Note: Descriptions are shown in the official language in which they were submitted.
WO 91/19419 PCT/L7S91/04092
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PASTEURELLA MULTOCIDA TOXOID VACCINES
Field of the Invention
This invention is generally in the field of
veterinary vaccines, vaccine compositions, and methods of
producing same. More particularly, this invention
relates to vaccine compositions and methods for
protecting animals against diseases associated with
infection by toxigenic strains of Pasteurella multocida.
Background of the Invention
Pasteurella multocida has been associated with
disease in many species of animals, including man and
bovine, ovine and porcine animals. It typically affects
the nasopharyngeal regions and lungs of infected animals.
For example, toxigenic strains of P. multocida, capsular
type A or D, cause atrophic rhinitis in swine. Atrophic
rhinitis (AR) results in severe necrosis of the epithelia
of the upper respiratory tract as well as deformities and
atrophy of the turbinates and snouts of pigs.
The pathogenicity of P. multocida is due in
large part to the production of a potent necrotizing
toxin, also called dermonecrotic toxin (DNT), which will
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be referred to hereinafter as "the toxin". The toxin has
been characterized as a heat-labile protein with a
molecular weight of approximately 140,000 to 160,000.
P. multocida is distinguishable from other
species of Pasteurella on the basis of its growth
characteristics, as follows: hemolysis: negative (90%);
growth on MacConkey's agar: negative; indole production:
positive; urease production: negative; and mannitol
metabolism: positive. See, Zinsser, Microbiology, edit.
by Joklik et al., Appleton-Century-Crofts, New York,
1980, pages 791-793.
Currently available vaccines for protecting
animals from diseases associated with infection by P.
multocida include inactivated toxigenic P. multocida
cells, inactivated preparations of partly purified P.
multocida toxin and combinations of P. multocida cell-
free preparations with other inactivated P. multocida
strains or B. bronchiseptica strains. [See, e.g., M.
Kobisch et al, Vet. Record, 124:57-61 (1989); and N. T.
Foged et al, Vet. Record, 125:7-11 (1989)]. These
vaccine preparations, however, are not fully protective
against disease because they fail to elicit effective
amounts of the antibody that neutralizes the toxin, known
as "antitoxin".
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There remains a need in the art of veterinary
practice for effective vaccines against infection of
animals by toxigenic P. multocida.
Summary of the Invention
The present invention provides novel vaccine
compositions and components which protect animals against
disease associated with infection by toxigenic
Pasteurella multocida. These vaccine compositions are
characterized by the ability to elicit significant
quantities of circulating antitoxin.
In a first aspect, this invention provides a
vaccine which comprises an immunogenic amount of a
stable, soluble, cell-free toxoid of P. multocida, and a
carrier suitable for internal administration. This novel
P. multocida toxoid is produced by a method including a
step of subjecting the toxin to varying pH and
temperature, which method is also a novel aspect of the
present invention. The terin "toxoid" describes a
preparation of the toxin that has been inactivated
("toxoided") by a process that abolishes its toxicity
without destroying its ability to induce the production
of the specific neutralizing antitoxin.
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In another aspect, the invention provides a
novel vaccine composition containing an immunogenic
amount of a whole Pasteurella multocida bacterin with
cell-bound toxoid. This composition can induce in a
previously unvaccinated animal a superior antitoxin
response compared to the free, soluble toxoid. This
composition is also preferably associated with a carrier
suitable for internal administration.
In still another aspect, the invention provides
a novel vaccine composition comprising immunogenic
amounts of (1) a whole Pasteurella multocida bacterin
with cell-bound toxoid which, upon internal
administration to an animal, induces an antitoxin
response, and (2) the free toxoid of P. multocida. This
vaccine composition produces an unexpected synergistic
antitoxin response, much greater than the sum of the
separate effects of the two components. A carrier is
also desirably associated with this composition.
In a further aspect the above three vaccine
compositions may be varied by combination with an
immunogenic amount of one or more additional antigens.
Such additional antigens may include, among others, a B.
bronchiseptica bacterin or an Erysilpelothrix
rhusiopathiae bacterin. Other conventional vaccine
components may also be added to the vaccine compositions
of this invention.
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Another aspect of this invention includes a
vaccine dosage unit of each of the above vaccine
compositions. One embodiment of the invention includes a
vaccine dosage unit comprising 0.5 to 3 mL of a sterile
5 solution containing an immunogenic amount of between
about 80 to about 1000 relative toxoid units (RU) of a
multocida toxoid. Another embodiment includes a dosage
unit of 0.5 to 3 mL of a sterile suspension of an
immunogenic amount of between 0.5 to 8 optical density
units (OD) measured at 625 nm of a P. multocida bacterin
with cell-bound toxoid which upon internal administration
to an animal induces an antitoxin response. Still
another embodiment is a dosage unit comprising 0.5 to 3
mL of a sterile mixture of the free and cell-bound
toxoids. A further embodiment is a dosage unit
comprising 0.5 to 3 mL of a sterile mixture of
immunogenic amounts of the free and cell-bound toxoids
and one or more additional antigenic components.
In yet another aspect, the invention provides a
method for detoxifying the P. multocida toxin to prepare
a free, soluble immunogenic toxoid which comprises
incubating the toxin at a pH greater than 9 for at least
12 hours.
A furtheraspect of the invention provides a
method for detoxifying a whole P. multocida culture in
which the toxin is completely converted to a stable
toxoid within the bacterial cells, for use as a vaccine.
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This method involves treating the culture with a suitable
concentration of formaldehyde, at a suitable temperature
and for a sufficient time.
Yet a further aspect of this invention is a
method for vaccina-ting an animal against P. multocida
which comprises internally administering to the animal an
effective amount of one or more of the vaccine
compositions descxibed above.
Other aspects and advantages of the present
invention are described further in the following detailed
description of preferred embodiments thereof.
Detailed Description of the Invention
The present invention provides vaccine
compositions useful in the prophylaxis of diseases
resulting from infections with toxigenic P. multocida,
non-toxigenic strains of P. multocida, and other
pathogenic organisms. Such diseases include atrophic
rhinitis (AR), pleuritic and pneumonic pasteurellosis,
and erysipelas, among others.
One embodiment of this invention is a vaccine
which comprises an immunogenic amount of a free, soluble
P. multocida toxoid in a suitable carrier. The toxoid
of this invention is prepared generally by extracting
toxin from the bacterial-eells and causing a partial
denaturation by incubating the cell-free toxin for about
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12 to 24 hours at a pH greater than 9, at an incubation
temperature of between about 12 C to about 19 C.
More specifically, the free toxoid of this
invention is prepared as follows: A selected toxigenic
P. multocida strain is grown in a suitable culture
medium. At the end of the growth cycle, the toxin is
liberated from the cells by conventional physical or
chemical means e.g., french press or sonic disruption,
and cellular debris is removed by centrifugation and
filtration. The cell-free extracted toxin is then
incubated, preferably at pH of about 10.5, at ambient or
slightly cooler temperature for preferably 18 hours.
Following this incubation, the pH is adjusted to
neutrality. This process results in complete
detoxification of the toxin, providing a toxoid soluble
in aqueous solutions (e.g. phosphate buffered saline,
tris buffered saline).
The soluble P. multocida toxoid preparation of
this invention is both antigenic and immunogenic.
Specifically, the soluble toxoid can elicit antibodies
that can bind to the toxin, and neutralize its toxicity.
Further, the soluble toxoid of this invention is
characteristically stable at 4 C for at least 24 months,
which is a highly advantageous commercial characteristic,
indicating that this vacc-ine may be stored for later use.
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As another embodiment of this invention there
is provided a whole bacterin-toxoid of P. multocida which
contains the toxoid encapsulated and stabilized within
the bacterial cell. The bacterin toxoid is prepared from
a culture that is still growing exponentially and that
has not yet begun to release the toxin into the growth
medium. Formalin (formaldelhyde solution USP) is added
at a concentration of 0.5% v/v and inactivation is
continued at about 37 C for 4 days. Other formalin
concentrations may be employed in this method. However a
higher concentration will require a shorter inactivating
incubation period, and a lower concentration will require
a longer inactivating incubation period. One of $kill in
the art can readily determine these parameters based on
this disclosure. The toxoid is thereby encapsulated
within the bacterial cell. The dead bacterial cells,
with the toxoid sequestered safely within, are ideal
antigenic particles for presentation to those host cells
that mediate the immunizing process. This is especially
important for animals that have not previously been
exposed to the toxin or the toxoid and that totally lack
antitoxin.
In the P. multocida bacterin-toxoid of this
invention the cell-bound toxoid is remarkably stable.
Loss of antigenic potency was undetectable after storage
at 4 C for more than two years.
