Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W095/25742 PCTAKs5/00185
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--1--
PAST~U~r ~ A~R~R ANTIGEN8 AND RELATED VACCIN~8
~ackground of the Invention
Animal vaccines designed to protect against pneumonias
caused by Pasteurellaceae are generally produced from
inactivated whole bacteria or extracts of bacterial
cultures. The protective potential of potassium thiocyanate
extracts of culture-grown Pasteurella multocida have been
investigated in mice, chickens, cattle and rabbits. These
extracts contained protein, hyaluronic acid,
lipopolysaccharide, DNA and RNA, making interpretation of
the protective component difficult. Although some cross-
protection has been observed, protection was mainly against
homologous challenge.
The immunogenic outer membrane proteins expressed by a
rabbit isolate of Pasteurella multocida grown in culture
have also been investigated. The major antibody response
appeared to be directed against outer membrane polypeptides
having molecular masses of 27 kD, 37.5 kD, 49.5 kD, 58.7 kD
and 64.4 kD (Lu et al. (1988) Infect Immun 56:1532-1537).
Further work demonstrated that a monoclonal antibody
specific for the 37.5 kD protein could passively protect
mice and rabbits from challenge, if the isolate used for
challenge expressed the antigenic determinant recognized by
the monoclonal antibody. However, not all isolates tested
expressed the antigenic determinant (Lu et al. (1991) Infect
Immun 59:172-180).
Most investigations that concern the cross-protective
capacity of Pasteurella multocida Type A have used
serotypes and isolates that infect poultry. Cross-
protective antiserum made in turkeys by inoculatinginactivated in vivo grown bacteria was used for passive
immunization/ and results showed this antiserum passively
protected young turkeys against heterologous challenge
(Rimler RB (1987) Avian Diseases 31:884-887). In an attempt
to determine the nature of these cross-protection factors in
Pasteurella multocida, investigators have shown that
wossl2s742 PCT~B95/00185
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complete lysis or partial solubilization of in vivo grown
bacteria followed by gradient centrifugation resulted in a
fraction that could provide partial protection against
homologous and heterologous challenge. Four protein bands
with molecular weights of approximately 74 kD, 65 kD, 39 kD
and 30 kD were suggested as being responsible for cross-
protection (Rimler RB et al. (1989) Avian Diseases
33:258-263). Additional studies have shown that organisms
recovered from bacteremic birds contain outer membrane
lo proteins of similar sizes. However, these polypeptides were
not detected in bacteria of the same isolate cultured in a
standard, enriched media.
Antibodies specific for polypeptides of approximately
153 kD, 179 kD, 192 kD and 204 kD recovered from in vivo-
grown bacteria that were detergent insolublè have also beendemonstrated to provide passive cross-protection against
heterologous challenge in poultry (Wang CL et al. Abstract
#32, p. 8, Conference of Research Workers in Animal
Diseases, November 9-10, 1992, Chicago, IL). This antiserum
was adsorbed against culture grown bacteria to remove
antibodies that reacted with antigens in the culture grown
preparation. These studies suggested that the regulation of
protein expression in Pasteurella multocida may depend upon
the local environment, and antigens that may be important
immunogens might not be expressed during in vitro growth.
These antigens may only be up-regulated during the infection
process as the organism invades its natural host.
The use of minimal medium for the production of in
vivo- expressed antigens is based on the idea that bacteria
grown in a mammalian host experience nutrient deprivation.
Numerous investigators have reported that certain
auxotrophic mutants of pathogenic bacterial species are
avirulent. Mutants with defects in the biosynthesis of
purines, aspartic acid, p-aminob~n7Oic acid, aromatic amino
acids, diaminopimelic acid, arginine and pyrimidines have
been found to be avirulent in such disparate species as
W095/25742 PCTAB95/00185
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Salmonella typhi (Brown, R.F. and Stocker B.A.D. (1987)
Infect. Immun. 55:892-898), Bacillus anthracis (Ivanovics et
al. (1968) J. Gen. Microbiol. 53:147-162), Escherichia coli
(Kwaga et al. (1994) Infect. Immun. 62:3766-3772),
Pasteurella multocida (Homchampa et al. (1992) Molec.
Microbiol. 6:3585-3593) and Yersinia enterocolitica (O'Gaora
et al. (1990) Microb. Pathogenesis 9:105-116). All of these
reports suggest that mammalian hosts stringently limit the
availability of essential nutrients to bacteria. These
lo results also suggest that bacteria must activate numerous
biosynthetic pathways to replicate inside a host and cause
a disease. In vivo expression technology (IVET), a
methodology which selects for bacterial genes that are
specifically induced in host tissues, has provided evidence
of a nutritionally-exacting environment in a host (Mahan et
al. (1993) Science 259:686-688; Mahan et al. (1995) Proc.
Natl. Acad. Sci. USA 92:669-673). IVET studies have
demonstrated that the Salmonella typhimurium carAB and pheST
genes are specifically induced in vivo. Expression of the
carAB operon results in the increased biosynthesis of
arginine and pyrimidines. Induction of the pheST operon
(which encodes two subunits of phenylalanyl-tRNA synthetase)
is believed to be a response to the depletion of a charged
tRNA, indicating starvation for the aromatic amino acid
phenylalanine.
The in vivo activation of microbial biosynthetic
pathways provides essential nutrients to bacteria which they
are unable to acquire from the host. However, essential
mineral requirements cannot be produced biosynthetically and
therefore must be obtained from the host. Among these
minerals are calcium, magnesium, iron, zinc, copper,
manganese and cobalt. The inability to biosynthesize these
mineral requirements puts bacteria into a severe nutritional
crisis. Metal ion transport has been best studied in iron
acquisition. Since bacteria are unable to biosynthesize
their own iron, iron restriction places bacteria into a
wossl25742 PCT~B95/00185
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86~ Z~
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severe nutritional crisis. Under such iron deprivation,
nearly all bacterial species induce several distinct systems
involved in iron acquisition (Cox, C.D., "Importance of iron
in bacterial virulence", T.J. Beveridge and R.J. Doyle
(eds.), Metal Ions and Bacteria, John Wiley & Sons, Inc.,
New York, N.Y., 1989, p. 207-246). Most of these iron-
acquisition systems include outer membrane proteins
specifically expressed in response to iron starvation. In
addition to forcing-bacteria to biosynthesize many of their
own essential growth factors, the mammalian iron-withholding
defense system requires bacteria to induce numerous systems
for the acquisition of this essential nutrient from the in
vivo environment.
For example, Pasteurella multocida isolates of turkey
origin cultured in media containing iron chelators, express
novel outer membrane proteins that are not synthesized in
standard, enriched culture media. Several investigators
have suggested that these novel proteins are the cross-
protective factors observed in in vivo-grown bacteria. This
relationship between the iron regulated outer membrane
proteins and the outer membrane proteins of in vivo grown
bacteria was investigated by comparison of their protein
profiles by SDS-PAGE. These analyses demonstrated that
several proteins identified from bacteria grown in media
containing an iron chelator were similar to those expressed
by in vivo-grown bacteria isolated from turkeys, but were
absent from bacteria grown in st~nA~rd, enriched media.
