAMPI.E 2 ~.
Ide tification of Av~ rulent TnE~oA Mutants
The transposition insertion mutants PmTn-294
and PmTn-396 were screened for resistance to complement~
mediated lysis and for ~irulence in turkeys. Re~istance
to complement mediated lysis correlates with ~irulence
in vivo~ The parent P-1059 wild strainsl Pm-P1059 and ¦
Pm-P1059(SmR), were resistant to complement-mediated
lysis and cau~ed fatal dise~se in 100% of turkeys within
18 hours.
Both PmTn-294 and PmTn-396 were susceptible to I -
35 complement mediated lysisO About 1 x 108 cells/ml of :~
PmTn-294 and PmTn-396 cells were incubated with 5 ml of
turkey plasma containing complement and incubated for
:``' ``
,.
~ W094/11024 2148~29 PCT/US93/1060~ ~
23 j-
l hour at 40C. After incubation, a sample of the
PmTIl-294 and PmTn-396 cells was serially diluted and
plated. After 24 hours of incubation, the number of
via~le bacteria present after treatment with complement
5 containing turkey plasma was determined by plate counts. J`` ;
~oth the PmTn~294 and PmTn-396 showed a 3-fold decreas~
in viable cells after treatment with complement when
compared tG the control complement resistant Pm-Pl059
strain. -
For in vivo virulence testing, groups of five
l~week old turkey poults were inoculated intravenously
with 5 x 104 colony forming units (CFU) of transposon ~;
insertion mutants or the virulent Pm-Pl053 strain. The
poults were observed for 8 weeks for the presence of - ~-;
15 disease. All dead turkeys were subjected to postmortem --
and bacteriological examination to establish the
presence of pasteurellosis disease. One hundred percent
(100%) of the poults infected with the virulent Pm-Pl059
strain died and 100% also showed symptoms of the disease
before death. However, infection of poults with either
PmTn-294 or PmTn-396 did not result in death or
development of the disease. The avirulent mutants are
being characterized further to determine the site of the ~-
transposon insertion by Southern hybridization. -~
~XAMP~E 3 `~
Ide~tificatio~ of the Locatio~ of
a Virule~ce ~ene of P. Multo~ida : -~
:
'
To identify the location of a virulence gene 1 -
inactivated by insertion of the TnphoA transposon and to
confirm the presence of the TnphoA transposon in the two
insertion mutants, genomic DNA ~as analyzed by Southern
blot hybridization by standard methods.
DNA was obtained from the wild-type virulent
P. multocida Pm-Pl059, the recipient streptomycin-
resistant P. multocida l059 strain (Pm-Pl059 SmR), the
donor E. coli strain carrying pRT733 (TnphoA
:
W094/1]024 PCT/US93/106~
9 1; ~
transposon) r PmTn-29.4~(TnphoA insertion mutant), and
PmTn-396 (Tnpho~ ~sertion mutant) was digested with ¦
either KpnI or EcorV. DNA restriction fragments were~
separated by gel electrophoresis and probed using a
S EcorI-XhoI digested 1.3 kb fragment or DraI-HpaI
digeE.t~ed 7 kb fragment.from pRT733. The restriction ma? :~
of the TnphoA transposon is shown in Fig. 1. The 1.3 kb
probe is a EcorI-XhoI fragment having a DNA sequence -
located between two portions of the left insertion 50 ~
10 sequence of the TnphoA transposon. The 7 kb probe is a .. ~.