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For purposes of this invention, any toxigenic
strain of P. multocida may be used to provide the free
toxoid or the bacterin-toxoid of this invention. The
free or cell-bound toxoids described above can be derived
from any strain of P. multocida which elaborates
dermonecrotic toxin. Several such strains are available,
e.g., from the American Type Culture Collection,
Rockville, Maryland or from a variety of veterinary
colleges or laboratories. The strain used below in the
examples is P. multocida, type D, strain 8, which is
available, upon request, from the University of Illinois.
Suitable culture media for use in growing the
P. multocida cultures may be selected by one of skill in
the art, but preferably includes, without limitation, the
medium described by Herriott et al, "Defined Medium for
Growth of Hemophilus Influenzae", J. Bact., 101:'513-516
(1970).
The above described novel free toxoid and whole
bacterin-toxoid may be employed separately in vaccine
compositions for induction of an antitoxin response that
will prevent the pathological changes characteristic of
atrophic rhinitis caused by toxigenic P. multocida. In a
vaccine composition, an immunogenic amount of.the free
toxoid or the bacterin-toxoid is desirably mixed with
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suitable conventional vaccine adjuvants and physiologic
vehicles for injection into mammals, especially swine.
A more preferred vaccine composition is
provided by a synergistic combination of the free toxoid
5 and the whole bacterin-toxoid described above. The
combination vaccine of this invention combines the whole
bacterin-toxoid with the soluble toxoid, both vaccine
components prepared as described above. No other toxoids
or vaccines are prepared in this manner. Such a
10 combination vaccine is prepared by mixing an immunogenic
amount of free toxoid and an immunogenic amount of
bacterin-toxoid with suitable adjuvants and physiologic
vehicles for injection into mammals. Preferred.adjuvants
include amphigen and aluminum hydroxide gel.
In vaccination experiments with animals, as
reported below in Examples 8 and 10, these two vaccine
components have been found to act synergistically in a
single vaccine preparation. The "combination vaccine"
produces in the vaccinated animal a surprisingly greater
effect than that expected by simply adding the effects of
each toxoid component administered separately. This
combination vaccine stimulates a remarkable production of
antitoxin in tested animals. This combined effect can
also be generated by sequentially administering the
bacterin-toxoid vaccine,-followed by an injection of the
soluble toxoid vaccine.
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While not wishing to be bound by theory, it is
presently believed that the bacterin-toxoid vaccine
primes the animals, particularly immunologically naive
animals incapable of responding to soluble toxoid. A
second dose of the bacterin-bound toxoid induces a
moderate secondary response. Once primed by the toxoid-
rich cells of the bacterin-toxoid, however, the anima].s
are very responsive to the soluble free toxoid. Just as
the bacterin-toxoid is a superior priming agent, the
soluble toxoid has been observed to be a superior
booster.
Still other preferred vaccine compositions of
this invention result from combining the free toxoid
and/or the bacterin-toxoid of this invention with other
vaccinal agents. An illustrative example is a vaccine
composition formed by the combination of a whole cell B.
bronchiseptica bacterin with the P. multocida bacterin-
toxoid. Alternatively, the P. multocida bacterin-toxoid
is illustrated in further combination with E.
rhusionathiae. Other possible vaccinal agents which may
be combined with the vaccine components of this invention
include, without limitation, Escherichia coli,
Streptococcus suis, Mycoplasma hyopneumoniae,
Actinobacillus pleuropneumoniae, Clostridium perfrinaens
types C and D toxoids, Pseudorabies Virus Vaccine
(modified live virus and/or killed virus), Rotavirus
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Vaccine (modified live virus), Coronavirus Vaccine
(modified live virus).
Vaccines of the invention may be prepared as
pharmaceutical compositions containing an effective
immunogenic amount of the free toxoid and/or the whole
bacterin-toxoid, as active ingredients in a nontoxic and
sterile pharmaceutically acceptable carrier. A preferred
embodiment of the vaccine of the invention is composed of
an aqueous suspension or solution containing the free
toxoid and/or bacterin-toxoid, preferably buffered at
physiological pH, in a form ready for injection.
Alternatively or additionally, the free toxoid
and/or bacterin-toxoid can be admixed or adsorbed with a
conventional adjuvant. The adjuvant is used as a non-
specific irritant to attract leukocytes or enhance an
immune response. Such adjuvants include, among others,
amphigen, aluminum hydroxide, muramyl dipeptide, and
saponins such as Quil A.
In yet another exemplary alternative, the free
toxoid and/or bacterin-toxoid can be administered with
another immunostimulating preparation, such as B.
bronchiseptica or E. rhusiopathiae bacterins prepared by
known techniques.
* Trademark
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For purposes of this invention an immunogenic
amount of free soluble toxoid, when administered as the
sole active ingredient, may be defined in terms of
relative toxoid units ("RU"). The value of RU was
determined empirically based on an estimate of the amount
of toxoid which, when inoculated into mice, would elicit
an immune response that protected mice against the lethal
effects of intraperitoneal inoculation of approximately
30 LDso of purified toxin. In this system, an antigen
extinction study was performed and a PD$o determined. A
PD50, which is a calculated value, is defined as the
amount of toxoid required to protect 50 percent of the
mice from challenge with a defined amount of toxin, in
this instance, 30 LD50 of purified toxin. Thus one RU is
approximately equal to one mouse PD50. Thus, a range of
immunogenic amounts of free soluble toxoid is between
about 50 to about 1000 RU. More preferably the
immunogenic amount ranges between about 50 to about 650
RU. Another preferred range is between about 80 to about
450 RU. Still another preferred range is between about
80 to about 150 RU. Another.immunogenic amount of the
free toxoid, as defined by weight, ranges between about
16.2 and about 32.4 g toxoid.
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For purposes of this invention an immunogenic
amount of bacterin-toxoid, when injected as the sole
active ingredient, is defined as an optical density
(O.D.) of between about 0.5 and about 8 per ml, more
desirably, about 1 to about 4 per ml. Still another
preferred range is an O.D. of between 1 to about 3 per
ml. As used throughout this specification, the terms
O.D., optical unit (OU) or absorbency unit are
interchangeable and are measured at 625 nm in a
Spectronic 20 spectrophotometer unless otherwise
specified.
In a vaccine composition containing both
components, the same immunogenic amounts may be employed.
Alternatively, due to the synergy of the components when
combined, the immunogenic amount of the free soluble
toxoid may range between about 100 and about 150 RU and,
more preferably between about 100 to about 120 RU. The
immunogenic, amount of the bacterin-toxoid in combination
with the free toxoid may be in the lower O.D. ranges,
from about 1 to about 3, and more preferably, about 2.
In such a combination vaccine, the bacterin-toxoid may be
reduced to approximately 1.875.O.D. or absorbency units.
Other appropriate therapeutically effective
doses can be determined readily by those of skill in the
art based on the above immunogenic amounts, the condition
being treated and the physiological characteristics of
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the animal. It is preferred that the vaccine of the
invention, when in a pharmaceutical preparation, be
present in unit dosage forms. Accordingly, a
pharmaceutical preparation provides a unit dosage of
5 between 0.5 to 3 mis of a sterile preparation containing
an immunogenic amount of the active ingredients, whether
the active ingredient is the free toxoid only, the cell-
bound bacterin-toxoid only, or a combination thereof. In
the presence of additional active agents, these unit
10 dosages can be readily adjusted by those of skill in the
art.
The presently preferred formulation, which
appears to give maximum synergy is a Bordetella
bronchiseptica, Erysipelothrix rhusiopathiae, Pasteurella
15 multocida bacterin toxoid combination which contains
approximately 100 RU free toxoid with approximately 1.875
absorbency units cell-bound toxoid.
A desirable dosage regimen involves
administration of two doses of desired vaccine
composition, where the antigenic content (i.e.,
immunogenic amount) of each fraction is desirably as
stated above. The mode of administration of the vaccines
of the invention may be any suitable route which delivers
the vapcine to the host. However, the vaccine is
preferably administered s-ubcutaneously or by
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intramuscular injection. Other modes of administration
may also be employed, where desired, such as
intradermally or intravenously.
Present investigations with swine employ
intramuscular injection of two doses of vaccine
administered to the subject animal at least two weeks
apart. These studies have shown that, for each of the
above described vaccine compositions, a primary
immunization of newborn animals is desirably initiated at
about one week of age with a booster dose at weaning age.
For primary immunization of pregnant dams, two doses are
recommended with the last dose administered two weeks
before farrowing. A booster dose is recommended prior to
each subsequent farrowing. Semi-annual revaccination is
recommended for boars.
It will be understood, however, that the
specific dose level for any particular patient will
depend upon a variety of factors including the age,
general health, sex, and diet of the patient; the species
of the patient; the time of administration; the route of
administration; synergistic effects with any other drugs
being administered; and the degree of protection being
sought. Of course, the administration can be repeated at
suitable intervals if necessary or desirable.