These proteins had molecular masses of 76 kD, 89 kD, and 94
kD (Choi KH et al. (1989) Am J Vet Res 50:676-683; Choi-Kim
et al. (1991) Vet Microbiology 28:75-92; Donachle, W., UK
Patent Application 2 202 851 A).
Other researchers have examined cell-free culture
filtrates from iron restricted cultures. These filtrates
contain secreted antigens, rather than membrane associated
antigens. Cell-free culture filtrates from either stAn~Ard,
enriched or iron restricted medium protected against
woss/25742 pcTnBsslool85
-5- ~l~6~ Zl
homologous challenge in turkeys, but did not protect against
heterol~gous challenge.
Antigens from the Pasteurella multocida and
Actinobacillus pleuropneumoniae isolated directly from the
pleural cavities of infected swine or from a minimal medium
formulation have now been identified. These antigens are
proteins which are up-regulated during infection in a host
animal and are not observed during culture in standard,
enriched media. It has now been found that these antigens
are up-regulated in minimal medium formulations. However,
these antigens are absent or only weakly expressed during in
vitro cultivation in standard, enriched media. An immune
response to these newly identified antigens invokes
protection against heterologous challenge. Therefore, these
antigens are useful in the production of an effective
vaccine providing cross-protection between multiple isolates
of the same species.
8ummary of th- Inv-ntion
An object of the present invention is to provide
antigens of the Pasteurella, Actinobacillus and Haemophilus
species of bacteria capable of being up-regulated during
infection in a host animal and in minimal medium
formulations which provide protection against infections
caused by these species.
Another object of the present invention is to provide
vaccines comprising antigens of the Pasteurella,
Actinobacillus and Haemophilus species of bacteria capable
of being up-regulated during infection in a host animal and
in minimal medium formulations which provide protection
against infections caused by these species.
Yet another object of the present invention is to
provide a method of immunizing healthy animals against
infections caused by Pasteurella, ACtinQhACillUs and
Haemophilu~ species of bacteria which comprises
administer;ng to a healthy animal an effective amount of a
W095/25742 PcTnB95/00185
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vaccine comprising antigens of the Pasteurella,
Actino~acillus and Haemophilus species of bacteria capable
of being up-regulated during infection in a host animal and
in minimal medium formulations which provide protection
against infections caused by these species.
9rief D-scription of Figur-J
Figure 1 is a graph depicting a passive immunity study
in mice. All mice received 0.5 ml, i.p., of an immune serum
followed four hours later by 100 to 200 CFU of virulent
Pasteurella multocida 16926.
In Figure lA immune serum was collected from a pig
following a primary infection with P. multocida isolate
8261. Group A (-) serum is a 1:10 dilution of this immune
serum; Group B (---)serum is a 1:10 dilution of this immune
serum adsorbed with detergent solubilized, standard,
enriched media grown isolate 8261; and Group C (...) is
preimmune serum diluted 1:10.
In Figure lB immune serum was collected from a pig
infected with isolate 8261 followed by a secondary exposure
to P. multocida isolate 16926. Group D (- -) serum is a
1:10 dilution of this immune serum; Group E (- -) serum is
a 1:10 dilution of this immune serum adsorbed with detergent
solubilized, standard, enriched media grown isolate 8261;
and Group C (c) is preimmune serum diluted 1:10.
Figure 2 is a graph depicting a second passive immunity
study in mice. All mice received 0.5 ml, i.p. of an immune
serum followed four hours later by 100 to 200 CFU of
virulent P. multocida 16929. Immune serum was collected
from a pig 7 days following a secondary exposure to P.
multocida isolate 16926. Group 1 (- -) is preimmune serum
diluted 1:10; Group 2 (-) is immune serum diluted 1:10;
Group 3 (---) is immune serum, treated with detergent and
purified, diluted 1:10; Group 4 (...) is immune serum,
adsorbed with detergent-solubilized bacteria and purified,
diluted 1:10; and Group 5 (- - ) is lung washing fluids
wossl25742 PCTAB95/00185
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~7~ 21 861 21
collected 18 hours following secondary exposure to isolate
16926.-
D-tailed DeQcription of the Invention
The Pasteurellaceae family of bacteria contains species
of the genera Pasteurella, Actinobacillus, and ~aemophilus.
Recent work on the phylogeny of the Pasteurellaceae family
confirmed the grouping of these three genera into this
family (Dewhirst et al. (1992) J. Bacteriol. 17~:2002-2013).
The various species within the Pasteurellaceae family fall
into four large clusters, each cluster containing species of
three different genera. Examples of species within this
family include, but are not limited to, the animal pathogens
P. multocida, A. pleuropneumoniae, P. haemolytica, H.
somnus, and A. suis.
Pasteurellaceae infections in animals result in
symptoms similar to those resulting from virulent septic
pneumonia. Death is generally due to endotoxic shock and
respiratory failure. High mortality rates can occur with
the acute form of these infections, however, subacute and
chronic forms which result in pleuritis are more common.
Treatment of field infections is difficult and often
unsuccessful due to widespread antibiotic resistance.
Therefore, it is preferred to prevent the infection in
animals through use of a vaccine. There has been
difficulty, however, in achieving a vaccine which will
provide protection against different isolates of a species
of bacteria within the Pasteurellaceae family.
Cross-protection against different isolates of a
species of bacteria within the Pasteurellaceae family seems
to be dependent upon the ability of the host to mount an
immune response against bacterial proteins exclusively
expressed under the influence of microenvironmental
conditions encountered during infection. Most vaccines
designed to protect swine against pneumonias caused by these
bacteria are prepared from inactivated whole bacteria.
Wossl25742 PCTAB95/00l85
~1 861 ~1
However, these vaccines only provide protection against
homologous isolates or serotypes (serotyping based on
capsular or lipopolysaccharide type). In contrast, it was
found that pigs that recover from an active infection are
generally quite resistant to reinfection, regardless of the
isolate or serotype. In addition, it is difficult to
incorporate crude bacterial products into large combination
vaccines because of the potential of untoward systemic and
site reactions that may result from immunization with
combinations of crude bacterial products. The present
invention provides antigens from the Pasteurella,
Actinobacillus and Haemophilus species of bacteria that are
up-regulated during infection in a host animal or in minimal
medium formulations but which are absent or only weakly
expressed during in vitro cultivation of the bacteria in a
standard, enriched media, said antigens being capable of
providing protection against an infection by these species
of bacteria. These antigens provide the basis for effective
vaccines designed to cross-protect animals from infection by
multiple isolates of one of the Pasteurella, Actinobacillus
or Haemophilus species of bacteria.