DraI-HpaI probe encoding portions of the left insertion -.
sequence and the right insertion seguence and the
kanamycin resistance gene. ~
The results of the Southern blot hybridization , ~:
15 are shown in Fig. 2. DNA from the Pm-PlOS9, rPcipient ::-
Pm-P1059 SmR, and the PmTn-294 digested with KpnI did not ;-
hybridize with the 1.3 kb probe. Howe~er, DNA digests .:~
from the donor E. coli carrying pRT733 ~nd PmTn-396 ~
showed identical fragments which hybridized with the ... :
1.3 kb probe. DNA from the Pm-P1059 and rec-ipient
Pm-P1059 SmR digested with EcorV also did not hybridize ~.
with the 1.3 kb probe.~ In contrast, DNA from the ..
transconjugant PmTn-396 digested with EcorV showed two !~'''
- bands at 10.9 kb and 9.4 Kb, which hybridized with the
1.3 kb probe. One band at 9.4 kb from PmTn-294 also
hybridized with the 1.3 kb probe. The DNA EcorV digest `-.
of the pRT733 donor strain showed identical fragments as
that of PmTn-396 which hybridized with the probe. The -.
same results were obtained when the digests were probed --:
with the 7 kb DraI-HpaI fragment from pRT733. --
The results indicate that avirulence is
associated with the insertion of all or a portion of the
TnphoA in a 9 . 4 kb EcorV fragment of the genomic DNA of 6
~ . ViruIence gene or genes present in this . ... :
35 region and inactivated by this insertion will be mapped .. ~-~
by additional restriction enzyme digestion and sequenced :~
, ~ .
21~8829
; WO94/11024 ^ PCT/US93/10600
. 25
by standard methods, as described in San~rook et al.,
cited supra. ~ :~
All patents and publications cited herein are
hereby incorporated by reference. While the present ~ ~
5 invention has been described in connection with the ~, -
preferred embodiment thereof, it will be understood man-y
modifications will be readily apparent to those s~illed ..
in the art, and this application is intended to cover
any~adaptations o.r variations thereof. It is manifestly ~.
lO intended this invention be limited only by the claims .
and equivalents thereof. ~
~,
,.:
~.
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.
7 ~ ~
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1 (
WO94/11024 PCTIUS93~10600 '~ r
?,~4QoQo~19 ~ ~
26
~ .~SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Regents of the University of ~inneso~a
Morrill: Hall
100 Church Street S.E.
Minneapolis, MN 55455 .
U.S.A. .
(ii) TITLE OF INVENTION: COMPOSITION PROTECTIVE AGAINST
P. ~LTOCIDA PASTEURELLOSIS INFECT10N
(iil) NUMBER OF SEQUENCES: 1 .
(iv) CORRESPONDENCE ADDRESS: -:-
(A) ADDRESSEE: ~erchant & Gould -
~B) STREET: 3100 Norwest Center ~.:
(C) CITY: Minneapolis
(D) STATE: MN
(E) COUNTRY: USA
(F) ZIP: 5S402 l.
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Flop~y disk
(B) COMPUTER: IBM PC ~ompatible --.
(C) OPERATING SYSTE~: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version ~1.25 .
(vi) CURRENT APPLICATION DATA~
(A) APPLICATION NUMBER~
(B) FILING DATE: .
(C) CLASSIFICATION: ~-
~vii) PRIOR APPLICATION DATA~
(A) APPLICATIOM NUMBER: US 07/973,070 ~:-
(B) FILING DATE:: 06-NOV-1992
~C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: -
(A) NAME: Woessner, Warren D.
(B) REGISTRATION NUMBER.: 30,440 ; -~
(C) REFERENCE/DOCKET NUNBER: 600.256-WO-01 ~ -
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 612-332-5300 ~ :.
(B) TELEFAX: 612-332-9081 ~ -~
i~ -
; ~
21488~9
., WOg4J~1024 - PCT/US93/10~00
27
(2) INFO~MATION FOR SEQ ID NO.l: ~.
(i) SEQUENCE CHARACTERISTICS~
(A) LENGTH: 64 base pairs . :-
~) TYPE: nucLeic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear ;:~
(ii) MOLECULE TYPE: DNA (genomic)
(vii) IMMEDIATE SOURCE:
(B) CLONE: Termini of insertion sequence 50
of the Tn5 transposon -~
~.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CTGACTCTTA TACACAAGTA GCGTCCTGAA CG . . .32
. . . GCGCAGGGGA TCAAGATCTG ATCAAGAGAC AG 64 -:~
.
'
.