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The specific mechanism of protection induced by
the vaccine compositions of the present invention is the
induction of toxin-neutralizing antibody (antitoxin) in
vaccinated animals, as indicated by the in vivo animal
tests described below.
The examples which follow illustrate preferred
methods for preparing the free soluble toxoid and
bacterin-toxoid of the invention and for preparing and
testing a variety of vaccines containing these novel
components. These examples are illustrative only and do
not limit the scope of the present invention.
EXAMPLE 1 - PREPARING PASTEURELLA MULTOCIDA TOXOID
A. Culturina the P. multocida
P. multocida type D (strain 8) [Dr. Ross
Cowart, University of Illinois, Urbana, Illinois) is
subcultured in a modified chemically defined synthetic
medium for one day. The medium is described by Herriott
et al, J. Bact., 101:513-516 (1970).
The pH of the assembled medium is adjusted
to 7.3 + 0.2 with sterile NaOH. Cells from this culture
are transferred to fresh synthetic medium and this
culture, when grown, is combined with a cryopreservative
and stored at -70 C. Production cultures are grown to
harvest during incubation at approximately 36 1 C for
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between 3 and 24 hours following inoculation. The
dissolved oxygen content of the culture is maintained by
aeration with sterile air and by agitation. Sterile
antifoam solution is used to control foam. The pH of the
culture is maintained at 7:3 0.2.
At the end of the growth cycle,
multocida cultures are examined and cell density is
determined by absorbance at 650 nm. Agitation is then
decreased, and aeration and pH control are discontinued.
The toxin content of the lysate is
measured by mouse lethality (LD50) and by the Enzyme-
linked Immunosorbent.Assay (ELISA) described below in
Example 4.
B. Pre-detoxification treatment
Following growth of the organism, sterile
merthiolate is added to the culture in an amount less
than or equal to 0.01 percent weight per volume. Culture
fluids may be aseptically transferred through closed
connections to a sterile closed container. The container
is connected through closed fittings to an apparatus used
to physically lyse cells and release cellular contents,
e.g., a "GAULIN" model l5M laboratory homogenizer.
Bacterial cells in the culture fluid are
lysed by continuous passage through the pressure chamber
of the homogenizer. This subjects the cells to an
immediate pressure drop from between an initial pressure
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of between 2000 and 5000 psi to ambient pressure of 15
psi. The lysed cells are aseptically deposited into
another closed container.
The lysate is clarified by sequential
steps of centrifugation and/or microporous filtration.
Clarified solutions may be concentrated before or after
filter sterilization. Ethylenediaminetetraacetic acid
(EDTA), in an amount up to a final concentration of 5 mM,
and glycerol, in an amount up to a final concentration of
1.0% (vol/vol), are added before concentrating and
filter-sterilizing, to prevent aggregation of the
concentrated proteins.
C. Detoxification
Sterile 5 N NaOH is slowly and aseptically
added to sterile toxin to increase the pH to
approximately 10.55 0.10 pH units. At this pH, the
detoxification occurs as the mixture is allowed to stir
slowly at approximately 15 1 C for.between 15 and 24
hours. The pH is not adjusted thereafter until
detoxification is complete or aliquots are taken to
measure residual toxicity. Sterile 5 N HC1 is then
slowly and aseptically added to adjust the pH to 6.80 +
0.20 pH units.
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At two-hour intervals beginning 16 hours
after the pH is adjusted to 10.55, an aliquot is taken.
Residual toxicity of each aliquot is measured and
expressed in mouse LD50's per mL. A preparation with an
initial value of nearly 10,000 LDso's per mL is usually
detoxified 18 hours after adjusting the pH to 10.55,
without appreciable decrease in assayable antigen
content. Thereafter the pH is adjusted to 6.80 + 0.1
unit with 5N HC1. The toxoid is then stored at 2 to 7 C
until combined with other components and assembled into
vaccine compositions. If the injection of mice shows any
residual toxicity, the pH of the preparation is again
raised to 10.55, and the temperature to 15 C. After
several hours, depending on the degree of toxicity
detected, the preparation is neutralized, cooled, stored,
and tested once more.
EXAMPLE 2 - VACCINE FORMULATION
An illustrative toxoid vaccine formulation
according to the invention was made by preparing the
soluble free toxoid as described above in Example 1.
The buffer used to prepare the vaccine
compositions is sterile saline at a neutral pH. Sterile
aluminum hydroxide gel is used as adjuvant and added at a
level sufficient to adsorb toxoid, generally 12% 1%
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(vol/vol). The vaccine compositions are prepared by
thoroughly mixing, then dispensing the indicated amount
of toxoid and aluminum hydroxide gel into a 500 ml
beaker. Sterile saline is then added. This mixture is
stirred and stored at 4 C. Dosage amounts of 2 ml/dose
are desirable, which provides about 450 relative toxoid
units per dose.
Table I illustrates the formulation of two
free-toxoid vaccines according to the invention.
Table I
Experimental Lot Component Total Volume
A Toxoid Concentrate 150.0 ml
Aluminum Hydroxide Gel 36.0 ml
Sterile Saline 114.0 ml
Total 300.0 ml
B Toxoid Concentrate 235.0 ml
Aluminum Hydroxide Gel 41.0 ml
Sterile Saline 304.0 ml
Total 580.0 ml
These free-toxoid vaccine formulations are
useful as an aid in prevention of atrophic rhinitis in
swine caused by P. multocida infections. An exemplary
test of the free toxoid vaccine is performed by injecting
the formulations into swine (pigs and dams) as described
below.
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EXAMPLE 3 - VACCINATION EXPERIMENTS
Using the formulations of Example
vaccinations were administered intramuscularly to pigs
and dams selected at random according to the following
protocols. In each test after vaccination the animals
were challenged with purified toxin at a dose known to
consistently induce clinical signs of atrophic rhinitis
in pigs. Toxicity of DNT was evaluated in mice before
and after challenge. The total dose of toxin each pig
received was 8.4 g, or 50 mouse LD50. Toxin was
administered in three equal doses over a three day period
beginning approximately two weeks following vaccination.
Results of the challenge were evaluated
approximately 28 days following the first dose of toxin.
The percent weight gain was calculated by the number of
pounds gained in the 28 days following challenge divided
by the weight, in pounds, at challenge. Nasal turbinate
atrophy was evaluated by cross-section of the snout at
the first premolar tooth as follows: score 0, normal;
score 1, minimal atrophy; score 2, moderate atrophy;
score 3, substantial atrophy; score 4, near complete
atrophy; and score 5, complete atrophy.
Protocol I: Four gilts were vaccinated with a
2 ml dose of P. multocida free toxoid (A), described
above in Example 2. Two-gilts failed to farrow because
of an infection of porcine parvovirus and were removed
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from the facility as soon as disease was evident. Pigs
born of the two remaining gilts were vaccinated at 13
days of age (gilt 637, 7 pigs) and 9 days of age (gilt
638, 4 pigs) with a 2 ml dose of P. multocida free toxoid
(B), described in Example 2. Second vaccinations were
administered to all pigs two weeks later. Pigs were
challenged with a dose of toxin two weeks following the
second vaccination. Gilts from the same herd with
farrowing dates similar to vaccinated gilts provided
contemporary unvaccinated control pigs.
Following challenge, vaccinated and
unvaccinated control pigs were commingled until they were
slaughtered for final scoring. Table II illustrates the
effects of challenge on pigs which were farrowed from
dams vaccinated with two doses of vaccine A, and which
were themselves vaccinated (VX) with two doses of free
toxoid vaccine 5, compared to unvaccinated (NonVX)
animals. These results show significantly lower snout
scores and significantly better weight gains in the
vaccinated group.
Table II
Weight at Weight at Weight Weight Mean
Group No. Challenge Slaughter Gain Gain Snout
(lb) % Score
VX 10 26.20 39.60 13.40 54.27 1.00
Non-VX S 22.88 31.56 8.69 35.30 2.34
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Protocol II: Four gilts were vaccinated with a
2 ml dose of vaccine A. One gilt failed to farrow
because of an infection of porcine parvovirus and was
removed from the facility as soon as disease was evident.
Pigs from remaining gilts were challenged with toxin as
follows: 9 pigs from one gilt at 10 days old; 2 pigs from
a second gilt at 12 days old; and 6 pigs from a third
gilt at 4 days old. Gilts from the same herd with
farrowing dates similar to vaccinated gilts provided
contemporary unvaccinated control pigs.
Vaccinated and unvaccinated control pigs were
challenged prior to weaning and thereafter commingled
until slaughtered for final scoring. Table III
summarizes the effects of challenge on pigs farrowed by
dams which received two doses of vaccine A. The data are
presented (a) independently of litter, and (b) by litter
averages.