Examples of host animals for these bacteria include,
but not limited to, swine, bovine, ovine, avian and equine
species. By "minimal medium formulations" it is meant a
culture media specifically designed to provide a sufficient
concentration of nutrients to bacteria to allow for
synthesis of antigens up-regulated in vivo in a host while
removing all undefined components. In contrast, st~n~Ard,
enriched laboratory media are designed to allow optimal
growth of bacteria and thus include an excessive levels of
nutrients when compared to the minimal growth requirements
for each species. Complete Heamophilus Pleuropneumoniae
(HP) medium is an example of a st~n~rd, enriched medium.
Undefined components in these media such as yeast extract,
tryptone, peptone or brain heart infusion all contain high
levels of complexed nitrogen, amino acids, vitamins, iron
wossl2s742 PCTAB95/00185
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and other minerals which provide an excellent nitrogen
source and general nutritional supplement so that little
metabolic demand is placed on the bacteria. In contrast,
culture media used in the present invention were designed to
S supply the bacteria with the minimum level of essential
nutrients necessary to support growth, thus mimicking the
environment encountered when bacteria invade the host
organism. Components of the minimal media used in the
present invention comprise basal salts (elemental
requirements), carbon sources, special nutritional
requirements of the Pasteurellaceae, and nonessential
optimizing supplements. Examples of basal salts include,
but are not limited to, potassium phosphate, potassium
sulfate, magnesium chloride, ammonium chloride, calcium
chloride and sodium chloride. Examples of elemental
requirements include, but are not limited to, potassium,
sulfur, phosphorus, sodium, chloride, and calcium. Examples
of carbon sources include, but are not limited to, glycerol
and lactic acid. Glucose, galactose, fructose, mannose,
sucrose, mannitol, and sorbitol can also be utilized by the
Pasteurellaceae. However, because of the fermentative type
of metabolism of these organisms, acid can be produced
during catabolism of sugars resulting in a lower yield of
bacterial cells in the culture. Thus, use of the non-
fermentable carbohydrates glycerol and lactic acid, which donot lead to acid accumulation in cultures, is preferred. In
addition, since members of the Pasteurellaceae are not
prototrophic in that they are unable to grow in a mineral
salts medium with a single carbon source, special
nutritional additives are required. For example, these
species require organic nitrogen sources and may require
several amino acids, B vitamins, ~-nicotinamide, adenine
nucleotides, or protoporphyrin and its conjugates. To
satisfy these requirements, the minimal medium may comprise
arginine, aspartic acid, cystine, glutamic acid, glycine,
leucine, lysine, methionine, serine, tyrosine, inosine,
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1 o 1 ~ ~ 1 2
uracil, hypoxanthine, thiamine, pantothenate, and
nicotinamide. For the propagation of Actinobacillus and
Haemophilus species ~-NAD, protoporphyrin or its conjugates
can also be added as needed. The addition of several
components were also found to enhance the cell yield of
P. multocida and A. pleuropneumoniae. These non-essential
optimizing supplements include a buffer such as HEPES to
increase buffering capacity, a nitrogen source such as
glutamic acid and Casamino acid (a semi-defined acid
hydrolysate of casein, Difco Laboratories Ltd, West Molesey,
Surrey KT8 OSE U.K.), and an organic sulfur source such as
L-cysteine. In addition, these minimal media formulations
contains no iron, zinc, copper, manganese or cobalt salts or
complex biological materials containing these minerals. The
sole source for these minerals is the trace amounts which
may be present as trace contaminants in the various other
defined components or water. The laboratory analyses of
trace minerals in these minimal media formulations are
provided in the following table.
WO 95/25742 PCT/IB95/00185
- 2 1 8 6 1 2 1
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W095/25742 PCTAB95/00l85
2 ~ ~; 2 1 8 ~ 1 2 1
-12-
In one embodiment of the present invention, a cultured
Pasteurellaceae species such as Pasteurella multocida of
Actinobacillus pleuropneumoniae bacteria is administered to
a host animal, preferably a pig, by injection into the
pleural cavity.
The host animal is euthanized approximately 12 to 18
hours later and the pleural fluids are collected. Large
cellular debris is removed from the fluids, and in vivo-
grown bacteria are recovered by centrifugation. The
resulting bacterial pellet is washed several times in buffer
and centrifuged. The bacterial pellets are then resuspended
in buffer and stored at -70C.
In another embodiment Pasteurellaceae bacteria such as
P. multocida or A. pleuropneumoniae are grown in a minimal
medium formulation. Cultured bacteria werè centrifuged to
concentrate the bacteria and remove media components.
Bacterial pellets were resuspended in PBS to an optical
density of approximately 3.0 and frozen at -70C until
analyzed.
Proteins expressed by the in vivo ~Lown bacteria or bacteria
grown in a minimal medium formulation are separated by gel
electrophoresis. Those proteins which are strongly
expressed can then be identified by transferring part of the
gel to nitrocellulose for Western blot analysis using immune
serum that has been adsorbed against in vitro-grown bacteria
in a standard, enriched media or using antibody recovered
from the local site of infection. The term "strongly
expressed" refers to proteins up-regulated or expressed
during infection in a host animal or in bacteria grown in a
minimal medium formulation which are absent or only weakly
expressed during conventional in vitro cultivation in
standard laboratory medium. The term "weakly expressed"
refers to expression in amounts so minor that the antigen is
incapable of providing protection against a Pasteurellaceae
infection. Conventional in vitro cultivation refers to
bacteria inoculated into a standard, enriched medium such as
W095/25742 pcTnB95lool85
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-13-
complete Heamophilus Pleuropneumoniae (HP) medium containing
supplements. The inoculate is incubated at 37C for several
hours, preferably in a shaking incubator. The bacteria are
centrifuged at 10,000 x g to remove culture medium and
resuspended in sterile PBS (10 mM phosphate, 0.87% NaCl, pH
7.2) prior to use. At least eleven antigens were identified
from the Western blot analysis of in vivo grown bacteria
that are absent from Pasteurella multocida grown in vitro
using in a standard, enriched media. Western blot analysis
of bacteria grown in vitro in a minimal medium formulation
demonstrated that the antigenic profile of bacterial
proteins produced in this minimal medium formulation was
identical to the antigenic profile produced in a host animal
infected by the bacteria. The corresponding protein bands
had molecular weights of approximately 115 kD, 109 kD, 96
kD, 89 kD, 79 kD, 62 kD, 56 kD, 53 kD, 45 kD, 34 kD and 29
kD.
The corresponding protein bands for each antigen are then
excised from the gel, re-isolated by gel-electrophoresis and
transferred onto sequence membranes for N'-terminal amino
acid sequencing. The N'-terminal amino acid sequence of a
34 kD antigen is as follows:
Ala Thr Val Tyr Asn Gln Asp Gly Thr Lys Val Asp Val Asn Gly
Ser Val Arg Leu Leu Leu Lys Gly Glu Lys Asp Pro Arg Arg Asp
Leu Met Met Asn Gly (SEQ ID NO: 1)
The N'-terminal amino acid sequence of 29 kD antigen is as
follows:
Ala Asp Tyr Asp Leu Lys Phe Gly Met Val Ala Gly Pro Ser Ala
Asn Asn Val Lys Ala Val Glu Phe Ile Ala (SEQ ID NO: 2)
The N'-terminal amino acid sequence of a second 29 kD
antigen is as follows:
Lys Phe Lys Val Gln Ile Ala XXX XXX XXX XXX Gln Asp Ile Asn
Gln Tyr Tyr Ala Gly Asp Ala Ala Phe Val (SEQ ID NO: 3)
The ability of these antigens to invoke a protective immune
response against Pasteurellaceae was verified in passive
transfer experiments. Antibodies to the bacteria were
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isolated from infected pigs. Those antibodies that were
rendered specific for the antigens of the present invention
by adsorption were administered to mice. These mice were
then challenged with a virulent heterologous isolate of
Pasteurella multocida. Control mice not receiving the
antibodies developed infections while those receiving the
antibodies did not. Thus, antibodies developed as an immune
response against these antigens provided cross protection
against heterologous isolates of Pasteurella multocida.
In additional experiments, vaccines were prepared from
P. multocida cultured in standard, enriched media containing
yeast extract and from bacteria cultured in a minimal medium
formulation. The vaccine prepared from bacteria cultured in
standard, enriched media was unable to protect mice from
either homologous or heterologous challenge of P. multocida.
In contrast, mice immunized with the vaccine prepared from
bacteria cultured in a minimal medium formulation had much
better survival rates. These experiments demonstrate that
growth of bacteria under conditions that limit the
availability of nutrients and trace elements such as iron,
copper, zinc, manganese or cobalt result in the expression
of antigens normally up-regulated by the host environment
and that they play an important role in protection. The
ability to produce these antigens by growth in minimal media
results in the ability to produce more efficacious vaccines
than are currently available.
In the present invention, Pasteurella multocida
antigens, which are capable of being up-regulated during
infection in a host animal and in a minimal media
formulation which provide protection against a Pasteurella
infection, are identified. In one embodiment, a Pasteurella
multocida antigen has a molecular weight, as determined by
gel electrophoresis, of approximately 115 kD. In another
embodiment, a Pasteurella multoclda antigen has a molecular
weight, as determined by gel electrophoresis, of
approximately 109 kilodaltons. In yet another embodiment,
Woss/25742 PCTAB95/00185
-15- 21 g61 21
a Pasteurella multocida antigen has a molecular weight, as
determrned by gel electrophoresis, of approximately 96
kilodaltons. In yet another embodiment, a Pasteurella
multocida antigen has a molecular weight, as determined by
gel electrophoresis, of approximately 89 kilodaltons. In
yet another embodiment, a Pasteurella multocida antigen has
a molecular weight, as determined by gel electrophoresis, of
approximately 79 kilodaltons. In yet another embodiment, a
Pasteurella multocida antigen has a molecular weight, as
determined by gel electrophoresis, of approximately 62
kilodaltons. In yet another embodiment, a Pasteurella
multocida antigen has a molecular weight, as determined by
gel electrophoresis, of approximately 56 kilodaltons. In
yet another embodiment, a Pasteurella multocida antigen has
a molecular weight, as determined by gel electrophoresis, of
approximately 53 kilodaltons. In yet another embodiment, a
Pasteurella multocida antigen has a molecular weight, as
determined by gel electrophoresis, of approximately 45
kilodaltons. In a preferred embodiment, a Pasteurella
multocida antigen has a molecular weight, as determined by
gel electrophoresis, of approximately 29 kilodaltons and an
N'-terminal amino acid sequence comprising SEQ ID NO: 2. In
a second preferred embodiment, a Pasteurella multocida
antigen has a molecular weight, as determined by gel
electrophoresis, of approximately 29 kilodaltons and an N'-
terminal amino acid sequence comprising SEQ ID NO: 3. It is
also preferred that a Pasteurella multocida antigen has a
molecular weight, as determined by gel electrophoresis, of
approximatel~y 34 kilodaltons and an N'-terminal amino acid
sequence comprising SEQ ID NO: l.
Antigens up-regulated during infection in a host animal
and in a minimal media formulation but not in bacteria grown
in vitro in a standard, enriched media were also identified
for isolates of Actinobacillus pleuropneumoniae. A.
3 5 pleuropneumoniae is member of the Pasteurellaceae family
which exists in the most distinct phylogenetic cluster from
Wossl2s742 PCT~B95/00185
` i' al;`' ; -16- 2~ 861 21
that of P. mul tocida . The most prominent antigenic
dif~erence between the in vivo or minimal media bacteria and
the bacteria cultured in a standard, enriched media was the
presence of additional bands of approximately 60 kD to 65 kD
in the in vivo and minimal media preparations.
In addition to being produced by bacteria grown in vivo
in a host animal or in minimal medium formulations, the
antigens of Pasteurellaceae can be produced recombinantly
using techniques well-known to those skilled in the art or
induced in culture via genetic manipulation.
Antigens of the present invention may be incorporated
into a vaccine and administered to a healthy animal in an
effective amount to protect against infection by bacteria of
the Pasteurellaceae family. Upon administration, the
antigens in the vaccine will invoke an immune response
resulting in the healthy animal forming antibodies against
the bacteria. "Effective amount" refers to that amount of
vaccine which invokes in an animal an immune response
sufficient to result in production of antibodies to the
antigens. The animal will then be protected from any
subsequent exposure to an isolate of Pasteurellaceae.
In a preferred embodiment a vaccine for the
Pasteurellaceae bacteria Pasteurella multocida comprises at
least one antigen having a molecular weight of 34 kD and an
2S N'-terminal amino acid sequence comprising SEQ ID NO: l or
a molecular weight of 29 kD and an N'-terminal amino acid
sequence comprising SEQ ID NO: 2 or SEQ ID NO: 3. The
antigen can be dissolved or suspended in any
pharmaceutically acceptable carrier. Examples of
pharmaceutically acceptable carriers include, but are not
limited to, normal isotonic saline, standard 5% dextrose in
water or water, preferably adjuvanted. Examples of
adjuvants include, but are not limited to, Quil A,
Alhydrogel, and Quil A and 5% Alhydrogel in tissue culture
media. The vaccine can be administered subcutaneously,
intramuscularly, intraperitoneally, intravitreally, orally,
wossl25742 PCT~B95/00185
-` ~; 21 861 2~
intranasally or by suppository at doses ranging from
approxi~ately 1 to 100 ~g/dose.
In another embodiment, the antigens of the present
invention produced in vivo or in bacteria grown in vitro in
a minimal media formulation, recombinantly or via genetic
manipulation or under specialized culture conditions can be
added to whole culture grown bacteria to produce an
effective vaccine. Addition of these antigens to the
culture grown bacteria increase the efficacy of the
resulting vaccine.
This invention is further illustrated by the following
nonlimiting examples.
.