These results show significantly lower snout
scores and significantly better weight gains in the
vaccinated group. These observations indicate that two
doses of vaccine A given to dams induced the production
of antitoxin that was passively transferred to otherwise
susceptible pigs. Furthermore, the duration of passive
protection was at least 10 to 12 days.
CA 02086258 2000-06-14
Table III
(a)
Weight at Weight at Weight Weight Mean
Group No. Challenge Slaughter Gain Gain Snout
5 (lb) o Score
VX 15 6.87 21.00 14.13 205.83 3.02
Non-VX 5 8.20 16.40 8.20 100.00 3.70
10 (b)
vx
Gilt 629 7 8.71 21.50 12.79 147.09 3.68
Gilt 639 2 8.00 29.25 21.25 268.65 2.38
Gilt 633 6 4.33 17.67 13.33 310.28 2.46
Gilt Avg 7.02 22.81 15.79 242.01 2.84
Non-VX 5 8.20 16.40 8.20 100.00 3.70
EXAMPLE 4 - ELISA TO OUANTIFY ANTIBODY
Pig sera and colostrum samples from the
experiments of Example 3 were tested for antibodies
against the toxin by a kinetic ELISA. Briefly, purified
toxin (250 ng/well) in 0.1 M sodium borate, pH 9.1, was
adsorbed to flat-bottom 96 well Nunc microtiter plates
overnight at 4 C. Plates were then blocked at 37 C for
minutes with 10% nonfat dried milk in PBS with 0.05%
Tweeri 20 (blocking buffer). Blocking buffer was rinsed
*
from the plates with two PBS/0.05% Tweeri 20 (PBS/Tween)
* Trademark
WO 91/19419 PCT/US91/040Q2
26
rinses, followed by a PBS rinse. Sera were diluted 1:100
in blocking buffer, and 50 l samples were added to each
of four wells. Plates were incubated for 60 minutes at
37 C, and then rinsed as above.
Goat-anti swine IgG (heavy and light chain
specific)-horseradish peroxidase (Kirkegaard and Perry
Laboratories, Gaithersburg, MD] was diluted 1:500 in
blocking buffer, and added (50 l) to each well.
Following a 60 minute incubation at 37 C, plates were
rinsed as above. ABTS substrate (2,21-axino-di-3-ethyl-
benzthiazoline sulfonate) [Kirkegaard and Perry] was
added, and plates were read immediately on a Vmax ELISA
reader at 405 nm (Molecular Devices Corporation, Palo
Alto, CA]. Each well was read eight times during a one-
minute interval, and the rate of the enzymatic reaction
was calculated.
Rates were calculated as the change in milli
units of optical density (mOD) per minute. Thus a
reading of 10o moD per minute would be equal to an OD of
1.0 in 10 minutes. Values were then corrected for the
amount of serum used per well and reported as mOD/min/ml
of serum. For instance, if 50 l of serum produced a
reading of 100 mOD per minute, the reported value would
be 2,000 mOD units per minute per ml.
WO 91/19419 PCT/US91/04092
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27
The following controls were included on each
ELISA plate. (1) Serum control: each diluted pig serum
was placed in a well that did not contain antigen, then
exposed to all subsequent reagents to check for non-
specific adsorption to the plate. At the dilution of pig
sera (1:100) used, no color greater than that obtained in
the negative serum control was seen. (2) Negative pig
serum control: each plate included three wells of a
known negative pig serum diluted 1:100 in blocking
buffer. (3) Positive pig serum controls: serum
containing specific antibodies to the toxin was diluted
in negative pig serum to obtain sera containing high,
moderate, and low concentrations of specific antibody.
These three sera were diluted 1:100 in blocking buffer
and placed in triplicate on each plate. Background, or
non-specific reactivity, was determined in wells that
contained all reagents except pig serum.
Table IV below summarizes the ELISA titers of
the dams and pigs vaccinated with toxoid vaccines A and
B, respectively, according to Protocol II (Example 4).
The table gives the geometric mean titers of sera taken
before the first and second dam vaccinations, of the
colostrum, and of sera taken before the first and second
pig vaccinations, challenge, and slaughter, as compared
to unvaccinated controls-(Non-Vx).
WO 91/19419 PCT/US91/04Q92
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28
Table IV
Geometric Mean ELISA Titers
ist 2nd 1st 2nd
Dam Dam Pig Pig
Group Vx Vx Colostrum Vx Vx Challenge Slaughter
VX 21.71 0 173.00 0.99 1.73 109.03 139.07
Non 25.35 12.34 83.38 1.45 1.72 .38 8.64
VX
These results indicate that two doses of
vaccine A given to dams, followed by two doses of vaccine
B given to their pigs, induced immunity to the toxin in
otherwise susceptible pigs.
From the same study (Protocol II, Example 4)
Table V summarizes the ELISA titers of vaccinated
(vaccine A) and unvaccinated dams and their unvaccinated
pigs.
WO 91/19419 PGT/US91/04092
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29
Table V
Geometric Mean ELISA Titers
lst 2nd
Dam Dam
A: Group Vx Vx Colostrum Challenge Slaughter
Vx 28.19 .76 104.31 7.98 22.53
Non-Vx 27.56 15.53 80.60 .19 .75
Individual ELISA titers Geometric mean titers
of litters at:
lst 2nd
Dam Dam
B: Group Vx Vx Colostrum Challenge Slaughter
Vaccinated
Gilt 629 21.80 5.80 70.60 1.66 29.15
Gilt 639 29.20 - 18.60 26.07 25.39
Gilt 633 35.20 - 154.10 36.43 16.03
Average 27.73 1.93 81.10 21.39 23.03
Unvaccinated
Gilt 636 23.40 10.80 66.80 - 12.40
Gilt 631 30.60 11.90 135.90 - 0.20
Gilt 626 19.80 17.60 76.70 - 9.40
Gilt 635 40.60 20.40 - - -
Gilt 632 27.60 19.60 60.60 4.60 -
Average 28.40 16.06 68.00 0.92 4.4
Table VI shows a summary of challenge-of-
immunity studies for dam and pigs vaccinated with various
doses (in relative toxoid units, RU) of free toxoid
preparations.
WO 91/19419 PCr/US91/040 2
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Table VI
RU Administered to: Significant Protection against
Dams Pigs No. Weight Loss Turbinate atrophy
5 876 32 307 70 10 Yes Yes
876 + 32 0 15 Yes Yes
391 52 0 10 No No
0 391 + 52 9 No No
10 The data shows significant protection of pigs
farrowed by dams vaccinated with two doses of a vaccine
containing 876 32 RU of free toxoid. In pigs or
pregnant gilts, two doses of experimental lots containing
between 300 and 400 RU/dose, did not appear to induce
15 protection.
EXAMPLE 5 - PREPARING A BACTERIN-TOXOID VACCINE
COMPOSITION
An embodiment of this composition includes a
bacterin-toxoid of P. multocida in which the toxoid has
20 been stabilized within the bacterial cell.
A culture of P. multocida, type D, strain 8,
is grown in the following medium: Tryptic Soy Broth
without Dextrose (Difco) 30 g; Yeast extract (Difco) 5
g; Dextrose 4 g; Deionized water to 1 liter; pH of
25 approximately 7; sterilized by autoclaving at 121 C.
WO 91/19419 PCT/US91/04092
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The culture is aerated with agitation to
maintain the dissolved oxygen concentration at
approximately 35% of saturation. The temperature is
maintained at 37 C, and the pH at 7 by the addition of
lON NaOH solution as needed. Towards the end of
exponential growth, aeration is discontinued and the
culture is inactivated by the addition of formaldehyde
solution (USP) to a final concentration of 0.5% v/v. The
culture is then held at 37 C for four days. Other
inactivating agents, such as beta-propriolactone,
glutaraldehyde, and binary ethyleneamine can be used in
place of formaldehyde.
A sample is withdrawn to test whether
inactivation is complete by administering the sample to
guinea pigs. Guinea pigs should be alive and healthy at
7 days after subcutaneous injection with 4 ml volumes of
the culture. At this point the toxin within the cells is
completely converted to toxoid, which is safe, very
stable and capable of inducing the production of
neutralizing antitoxins upon injection into animals.
The inactivated culture is centrifuged. The
sedimented bacteria are dispensed in sufficient
supernatant fluid to make a suspension with an OD of 4.2.
The suspension is then adsorbed with A1(OH)3 gel, 25% v/v,
thimerosol (0.01% w/v) is added as a preservative, and
the pH is adjusted to 6.5 0.2.
WO 91/19419 PGT/US91/0.4092
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This P. multocida bacterin-toxoid may be used
in vaccines as the sole vaccine component.
Alternatively, the bacterin toxoid may be employed in
vaccine compositions with other vaccine components.
Whether the bacterin-toxoid is used alone or in
combination, saponin (0.5 mg/ml) may be added as
adjuvant.
One example of a combination vaccine contains a
suspension of adsorbed P. multocida (O.D. 7.5 before
adsorption) mixed with equal volumes of similarly
adsorbed and preserved cultures of Bordetella
bronchiseptica (O.D. 4.2 before adsorption) and
Erysipelothrix rhusionathiae (O.D. 7.5 before
adsorption). This bacterin-toxoid vaccine, referred to
as Atrobac 3 (Beecham Laboratories), has a dose volume of
2 ml.
EXAMPLE 6 - A COMBINATION VACCINE
Combination vaccines may contain the bacterin
toxoid of Example 5, and/or the soluble toxoid of Example
1 with optional components, such as other inactivated
microorganisms, e.g., B. bronchiseptica and other strains
of P. multocida (P. multocida serotype A for protection
against pleuritic and pneumonic forms of pasteurellosis).
CA 02086258 2000-06-14
33
One exemplary combination vaccine employs the
P. multocida bacterin-toxoid described in Example 5 and
the P. multocida free toxoid described in Example 1.
Another combination vaccine may include the free toxoid
and bacterin toxoid of P. multocida type D, described
above, with an inactivated whole culture of B.
bronchiseptica.
Still another efficacious vaccine composition
against infection by P. multocida can be prepared by
combining the bacterin-toxoid vaccine composition,
Atrobac 3, described above in Example 5, and the soluble,
free toxoid of P. multocida prepared as described above
in Example 1.
One exemplary formulation for a combination
vaccine consists of the following components:
Component Vol/ds (ml) Vol (ml)
Atrobac 3~ 2.000 250.00
Free toxoid (650 U/ml) 0.242 30.25
oil/lethicin 0.100 12.50
Tween*80 0.056 7.00
Span'80 0.024 3.00
TOTALS* 3.000 375.00
* Trademark
WO 91/19419 PCT/US91/040-92
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o~1r~r
34
For emulsification, these components were
combined and emulsified, as a single batch, for 2
minutes. For production scale, it is anticipated that
metered in-line rather than batch combination is
desirable.
Other ingredients may be added to, or may
replace existing ingredients in, the specific formulation
above. For example, aluminum hydroxide gel may be
employed as an adjuvant in place of oil/lethicin.
EXAMPLE 7 - A COMBINATION VACCINE
Another combination vaccine was prepared as
follows: E. bronchiseptica, Strain 2-9 NADC [National
Animal Disease Center, Ames, Iowa] was subcultured six
times. P. multocida type A strain 169 was cultured in a
manner similar to that described for strain 8 in Example
1. For example, at the end of their respective growth
periods, cultures of B. bronchiseptica and P. multocida
type A strain 169 are inactivated by the addition of
beta-propiolactone (BPL). A second addition of BPL is
made 2 to 18 hours following the first. The final
concentration of BPL does not exceed 1:500 (0.2%). Each
culture is incubated at less than 20 C with constant
agitation for at least 12 hours.
::A 02086258 2000-06-14
For inactivated cultures of B. bronchiseptica
and P. multocida type A strain 169, sterile mineral oil
[Drakeol] containing 3.3% to 40% by weight of lecithin
[Central Soya] is added as adjuvant. In the final
5 product, the concentration of the oil fraction is
approximately 5% by volume. Tween*80 is added to a final
concentration of between 0.7 and 2.8%. An emulsifier is
added to a final concentration of about 0.3 to 1.2%
(e.g., Span 80). A selected paraben, e.g. methyl p-
10 hydroxyl-benzoate, propyl p-hydroxylbenzoate, or butyl p-
hydroxylbenzoate, may be added as an additional
preservative.
The P. multocida free toxoid is mixed with
sterile aluminum hydroxide gel (equivalent to 2% A1203) as
15 adjuvant. In the final product the concentration of
aluminum hydroxide gel is 12% by volume.
Concentration is performed by ultrafiltration
under aseptic conditions.
In an exemplary combination vaccine, the B.
20 bronchiseptica fraction contains at least 1500
nephelometric units per vaccine dose. The nephelometric
units are based on the value measured at the time of
harvest. The P. multocida type A strain 169 fraction
contains at least 3.4 absorbance units per dose. The
25 absorbency units are based on the value measured at the
* Trademark
WO 91/19419 P(.T/US91/04092
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36
time of inactivation. The P. multocida free-toxoid
fraction contains at least 450 relative toxoid units per
2.0 ml dose. Relative toxoid units are measured prior to
final product assembly.
One or more complete or partial bulk lots of
each fraction are combined with adjuvant and saline
diluent to obtain the standard antigen concentration.
These fractions are formulated prior to final
assembly by combining the B. bronchiseptica and P.
multocida type A strain 169 components (Fraction I),
preparing the free toxoid formulation (Fraction II), and
combining Fractions I and II (Vaccine) in the proportions
shown in Table VII below.
WO 91/19419 PCT/US91/04092
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37
Table VII
Component Vol/2.0 ml Total Vol (ml)
dose (ml)
FRACTION I:
B. bronchiseptica 0.250 6,250
P. multocida type A 169 0.500 L2,500
Oil/Lethicin 0.100 2,500
Saline 0.150 3,750
Totals 1.000 25,000
FRACTION II:
E. multocida Toxoid 0.300 7,500
A1(OH)3 gel 0.240 6,000
Saline 0.460 11,500
Totals 1.000 25,000
VACCINE:
Fraction I 1.000 25,000
Fraction II 1.000 25,000
Totals 2.000 50,000
CA 02086258 2000-06-14
38
EXAMPLE 8 - VACCINE TESTS IN ANIMALS
The vaccine compositions of Examples 2, 5, 6
and 7 are useful in the prevention of atrophic rhinitis
and pneumonia in swine caused by B. bronchiseptica and/or
P. multocida. During the vaccine tests, it was
surprisingly observed in the evaluation of the antibody
response in swine, that combining the P. multocida
bacterin-toxoid and free toxoid had more than an additive
effect on the induction of antitoxin, compared to use of
the bacterin-toxoid alone or the free toxoid alone.
In one experiment, groups of pigs were
vaccinated with Atrobac 3; which contains the P.
multocida bacterin-toxoid and preserved cultures of B.
bronchiseptica and E. rhusiopelothrix (Example 5), or
with the free soluble P. multocida toxoid of Example 1
only, or with a combination of bacterin-toxoid and
soluble toxoid as described in Example 6. Table VIII
demonstrates antibody response to vaccination with free
toxoid alone, with bacterin-toxoid alone and with a
combination of these two vaccine components. The ELISA
titers indicate a synergistic effect of this combination
vaccine. This combination vaccine composition is
believed to induce the best immunity in swine.
* Trademark
WO 91/19419 PCT/US91/04092
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39
The post vaccination sera were also assayed for
neutralizing antitoxin, the actual protective antibody,
by the method of Roberts and Swearingin, Am. J. Vet.
Res., 49: 2168 (1988). The antitoxin values show a
strong synergy of the free and cell-bound toxoids (Table
VIII).
Table IX demonstrates the results of another
experiment wherein a vaccine containing whole cell
inactivated cultures of P. multocida (PmD), soluble
toxoid and B. bronchiseptica inactivated whole cells
(Bb), was used in guinea pigs and serum antibody levels
measured by the EBL tissue culture assay [J.M. Rutteret
al, Veterinary Record, 114: 393-396 (1984)]. In this
experiment the combination vaccine dosageunit is 2
ml/dose. In this experiment 600 RU toxoid failed to
induce an appreciable anti-toxin response. In contrast,
600 RU toxoid combined with inactivated cultures of P.
multocida (PmD) induced an anti-toxin response level of
128. This demonstration serves as yet another example of
immunologic synergy for soluble toxoid and inactivated
cultures of toxigenic P. multocida.
WO 91/19419 PCT/US91/040.92
Table VIII
Serum Neutralizing
No. Bacterin Free Antibody Antitoxin
Pigs Toxoid Toxoid Adjuvant Levels units/ml
5 (ml) (RU) PRE POST POST
Vx Vx Vx
8 0 ml 200 A120H3 <10 13 <1
8 0 ml 200 Amphigen-A120H3 <10 16 <1
8 2 ml 0 A120H3 <10 93 20
10 8 2 ml 0 Amphigen-A120H3 <10 46 20
8 2 ml 120 A"0H3 <10 252 40
8 2 ml 120 Ambhigen-A120H3 <10 302 80
Table IX
Bacterin- Free Dose Serum Antibody
15 Toxoid Toxoid Fraction Adjuvant Levels
(RU) PRE-Vx POST-Vx
Bb+PmD 600 1/25 Amphigen-A120H3 < 2 128
Bb+PmD 300 1/25 Amphigen-A1ZOH3 < 2 4
Bb+PmD 0 1/25 Amphigen-A120H3 < 2 < 2
20 Bb 600 1/25 Amphigen-A120H3 < 2 4
Bb 300 1/25 Amphigen-A120H3 < 2 < 2
EXAMPLE 9 - A COMBINATION VACCINE
A further embodiment of the vaccine
25 co}npositions of this invention includes a combination
vaccine containing a B. bronchisebtica bacterin, a P.
multocida toxoid of Example 1, a P. multocida type A
strain 169 bacterin described in Example 7, and an E.
rhusiopathiae bacterin. This vaccine is useful for the
30 vaccination of healthy swine as an aid in prevention of
WO 91 / 19419 PCT/US91 /04092
p~ t,c) r=U
41 f~ ") ;3 ~ ~
atrophic rhinitis, erysipelas and pneumonia caused by B.
bronchiseptica, Erysipelothrix rhusiopathiae and P.
multocida.