EXAMPLE8
Example 1: Bacteri 1 isol~t-s ~nd growth conditions
Pasteurella multocida isolates 8261 and 16926 were
field isolates received from the Iowa State Veterinary
Diagnostic laboratory. Both isolates were serotype 3A. For
conventional in vitro growth, bacteria were inoculated into
Heamophilus Pleuropneumoniae (HP) medium (Gibco, Grand
Island, NY) containing supplements, and incubated for 6
hours at 37C in a shaking incubator. The bacteria were
centrifuged at 10,000 x g to remove culture medium and
resuspended in sterile PBS (10 mM phosphate, 0.87% NaCl, pH
7.2). For in vivo growth, 1 ml of cultured bacteria at a
concentration of 2 x 108 CFU/ml were administered to pigs by
transthoracic injection into the ~iAp~ragmatic lobe. Pigs
were euthanized 16 hours later and in vivo-grown bacteria
were recovered from the pleural fluids. The pleural fluids
were centrifuged at 250 x g to remove large cellular debris,
and then in vivo-grown bacteria were recovered by
centrifugation at 10,000 x g for 40 minutes at 4C. The
bacterial pellet was washed three times with sterile PBS by
centrifugation as above. Bacterial pellets were resuspended
in PBS and stored at -70C.
W095/25742 pcTnBsslool85
c`~a~ -18- 21861~1
Protein concentrations from both in vivo and in vitro
grown rsolates were determined using the BCA Protein Assay
(Pierce, Rockford, IL.) in accordance with the
manufacturer's instructions.
S Bxample 2: Convalesc-nt sera and antibody from immun-,
challenged pig~
Three groups of 6-8 week old pigs were infected with 5
to 10 x 106 colony forming units (CFU)/ml of PmA isolate
16926 or 8261 by transthoracic challenge. The pigs were
ailowed to convalesce for a period of between 10 days to one
month and then treated with antibiotics to clear any
residual infection. Following an additional period of one to
two months, the pigs were exposed to a heterologous isolate
of PmA (8261 or 16926) by the transthoracic route (1.5 - 2.5
x 10~ CFU/ml). The pigs were euthanized at day 0, 1, 2, 3,
4, or 7. Serum samples were collected from the pigs at the
time of primary infection, one month following primary
infection, at the time of second infection, and at the time
of euthanasia.
Lung washings were recovered from the pigs at the time
of euthanasia to obtain antibody from the local site of
infection. To recover antibody, the lungs and trachea were
rinsed with approximately 100 ml of sterile PBS. The lungs
were massaged, and then fluids containing the antibody were
poured into sterile tubes. The fluids were centrifuged at
250 x g for 20 minutes at 4C to remove large cellular
debris and red blood cells.
Bxample 3: Adsorption of antibody pr-parations with
d-t-rg-nt-solubiliz-d or whol- Pasteurella
multocida
Antibodies that recognize in vitro grown bacterial
antigens were removed by adsorption against either whole
bacteria or detergent-solubilized antigen. Fresh culture
grown bacteria (isolate 8261 or 16926) were obtained for the
whole bacteria adsorptions. Approximately 10 ml of bacteria
w095l25742 PCTAB95/00185
~ t r 2 1 8 6 1 2 1
--19--
(3 x 109 CFU/ml) were pelleted by centrifugation at 11,000
x g for 40 minutes at 4C (Beckman microfuge, Beckman
Instruments, Palo Alto, CA). The supernatant was removed
and o.1 ml of antiserum added. The mixture was incubated
overnight at 40C while being gently agitated. Following
incubation, the adsorbed material was centrifuged at 11,000
x g for 40 minutes and the supernatant was removed and added
into a fresh bacteria pellet. The final supernatant was
collected and stored at -20C for Western blot analysis.
For detergent solubilization, the culture grown
bacteria was solubilized in a solution containing 0.062 M
Tris, 0.069 M SDS and 1.09 M glycerol, pH 7Ø The antigen
preparation was then boiled for 10 minutes at 100C to
solubilize the bacteria. After the solubilized antigen had
cooled, it was mixed with an equal volume of antibody and
incubated overnight at 4C. The mixture was centrifuged at
20,000 x g for 40 minutes to pellet the precipitated
antibody-antigen complexes. The final supernatant was
collected and stored at -20C.
Exampl- ~: Purification of ~ntibody pr-p~rations using
ammonium sulfate pr-cipit~tion
Antibody preparations used for the passive immunity
study in mice were partially purified using ammonium
sulfate. Serum samples were diluted 1:3 in sterile PBS. A
solution of saturated ammonium sulfate was diluted to 90% of
saturation and then added drop-wise to the diluted serum
until a volume equivalent to the diluted serum was added,
resulting in a 45% ammonium sulfate precipitation of the
antibody. This solution was incubated on ice for 1 hour.
The mixture was centrifuged at 10,000 x g for 40 minutes at
- 4C to pellet the ammonium sulfate precipitate. The
supernatant was discarded and the pellet was resuspended to
the initial serum volume using sterile PBS. The ammonium
sulfate precipitation was repeated to further purify the
antibody. Following resuspension of the antibody, the
W095/25742 pcTnB95lool85
~ 20- ~ ~ 8 6 1 2 1
solution was dialyzed against three changes of PBS using a
12,000 ~ cutoff membrane tubing.
~xampl- 5: 8D~-PAGE and West-rn blot analysis
Proteins from PmA were separated on 10% sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
followed by electrotransfer and immunoblotting. Proteins
from PmA isolates 8261 and 16926, both from in vivo and in
vitro grown bacteria, were boiled for 10 minutes with 5X
reducing buffer (Pierce, Rockford, IL). The proteins were
then separated on 10% SDS gels at a concentration of 16 ~g
per lane. After electrophoresis (20 mA constant amperage),
proteins were transferred on blotting membrane (Immobilon,
Millipore, Bedford, MA) overnight at 4C (30 volts, constant
voltage).
The membrane was blocked with 10% non-fat dry milk in 10 mM
Tris, 0.9% NaCl, pH 7.2 (Tris-saline) and strips were
further incubated with appropriate dilutions of absorbed
antiserum for 1 hour at room temperature. Subsequently,
strips were rinsed 3 times with Tris-saline containing 0.1%
Triton X-100 and further incubated 1 hour with alkaline
phosphatase conjugated goat ~-swine IgG (H+L) (Kirkegaard
and Perry, Gaithersburg, MD) at a 1: 1000 dilution. After
3 washings with lX Tris-saline Triton-X (each for 5
minutes), the strips were immersed in 5-bromo-4-chloro-3-
indolyl-phosphate (BICP) at a concentration of 0.21 g/l and
nitroblue tetrazolium (NBT) at a concentration of 0.42 g/l
in an organic base/Tris buffer (Kirkegaard and Perry,
Gaithersburg, MD) for 10 min. The strips were rinsed in
distilled water to stop color development.