Cultures of B. bronchiseptica and P. multocida
type A strain 169 are inactivated and individually.
formulated into fractions as described in the above
example.
Cultures of E. rhusionathiae strain SE-9
(Dellen Labs, Omaha, Nebraska] are inactivated by the
addition of formaldehyde solution (37 percent) to a final
concentration of 0.35 to 0.45 percent. The E.
rhusiopathiae fraction is formulated in aluminum
hydroxide gel as described for the free-toxoid fraction.
The free toxoid is prepared and formulated as
described above in Examples 1 and 2.
Concentration is performed by ultrafiltration
under aseptic conditions.
Each fraction is combined with adjuvant and
saline diluent to obtain.the standard antigen
concentration. These fractions are formulated prior to
final assembly by combining the B. bronchiseptica and P.
multocida type A strain 169 components (Fraction III),
preparing the free toxoid and E. rhusiopathiae components
(Fraction IV) and combining Fractions III and IV
(Vaccine) in the proportions shown in the Table X below.
WO 91/19419 PCT/US91/04092
42
Table X
Component Vol/3.0 ml Total Vol (ml)
dose (ml)
FRACTION III:
B. bronchisebtica 0.250 6,250
P.. multocida type A 169 0.500 12,500
Oil/Lethicin 0.100 2,500
Saline 0.150 3,750
Totals 1.000 25,000
FRACTION IV:
rhusiogathiae 0.500 12,500
~. multocida 8 Toxoid 0.300 7,500
aluminum hydroxide gel 0.600 15,000
Saline 0.600 15,000
Totals 2.000 50,000
VACCINE:
Fraction III 1.000 25,000
Fraction IV 2.000 50,000
Totals 3.000 75,000
WO 91/19419 PCI'/US91/04092
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43
Each dose of vaccine contains at least 1500
nephelometric units of B. bronchiseptica, at least 3.0
absorbency units of E. rhusiopathiae, at least 3.4
absorbance units at 650 nm of P. multocida type A strain
169, and at least 450 relative units of P. multocida
toxoid. The absorbency units are based on the value
measured at the time of harvest.
EXAMPLE 10 - VACCINATION EFFICACY EXPERIMENTS
Five experimental vaccines according to the
present invention were evaluated for efficacy in animal
studies using pathogenic challenge of vaccinated and
unvaccinated animals. The experimental vaccines are
formulated essentially as described above and include the
following active components as set out in Table XI below:
Table XI
P. multocida
B. bronchi- Strain E. rhusio-
VX septica 169 Toxoid pathiae
C 170 mis 425 mls 646 mls 0
D 170 0 646 0
E 220 505 260 0
F 300 689 355 1035
G 200 591 250 350
WO 91/19419 PCT/US91/04092
!2C)c4)(' 44
N ..r :.) 'at 1+i t =.~
I. P. multocida type A Challenge
One experiment was conducted as follows: On
day 1 and day 14, ten pigs were vaccinated with a 2 ml
dose of Vaccine C and ten pigs with a 2 ml dose of
Vaccine D. Two weeks after the second inoculation, these
pigs and ten contemporary unvaccinated controls were
infected with P. multocida strain 169. Disease severity
was quantified by a clinical score at death or two,weeks
after infection (Table XII).
Table XII
Clinical Scores
VX No. Dead/Total Mean S.D. Signif.
C 10 1/10 4.0 2.75 p<.0025
D 10 5/10 9.0 1.41 none
NonVx 10 5/10 8.4 2.31 CONTROL
Tab1e XIII summarizes the agglutinin responses
(geometric mean titer (GMT)) of the three groups to P.
multocida, at firstand second vaccinations, at
challenge, and at death or slaughter.
WO 91/19419 PCf/US91/04092
45 20 8
Table XIII
AGGLUTININ TITERS (GM)
At Death or
VX 1st Vaccine 2nd Vaccine At Challenge Slaughter
C <4 16 138 939
D <4 <4 <4 36
NonVX <4 <4 <4 128
Following challenge, clinical signs in
susceptible pigs were consistently severe. Predominant
clinical signs included death (or moribundity), lameness,
and pleuritis and pericarditis with fibrinous deposits
and adhesions. These signs were abundant in all pigs
except those vaccinated with Vaccine C. Of the ten pigs
given Vaccine C, substantial protection was evident in
nine. Based on these observations, two doses of Vaccine
C given to susceptible pigs induced immunity to challenge
with P. multocida type A.
II. Toxin Challenge
Another experiment was performed as follows:
On day 1 and day 15, five pregnant dams were vaccinated
with Vaccine D (2 ml dose) and five with Vaccine E (2 ml
dose). Farrowings occurred beginning about two weeks
thereafter. Five pigs from each of the ten vaccinated
dams and five pigs from unvaccinated dams were given a
WO 91/19419 PCT/US91/04092
2 E)~~N ~c) 46
2 ml dose of Vaccine E. At weaning, vaccinated pigs and
unvaccinated pigs (littermates to vaccinated pigs
farrowed from unvaccinated dams) were challenged with
toxin. About one month thereafter, pigs were scored for
clinical signs of atrophic rhinitis.
A summary of weight gains and snout scores for
each group is shown in Table XIV below.
Table XIV
Group Mean Values
Number Wt. (lbs.) at: Wt Gain by: Snout
Group Dams Pigs Chal. Final (lbs) % Score
1 5 25 21.32 59.04 37.72 179.11 1.13
35 2 5 25 19.96 57.48 37.52 191.80 1.02
3 5 25 **21.76 51.62 29.67 138.97 2.83
***21.76 43.72 21.96 99.95
4 5 10 **21.20 52.43 31.00 144.91 2.57
***21.20 36.70 21.70 101.44
5 4 4 23.50 64.75 41.25 176.58 0.44
Group 1: Vaccinated: Dams - vaccine D; Pigs - vaccine E
Group 2: Vaccinated: Dams - vaccine E; Pigs - vaccine E
Group 3: Vaccinated: Dams - None; Pigs - vaccine E
Group 4: Unvaccinated: Dams and Pigs
Group 5: Sentinel
* Pigs in groups 3, 4, and 5 were farrowed from common
dams
** Values calculated using only pigs surviving challenge
*** Values calculated including pigs not surviving
challenge
WO 91/19419 PCT/US91/04092
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47
Pigs in groups 1 and 2 (Table XIII) gained
37.72 and 37.52 pounds per litter, or 179.11 and 191.80
percent, respectively. Susceptible pigs, groups 3 and 4,
gained 29.67 and 31.00 pounds, or 138.97 and 144.91
percent, respectively. The difference in weight gains
for susceptible pigs (groups 3 and 4, n=35) were compared
to weight gains for vaccinated and protected pigs (groups
1 and 2, n=50) by student "t" test. The difference was
very highly significant (p <<0.0005) showing increased
weight gain for groups 1 and 2 (Table XIII). The snout
scores for susceptible pigs (groups 3 and 4) were
compared to those for pigs in groups 1 and 2. There was
a marked decrease in the destruction of nasal turbinates
in groups 1 and 2. The difference was highly significant
(Student "t" test: p <0.0005). According to the
analysis of weight gains and nasal turbinate scores, pigs
in groups 1 and 2 were protected from challenge with
toxin (Table XIV).
Serum samples were collected immediately prior
to each dam and pig vaccination, from the dam at
farrowing, from pigs when challenged with,toxin (chal),
and from pigs when signs of AR were scored (slaughter).
Table XV summarizes the antibody titers of the dams and
pigs in the various treatment groups. N/A means the data
are not available.