Proteins that were strongly expressed by in vivo grown
PmA isolates 8261 and 16926 were identified by Western blot
analysis using immune serum that had been exhaustively
adsorbed against culture-grown bacteria. At least eleven
protein bands with molecular weights of approximately 115
kD, 109 kD, 96 kD, 89 kD, 79 kD, 62 kD, 56 kD, 53 kD, 45 kD,
W095/25742 PcT~Bsslool85
~ 21- 2 1 8 6 1 2 1
34 kD and 29 kD were identified from in vivo grown bacteria
that were absent from or poorly expressed by culture grown
PmA.
Using non-adsorbed sera, the differences in band
profiles could not be distinguished between the in vivo and
culture grown antigens. This indicates that the majority of
the antibody response mounted against an infection with
Pasteurella multocida is specific for antigens that are
expressed either when the bacteria are cultured in vitro or
when the bacteria are recovered from their natural host. In
contrast, the majority of antibodies that are not removed by
adsorption with cultured bacteria react only with in vivo
bacteria recovered from the host.
Exampl~ 6: D-t~rmin~tion of Mol~ ights of
Id-ntifi-d Proteins
The molecular weights of proteins identified to be
unique or up-regulated under in vivo growth conditions were
estimated by Whole Band Analysis using the BioImage Computer
System (BioImage/Millipore, Ann Arbor, MI). The weights
were estimated for the identified bands based on known
molecular weights markers. Both Rainbow colored protein
molecular weight markers (Amersham Life Science, Arlington
Heights, IL) and Bio-Rad SDS-PAGE broad range molecular
weight st~n~rds (Bio-Rad Laboratories, Hercules, CA)
stained with Coomassie blue were used as the st~n~rds of
comparison.
E~mpl~ 7: P~ssiv- Immunity in Mic-
- All antibody preparations used in the passive immunity
experiments were generated in swine against a primary
infection with P. multocida isolate 8261, and in some cases
were followed by a second infection with isolate 16926.
Serum collected prior to the primary infection was used as
a negative control for the passive immunity experiments.
Convalescent serum from pig 104 was collected either at 30
W095/25742 PCTAB95/00185
. 2186121
-22-
days following the primary infection or at 7 days following
the secondary exposure, and lung washings from pig 103 were
collected 18 hours following the secondary infection. A
secondary antibody response would be expected following re-
exposure with the second strain. However, the secondaryresponse should be limited to antigens that are common
between the two Pasteurella multocida isolates.
The serum antibody preparations were adsorbed with
detergent-solubilized bacteria and purified by ammonium
sulfate precipitation as described in Example 4. The
partially purified antibody preparations were standardized
by volume to represent a 1:10 dilution of the original serum
sample. Lung washings were not adsorbed or purified.
Mice were injected by the intraperitoneal route with
lS 0.5 ml of the antibody preparations. Four hours later the
mice were infected with isolate 16926 at a rate of 100-200
CFU/mouse. Mice were observed for 10 days following
challenge for clinical signs and mortality.
Both the antisera collected after the primary challenge
and the antisera collected after the secondary challenge
passively protected mice against the 16926 challenge (9 of
and 8 of 10 mice survived at least ten days post
infection, respectively). These antisera were also adsorbed
with detergent treated culture grown bacteria (8261).
Following adsorption, the antisera lost some of their
protective capacity, but the death rate in these groups were
still lower than in the control group that received
preimmune serum.
A second passive immunity study was performed to verify
the protective capacity of adsorbed antisera. Serum
collected following the secondary infection was used in this
study. All groups of mice given immune serum had better
survival rates than that of the control mice that received
preimmune serum. The group given non-treated immune serum
exhibited the highest survival rate (9 out of 10 mice
survived the ten day challenge period). The antisera that
woss/2s742 PCT~B95/00185
- ~186121
-23-
had been detergent treated and purified provided
intermediate protection. In these groups mice began dying
between 6 and 10 days following challenge. No difference in
survival time or mortality was seen between the mice that
S received detergent treated and purified antiserum versus the
mice that received antiserum that had been adsorbed with
detergent-solubilized culture grown bacteria and then
purified. Deaths occurring in these groups suggest that
either the total quantity of specific antibody was reduced
during purification, or that the half-life of the antibody
was shortened by the detergent treatment or purification.
In either case, the loss appeared to be non-specific.
Western blot analysis of the antibody preparations used in
these experiments demonstrated that purification in the
absence of adsorption did not remove antibodies that were
common to culture grown and in vivo grown bacteria and that
most reactivity to culture grown bacteria was removed by the
adsorption process. Adsorption of the immune serum was
performed using isolate 8261. However, Western blot
analysis demonstrated that the majority of reactivity
against culture-grown isolate 16926 was also removed and
that the remaining reactivity against the in vivo bands is
seen with both the 8261 and 16926 isolates. These
observations suggest that the antibodies that remained
following adsorption are specific for in vivo grown
bacterial antigens and that these antibodies are largely
responsible for protection against challenge.
Preimmune serum did not afford any passive protection
in this model. All mice in this group died between 1 and 3
days post challenge. While the preimmune pig serum reacted
with a few bacterial proteins in a Western blot analysis,
the reactions were weak (1:10 dilution). This would be
expected since the pigs used in these studies were not
specific-pathogen free. Although they may have had some
exposure to Pasteurella multocida or other pathogens with
cross-reactive antigens, they were susceptible to
Wo95/25742 PCTAB95/00185
21 8~1 21
-24-
Pasteurella multocida at the time of primary infection, and
did develop clinical disease following challenge.
Antibody recovered from the lung of an immune pig that
had been originally infected with isolate 8261 and then
exposed to a heterologous isolate of Pasteurella multocida
(isolate 16926) was also tested for its ability to passively
protect mice. Antibody was recovered from the lung 18 hours
after a secondary transthoracic exposure to Pasteurella
multocida. This antibody recovered from the local site of
exposure would have direct contact with invading bacteria.
Vascular leakage of specific antibody from the circulation
into the extracellular environment of the lung represents an
important immune mechanism that contributes to the
protection seen in immune pigs following secondary exposure.
Partial protection was seen in these mice, when compared to
the control group. Five mice died within the first 1 to 3
days after challenge, but the remaining S mice lived an
additional 4 to 5 days. Coomassie staining of the proteins
contained in the lung washing material suggested that the
majority of protein was immunoglobulin. However, Western
blot examination of the lung washing material indicated that
the specific antibody concentrations found in lung washings
were considerably lower than those recovered from serum.
Thus the decreased level of protection seen in these mice
can be explained by the decreased concentration of antibody
that was recovered from the lung.
~a~ple 8- Activ- Protection ~ t P. multocid~ in
~ic-
Bacteria P. multocida (isolate 8261) recovered from the
pleural cavities of acutely infected pigs were isolated asdescribed in Example 1. Isolate 8261 was also grown in
culture as described in Example 1. Both preparations were
inactivated with 0.3% formalin and adjusted to a
concentration of 1 x 109 colony forming units (CFU)/ml. Each
preparation was adjuvanted with a mineral oil emulsion
W095/25742 PCTAB95/00185
~ 25- 2 1 8 6 1 2 1
containing 5% aluminum hydroxide gel. Five-fold and twenty-
five-f~ld dilutions of the two vaccines were prepared by
diluting the original vaccine in adjuvant. All vaccine
preparations were administered by intraperitoneal injection
into CFl mice. Mice were vaccinated twice at a three week
interval with a 0.1 ml dose.
All mice were challenged with 50 to 100 CFU of virulent
Pasteurella multocida isolates 8261 or 16926 and observed
for 15 days. As shown in Table 2 below, when the highest
concentrations of vaccine were used, both preparations
protected mice from both homologous and heterologous
challenge. However, when less concentrated vaccines were
used, only the vaccine produced from in vivo grown bacteria
was able to protect the mice against virulent challenge.
Eight of ten mice vaccinated with 1 x 107 CFU equivalents of
in vivo antigens were protected against homologous
challenge, and seven of ten mice were protected against
heterologous challenge. In contrast, zero of ten mice
vaccinated with the least concentrated cultured bacterial
antigens survived homologous challenge and only three of ten
mice vaccinated with cultured bacterial antigens survived
heterologous challenge. None of the ten non-vaccinated mice
challenged with isolate 8261 survived, and only one of ten
non-vaccinated mice challenged with isolate 19629 survived.
This test of active immunity in mice demonstrated that
the addition of antigens that are up-regulated by in vivo
growth produced a vaccine that was between 5 and 25 times as
effective as a vaccine produced from bacteria grown in a
standard, enriched media.
W095/2~742 PCT~B95/0018S
``S. 3~ 26- 21~6121
TABLE 2
Number of SuMving Mice at each Vaccine
Do~e
Vaccine Cl~s"~ng~(1 x 108 CFU)2 x 10' CFUS x 10~ CFU
in vivo 8261 8261 9 7 8
Cultured 8261 8261 9 2 0
None 8261 0 NAb NA
in vivo 8261 16926 10 8 7
Cultured 8261 16926 10 5 3
None 16926 1 NA NA
NA is representative of not applicable.
Bxample 9: Partial amino a¢id s-guencing of prot-ins
To determine the N'-terminal amino acid seguence of the
proteins identified from in vivo grown bacteria, proteins
from in vivo grown bacteria isolate 8261 were separated on
10% SDS gels. Western blots were performed simultaneously
15 to identify the correct protein bands. Then, protein bands
were excised from the gels, electroeluted and transferred to
a PVDF membrane (ProBlott, Applied Biosystems).
Subsequently, protein bands (40-50 pmol each) were stained
with O.l % Coomassie blue R-250 (Bio-Rad, Hercules, CA).
Individual protein bands were excised from the membrane for
amino acid sequencing. Sequencing was performed on an
Applied Biosystems Model A vapor phase protein sequencer at
the Biotechnology Instrumentation Facility, University of
California, Riverside, CA.
5 ~x~mple lO: Product~on of monoclonal antibodi-s sp-cific
for antig-nJ
Balb C mice were immunized with in vivo grown P.
multocida as described in Example 8, and then reimmunized
with in vivo bacteria solubilized in SDS. Spleens from
immunized mice were fused with SP2/0 myeloma cells to
produce antibody-secreting hybridomas. A hybridoma cell
wos~/25742 PCTAB95/00185
'``'`'`~ ~; -27- 21 86~ 2~
secreting antibody specific for the l09 kD protein was
cloned twice by limiting dilution and designated as Mab PMA
3-l. The resulting monoclonal antibody was specific for the
l09 kD protein produced by in vivo grown bacteria and did
not react with bacteria grown in complete media.
A second monoclonal antibody was selected based on the
ability to bind the 29 kD protein from in vivo grown P.
multocida. Western blot analysis of in vivo grown and
cultured bacteria demonstrated strong binding to the 29 kD
protein of in vivo grown bacteria, and very little
reactivity to culture grown bacteria. This monoclonal
antibody producing cell was cloned by limiting dilution and
designated Mab PMA 3-2l.
Example ll: Expr-ssion of Ant~g-ns in Minimal Medium
For~ulation
Culture conditions were designed to supply P. multocida
with the minimum level of essential nutrients necess~ry to
support growth, thus mimicking the environment that might be
encountered when bacteria invade the host organism.
Formulations of minimal medium are shown in Table 3.
TABLE 3
CO~PONENT MEDI~ #l MEDI~M #2 NED~M #3
CARBON ~O~P~
glycerol 40 mM 40 mM 40 mM
25sodium lactate 20 mM 20 mM 20 mM
~u~
HEPES 50 mM 50 mM 50 mM
Amino Acids
L-arginine HCl0 0.0300% 0.0300% 0.0300%
30L-aspartic acid 0.0500% 0.0500% 0.0500%
L-cystine 2HCl 0.0260% 0.0260% 0.0260%
L-cysteine HCl, 0.0790%
anhydrous
W095/25742 PCT~B9S/00185
? i ~ 2186121
-28-
COMPONENT MEDI W #1 MEDIUM #2 NEDIVM #3
L-glutamic acid 0.1300% 0.1300% 0.1300%
L-glutamic acid-Na 0.1870% 0.1870%
glycine 0.0020% 0.0020% 0.0020%
L-leucine 0.0300% 0.0300% 0.0300%
5L-lysine HCl 0.0062% 0.0062% 0.0062%
L-methionine 0.0100% 0.0100% 0.0100%
- L-serine 0.0100% 0.0100% 0.0100%
L-tyrosine 2Na 2H20 0.0290% 0.0290% 0.0290%
8ALT8
10sodium chloride 0.0058% 0.0058% 0.0058%
potassium sulfate 0.1000% 0.1000% 0.1000%
potassium phosphate 0.2656% 0.2656% 0.2656%
dibasic
potassium phosphate 0.2720% 0.2720% 0.2720%
15monobasic
magnesium chloride 0.0187% 0.0187% 0.0187%
anhydrous
EDTA tetrasodium 0.0004% 0.0004% 0.0004%
salt
20ammonium chloride 0.0220% 0.0220% 0.0220%
calcium chloride 0.0022% 0.0022% 0.0022%
anhydrous
OT~ER COMPONENT8
nicotinamide S0 ~M 50 ~M 50 ~M
25Casamino acids 0.2000% 0.2000%
inosine 0.2000% 0.2000% 0.2000%
uracil 0.0100% 0.0100% 0.0100%
sodium hypoxanthine 0.0023% 0.0023% 0.0023%
thiamine-HCL 0.0004% 0.0004% 0.0004%
30D-Ca(II) 0.0004% 0.0004% 0.0004%
pantothenate
wos~/2s742 pcTnB95lool85
2 1 ~6 1 2~1
-29-
Growth of P. multocida in this media resulted in production
of the~same antigens as produced in vivo. Western blot
analysis demonstrated that the antigenic profile of
bacterial proteins produced in this minimal medium
formulation was identical to the antigenic profile of in
vivo grown bacteria as described in Example 5.