WO 91/19419 pCi'/US91/04092
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48
Table XV
Geometric Mean Titer (GMT) to each fraction for sera
taken at or before:
1st 2nd 1st 2nd
Dam Dam Pig Pig
Group Vx Vx Farrow Colostrum Vx Vx Chal Slaughter
1
Bb 21.11 294.06 147.03 776.05 25.64 19.97 22.05 7.16
PmA 16.00 18.38 10.56 10.56 <4 6.53 82.14 105.40
PmD 9.58 11.33 30.48 195.14 18.95 11.94 61.36 248.53
2
Bb 24.25 512.00 222.85 1552.00 28.65 17.88 14.72 18.38
PmA 16.00 73.52 73.52 168.90 32.90 13.93 65.80 75.59
PmD 6.13 9.99 32.36 245.14 28.56 16.49 106.02 272.76
3
Bb N/A N/A 9.19 42.22 5.90 7.78 17.67 14,.32
PmA N/A N/A 8.00 12.13 <4 8.69 91.77 100.43
PmD N/A N/A 7.56 12.48 5.03 5.06 5.88 24.60
4
Bb N/A N/A 9.19 42.22 5.28 4.67 4.29 4.88
PmA N/A N/A 8.00 12.13 <4 4.30 5.15 <4.00
PmD N/A N/A 7.56 12.48. 3.04 3.46 4.34 3.67
5
Bb N/A N/A 8.00 38.05 13.45 8.00 5.66 4.76
PmA N/A N/A 8.00 12.13 11.32 6.73 5.66 <4.00
PmD N/A N/A 7.56 12.48 - 2.73 4.19 4.40 1.67
WO 91/19419 PCT/US91/04092
49
For purposes of Table XV, the symbol Bb means B.
bronchiseptica; PmA means P. multocida type A (strain 169)
and PmD means P. mu].tocida toxin. The vaccine groups are as
defined in Table XIV.
These results show that two doses of Vaccine E did
not induce antibody to toxin when given to pigs farrowed
from unvaccinated dams. These pigs remained susceptible to
challenge with toxin. In contrast, two doses of Vaccine E
induced antibody to toxin in pigs farrowed from vaccinated
dams. From these data, vaccination of dams and pigs with
toxoid immunized pigs against the toxin of P. multocida.
III. Challenge with E. rhusiopathiae
Still an additional experiment was performed as
follows: On day 1 and day 14, eight pigs each were
vaccinated with vaccines F and G (see Table XI). On the
same dates eight pigs were vaccinated with two doses of
vaccine E. On day 1 four pigs were vaccinated with a single
dose of erysipelothrix bacterin. On about day 30, pigs were
challenged with virulent E. rhusionathiae.
A summary of response to challenge is presented in
Table XVI below.
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Table XVI
Number
Vaccine Normal Total Percent Result
F 8 8 100 3Sat
G 7 8 87.5 3Sat
'Exp. ER 4 4 100 3Sat
ZE 2 8 25 'Sat
Vaccinated: single.dose of an experimental Erysipelothrix
rhusiopathiae bacterin. Pigs in this group served as a
positive control.
2 Vaccinated: 2 doses vaccine E (no erysipelothrix). Pigs
in this group served as susceptible controls for challenge
with virulent E. rhusiogathiae and for comparison of
serologic response to unchallenged fractions.
3 Satisfactory protection of vaccinated animals as defined
in 9 CFR 113.04(e).
Satisfactory disease in susceptible swine, e.g.,
unvaccinated controls as defined in same regulation.
The results in Table XVI show that at least 75% of
the susceptible animals displayed clinical signs of
erysipelas, validating the challenge. At least 75% of the
animals vaccinated with two doses of vaccines F or G were
without clinical signs of erysipelas. These results
indicate that two doses of vaccines F or G provide effective
protection against challenge with virulent E. rhusiopathiae.
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51
At the end of the observation period, all
surviving pigs were treated with penicillin for 4 to 5 days
at 1,000 units per pound. Once signs of erysipelas were no
longer apparent, all pigs were challenged with P. multocida
toxin.
A summary of mean weight gains and snout scores
for each group is shown in Table XVII below.
Table XVII
Challenge Final Wt Gain'in: Snout
Vaccine Number Wt Wt' (lbs) Score
F 8 52.25 72.13 19.88 38.04 1.63
G 8 51.50 78.25 26.75 52.13 1.31
*Exp. ER 4 43.75 63.75 20.00 45.75 2.50
~*~- i~k R4.lq 61.91 1-1 . 4Z. 40 .14 -z . o0
Pigs in this group served as susceptible controls for
aha2lenqe with DN'T.
** Protection not expected (see Tabie xva),
Based on the results in Tables XIV and XV, two
doses of vaccine E (Table XI) were not predicted to induce a
protective immune response to challenge with DNT. However,
two doses of vaccine E were adequate to prime susceptible
pigs since a secondary response to toxin was evident
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following toxin challenge (Table XVIII). Similarly, pigs
vaccinated with the two vaccines ER and E did not respond as
well as pigs farrowed and nursing from vaccinated dams
(Table XV). However, all pigs vaccinated with vaccines F
and G demonstrated a primary response to vaccination and a
secondary response to toxin injection equal or superior to
the group vaccinated with vaccine E. Further, pigs given
vaccine E were very nearly protected from challenge. These
data indicate a freedom from immunological interference
between the fractions in experimental vaccines.
Serum samples were collected immediately prior to
each vaccination, when E. rhusiopathiae challenge was
administered (Erh Chal) when toxin challenge was given (PmD
Chal), and at slaughter. A summary of the serological
responses to fractions other than rhusiopathiae is
presented in Table XVIII below.
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Table XVIII
Geometric Mean Titer (GMT) to each fraction for sera
taken at or before:
1st Pig 2nd Pig Erh. PmD
Group* Vx Vx Chal. Chal. Slaughter
6 -
Bb** 5.19 16.00 47.61 40.03 26.25
PmA <4.00 8.72 256.00 166.00 172.57
PmD 1.33 7.63 26.19 20.10 468.71
7 -
Bb 6.87 16.00 36.71 34.90 34.90
PmA <4.00 29.34 139.58 107.63 128.00
PmD 0.66 4.77 23.30 35.70 512.63
8 -
Bb <4.00 4.00 4.00 <4.00 4.00
PmA <4.00 42.22 <4.00 8.00 <4.00
PmD 4.96 4.19 4.75 5.67 4.21
9 -
Bb 6.17 14.67 49.35 32.00 26.25
PmA 5.18 26.91 139.58 256.00 186.61
PmD 6.86 8.12 6.90 9.04 47.11
Group 6: Vaccinated, vaccine F (see Table XI)
Group 7: Vaccinated, vaccine G (see Table XI)
Group 8: Vaccinated, Ervsipelothrix rhusiopathiae bacterin
vaccine
Group 9: Vaccinated, vaccine E (see Table XI)
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Pigs farrowed by unvaccinated dams and vaccinated
with Bordetella bronchiseptica-Pasteurella multocida
bacterin-toxoid experimental Vaccine E were protected
against challenge with E. multocida type A (strain 169).
From this result it can be concluded that dam vaccination
with this formulation is not required in protecting pigs
against type A.
Experimental Bordetella bronchiseptica-
Erysipelothrix rhusiopathiae-Pasteurella multocida bacterin-
toxoid given to susceptible pigs protected at least 75%
against a valid challenge with virulent E. rhusiopathiae.
From this result it is concluded that two doses of this
bacterin-toxoid formulated as either a 3 ml dose or a 2 ml
dose induced effective immunity against erysipelas.
Example 11 - Combination Vaccine
Pigs were immunized in two, 2 mL doses with a
combination vaccine according to the invention containing
Bordetella bronchiseptica, Ervsipelothrix rhusiopathiae,
Pasteurella multocida Bacterin-Toxoid and P. multocida free
toxoid, which has been standardized to contain approximately
100 RU free soluble toxoid and 1.875 absorbency units (AU)
of cell-bound toxoid.
WO 91/19419 PC'T/US91/04092
The individual components of the vaccine were
prepared as described in the preceding Examples. A 2 mL
dose was formulated as follows. The Bordetella bacterin
containing 4.13 doses (6195 NU) per mL was adjusted to pH
5 6.5. Al(OH)3 gel (23.85 mL) and 0.615 mL thimerosal, 2.5%
v/v, with EDTA, 2.5% v/v, were added to 930 mL of the
Bordetella. The mixture was again adjusted to pH 6.5 and
was gently mixed 1 hour at approximately 25 C on a shaker.
Sufficient saline (581.9 mL) was added to make a final
10 volume of 1536.4 mL.
The Erysipelothrix, containing 4.7 opacity units
(optical density x volume in ml) per ml, as determined on
Spectronic 20D at 625 nm was clarified by centrifuging at
8000 g for 30 minutes. 1800 mL of the supernatant was
L5 concentrated by ultrafiltration with a 10,000 MW membrane to
a calculated equivalent of 12.5 opacity units per ml to give
a final volume of 677 mL. Next 222 mL A1(OH)3 gel and 3.55
mL thimerosal, 2.5% v/v, with EDTA, 17.5% v/v, were added to
666 mL of concentrated supernatant. The mixture was
20 adjusted to pH 6.6 and gently mixed for 1 hour at
appromately 25 C on a shaker.
WO 91 / 19419 PCT/US91 /04092
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P. multocida type A (2518 mL) which was BPL-
inactivated and contained 5.4 opacity units (OU) per mL
(Spectronic 70 at 650 nm), was centrifuged (4500 g for 1
hour) and the cells resuspended in supernatant fluid to 400
mL. Saline (800 mL) was added to obtain a calculated 11.33
opacity units per mL. Al(OH)3 gel (400 mL) and 6.4 mL
thimerosal, 2.5% v/v, with EDTA, 17.5% v/v, were added. The
mixture was adjusted to pH 6.5 and gently mixed 1 hour at
approximately 25 C on a shaker.
P. multocida Type D strain 4677 [SmithKline
Beecham) Cell-Bound Toxoid (3304 mL) containing 2.1 OU per
mL (Spectronic 70 at 625 nm) was centrifuged (4500 g for 1
hour), and the cells were resuspended in supernatant fluid to
222 mL. Saline (333 mL) was added to obtain a calculated
12.5 OU per mL. Al(OH)3 gel (185 mL) and 2.96 mL
thimerosal, 2.5% v/v, with EDTA, 17.5% v/v, were added. The
mixture was adjusted to pH 6.5 and gently mixed 1 hour at
approximately 25 C on a shaker.
For P. multocida Type D, strain 8, free toxoid,
250 mL Al(OH)3 gel and 4.0 mL thimerosal (2.5% v/v) with
EDTA (17.5% v/v) were added to 750 mL P. multocida toxoid
containing 540 relative units (RU) per mL. This mixture was
adjusted to pH 6.5 and gently mixed 1 hour at approximately
C on a shaker.
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A 2 mL dose contains the following amounts of the
components prepared as described in the above paragraph: 0.4
mL of the Bordetella bacterin, 0.8 mL culture-clarified
Erysipelothrix, 0.4 mL P. multocida type A, 0.2 mL P.
multocida type D cell-bound toxoid, 0.2 mL P. multocida type
D free toxoid. This dose was used in the following
challenge experiments.
Briefly described, following immunization, pigs
were challenged for immunity to E. rhusionathiae in
accordance with 9 C.F.R. 113.104e or against atrophic
rhinitis by sequential infection with virulent B.
bronchiseptica and P. multocida according to the method of
Kobish and Pennings, Vet. Rec., 124:57-61 (1989).
The results of these challenge studies
demonstrated that the magnitude of the response to this
formulation was greater than an additive effect of combining
the gs multocida free toxoid and bacterin-toxoid and
represented immunological synergy. In addition, the solid
protection induced against a severe challenge with
erysipelothrix showed that the removal of bacteria from the
E. rhusiopathiae fraction had no adverse effect on the
efficacy of this fraction.
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2
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A. Challenae for Immunity to E. rhusiopathiae
Twenty-eight crossbread feeder pigs acquired from
Sands, Inc. were used to evaluate the effectiveness of
decreased antigen levels on immunity of Bordetella
brochisei3tica-Ervsipelothrix rhusiopathiae-Pasteurella
multocida Bacterin Toxoid. Three groups of eight pigs
(treatment groups A, B and C, respectively) were injected
intramuscularly in the left side of the neck on day 0 with 2
mL of either a Full Dose, 1/2 Dose, or 1/10 Dose of the
above-described vaccine. The one half and one tenth dosages
were made by diluting the above formulation with sterile
PBS. On day 21, the pigs in treatment groups A, B and C
received a second intramuscular injection of their
respective treatment. With the second injection, the
injection sites were rotated to the right side of the neck.
On day 39, all pigs, treatment Groups A, B, C and control
group D, were challenged with E. rhusiopathiae via a 2 mL
intramuscular injection in the ham.
One animal in the group which received 1/10 dose
died for reasons unrelated to vaccination or challenge and
was removed from the test. One hundred percent of the non-
vaccinated animals displayed characteristic clinical signs
or erysipelas and 75% died. In contrast, 100% of the pigs
which received a full dose of vaccine were protected. From
this data it was concluded that vaccination with the vaccine
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of this example induced an immunity that withstood a very
strong challenge with virulent E. rhusiopathiae.
B. Challenge of Immunity for Atrophic Rhinitis
Six of eight naive crossbred late gestation sows
(Eberly Farms, Bradshaw, NE) were inoculated intramuscularly
approximately 3 and 5 weeks prior to farrowing on two
occasions with 2.0 mL of the vaccine formulation described
above. At farrowing, pigs in each litter were randomly
assigned to treatment groups. Treatment group A received
two, 2.0 mL intramuscular injections of the vaccine
formulation. The first injection was administered in the
left side of the neck when the pigs were one week old and
the second was administered in the right side of the neck at
weaning age. Treatment group B did not receive any test
substance. The pigs born of the two control sows served as
control pigs.
At four days of age, the pigs were intranasally
challenged with 1.0 mL of B. bronchiseptica (strain 2-9, 2 x
10$ organisms/mL) and again intranasally challengedwith 1.0
mL of P. multocida type D (strain 4677, 8 x 109
organisms/mL) at nine days of age. Five weeks after
infection with P. multocida type D, the pigs were
slaughtered and nasal turbinate atrophy measured.
WO 91/19419 PCT/US91/04092
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The non-vaccinated groups gained significantly
less weight than pigs in groups which included vaccination
treatment. Further, damage to nasal scrolls was uniformly
severe in non-vaccinated pigs farrowed from non-vaccinated
dams. In contrast, pigs born to vaccinated dams gained
weight at a rate expected of a healthy pig and had
significantly less turbinate atrophy. The results of the
challenge are summar'ized in Table XIX below in which Group A
represents vaccinated dams, vaccinated pigs; Group B
represents vaccinated dams, non-vaccinted pigs, and Group C
represents non-vaccinated dams, non-vaccinated pigs.
Table XIX
#/Group Weight(lbs) Avg. Signif- Snout Signif
Gp Dams Pigs initial Final Dai1v Gain icance Score icance
A 5 19 5.25 28.21 0.608 p<0.001 1.97 p<0.001
B 5 19 5.61 31.66 0.688 p<0.001 1.88 p<0.001
C 2 23 5.47 22.50 0.431 control 4.21 control
The serological response showed that measurable
antibodies to B. bronchisevtica, P. multocida type A, and
P. multocida DNT were present in colostrum samples from
vaccinated dams and not in colostrum samples from non-
vaccinated dams. Further, assay of antibody to these
fractions in serum taken from pigs at five days of age
(Bordetella challenge) showed transfer of maternal antibody.
WO 91/19419 PCT/US91/04092
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61
Table XX below shows antibody titers that were protective
against severe atrophic rhinitis caused by B. bronchiseptica
and P. multocida infections, regardless of whether
immunization was active or passive. In nonvaccinated pigs
farrowed from vaccinated dams, however, the results clearly
indicate that maternal antibody does not persist and that
vaccination of pigs is required for an immunity that endures
through the first few months of life.
Table XX
Geometric Mean Titer (GMT) for sera taken at or before:
Dams Pias
Dams Borde- Pasteur-
ist 2nd tella ella Final
Fraction Vx Vx Colostrum Challenge Challenge Bleeding
Vaccinated Dams Vacciriated Pigs
Bb 7.0 64.0 222.9 7.7 4.8 8.6
PmA 1.4 2.2 249.0 29.4 21.4 5.6
PmD 20.3 43.8 122.5 35.5 19.7 49.7
Non-Vaccinated Pigs
Bb 17.2 8.6 4.3
PmA 35.3 22.3 1.8
PmD 46.8 34.2 3.8
Non-vaccinated Dams Non-vaccinated Pigs
Bb 11.3 NA < 4 < 4 < 4 < 4
PmA 1.0 NA 19.0 < 4 < 4 < 4
PmD 14.4 NA 16.1 2.8 1.3 0.9
Bb = Bordetella bronchisentica
PmA = Pasteurella multocida type A
PmD = Pasteurella multocida DNT
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Numerous modifications and variations of the
present invention are included in the above-identified
specification and are expected to be obvious to one of skill
in the art. For example, use of other appropriate
inactivated pathogens, other than those of B. bronchiseptica
and E. rhusiopathiae, may be employed in the combined
vaccines of this invention. Similarly, other conventional
adjuvants and inactive vaccine components may be employed in
the formulations and selected by one of skill in the art.
The dosages and administration protocols for use of these
vaccine compositions may also be adjusted by one of skill in
the art based on the animal to be vaccinated, the disease
for which protection is desired and other related factors.
Such modifications and alterations to the compositions and
processes of the present invention are believed to be
encompassed in the scope of the claims appended hereto.