~ample 12: Active Protection of ~io- by Vaccine Produc-
~from bact-ria gro~n in minimal medium
formulation
P. multoclda isolate 8261 was cultured in a minimal
medium formulation as described in Example 11. Bacteria
were also cultured in a standard, enriched media as
described in Example 1 or harvested directly from the
pleural cavities of infected swine as described in Example
1. All preparations were inactivated with 0.3% formalin
with constant stirring for 24 hours at 37 C. The
inactivated bacteria were diluted to a pre-inactivation cell
count of 1 x 109 CFU/ml. The bacterial suspensions were
adjuvanted in a squalene emulsion containing Quil A and TDM.
Mice (female CF1, Charles River Laboratories) were
vaccinated with a 0.1 ml dose by the intraperitoneal route
in the lower right quadrant of the abdomen. Mice received
two vaccinations three weeks apart and were challenged with
either 380 LD50 (50% lethal dose) of P. multocida 8261 or 209
LD50 of isolate 16926 two weeks following the second
vaccination.
In this experiment, the vaccine prepared from P.
multocida cultured in a st~n~rd, enriched media containing
yeast extract was unable to protect mice from either
homologous (8261) or heterologous (16926) challenge. Only
one of ten mice survived the 8261 challenge, and only five
mice survived the 16926 challenge. In contrast, mice
immunized with vaccines prepared from in vivo grown bacteria
or from bacteria cultured in either of the minimal media
formulations had much better survival rates. These survival
W095/2~742 PCT~B95/00185
3 ~ Z 1 ~ 6 1 2 1
rates ranged from seven out of ten to complete protection
(ten of- ten mice).
TABL~
Number of Surviving Mice (n = 10)
5 VACCINE rU~TT~NGE WIT~ ~U~T.s.~NG~ ~ITH
Foa~uLATIoN 8261 16926
in vivo grown 8261 7 8
Standard, enriched 1 5
media
loMinimal media #1 8 9
Minimal media #2 7 10
~x~mple 13: Induction of "in vivo~ " antigens of
Acti nn~Cillus pleuropneumoniae
Bacteria were recovered from the pleural infusions of
pigs that died or were euthanized following acute infection
with Actinobacillus pleuropneumoniae. Fluids were collected
into bottles containing glass beads to remove fibrin clots
and then transferred to centrifuge bottles. Red blood cells
and other cellular debris were removed by centrifugation at
900 x g for 15 minutes at 4C. Bacteria were then recovered
from the supernatant fluid by centrifugation at 14,000 x g
for 15 minutes at 4C. The bacterial pellet was resuspended
in phosphate buffered saline (PBS) and washed three times by
centrifugation and resuspension as above. The final
bacterial pellet was resuspended in PBS to an optical
density of approximately 3.0 and frozen at -70C until
analyzed.
The same isolates of Act i noh~cillus pleuropneumoniae
were also cultured in defined media #1, defined media #3 and
complete HP media (see Table 3). All media contained 10 mM
nicotinamide adenine dinucleotide (NAD). Cu~tured bacteria
were centrifuged as above to concentrate the bacteria and
remove media components. Bacterial pellets were resuspended
W095/25742 PCT~B95100185
`~`' '~i'`~' ' -31- 2186121
in PBS to an optical density of approximately 3.0 and frozen
at -70 ~ until analyzed.
Western blot analyses were performed as described in
Example 5. Convalescent antiserum collected from a pig that
had been experimentally infected with A. pleuropneumoniae
(Serotype 5) eight weeks previously was used to develop the
immunoblot. The most prominent antigenic difference between
the in vivo or defined media bacteria and the bacteria
cultured in complete HP was the presence of additional bands
of approximately 60 kD to 65 kD in the in vivo and minimal
media preparations. Three different serotypes of
Actinobacillus pleuropneumoniae ( serotypes l, 5 and 7) were
grown in defined media and in complete HP media in order to
confirm these differences. In each case, several additional
lS bands were present in the defined media preparation that
were not present in the HP grown extract. These antigens
were also detected in in vivo preparation from the
respective serotype.
Similar bacterial preparations from serotype 5 were
probed with an antiserum that was specific for a transferrin
binding protein that is known to be up-regulated by iron
chelation and thought to function in acquiring complexed
iron during in vivo growth (Deneer and Potter, Infection and
Immunity (1989) 57(3):798-804). A heavy band at
approximately 60 kD was detected in the lanes containing in
vivo bacteria and in lanes containing bacteria grown in
defined media, but not in lanes contAining the bacteria
grown in complete HP media. This finding confirms that the
up-regulation of proteins seen during growth of
Actinobacillus pleuropneumoniae within the host also occurs
when these bacteria are cultured in defined media.
W095/25742 PCT~B95/00185
32 2 1 8 6 i 2 1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Pfeiffer, Nancy;
Ankenbauer, Robert;
Dayalu, Krishnaswamy Iyengar;
Isaacson, Wanda Kay;
Kaufman, Thomas James;
Li, Wumin
(APPLICANTS FOR UNl'l'~:U STATES OF AMERICA ONLY)
Pfizer Inc. (APPLICANT FOR ALL OTHER
COUNTRIES)
(ii) TITLE OF INVENTION: Pasteurellaceae Antigens and
Related Vaccines
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Pfizer Inc., Patent Dept.
(B) STREET: 235 East 42nd Street, 20th Floor
(C) CITY: New York
(D) STATE: New York
(E) COUNTRY: U.S.A.
(F) ZIP: 10017-5755
(v) COMPUTER ~n~RLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COh~ul~K: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: N/A
(B) FILING DATE: Herewith
(C) CLASSIFICATION-
WO 9S/25742 PCT/IB9S/0018S
6 1 2 1
-33-
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/216,202
(B) FILING DATE: March 22, 1994
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Lorraine B. Ling
(B) REGISTRATION NUMBER: 35,251
(C) REFERENCE/DOCKET NUMBER: PC9064A
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 573-2030
(B) TELEFAX: (212) 573-1939
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Ala Thr Val Tyr Asn Gln Asp Gly Thr Lys Val Asp Val Asn Glyl5
Ser Val Arg Leu Leu Leu Lys Gly Glu Lys Asp Pro Arg Arg Asp30
Leu Met Met Asn Gly 35
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: Amino Açid
W095/25742 PCTAB95/00185
2 ~ ~ ~ l 2 1
tD) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ala Asp Tyr Asp Leu Lys Phe Gly Met Val Ala Gly Pro Ser Alal5
5 10 15
Asn Asn Val Lys Ala Val Glu Phe Ile Ala 25
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Lys Phe Lys Val Gln Ile Ala XXX XXX XXX XXX Gln Asp Ile Asnl5
Gln Tyr Tyr Ala Gly Asp Ala Ala Phe Val 25
