U.S. patent application number 12/684335 was filed with the patent office on 2010-05-06 for multi component vaccine containing clostridial and non-clostridial organisms in a low dose.
This patent application is currently assigned to Bayer Corporation. Invention is credited to Sharon A. Bryant, Michael J. McGinley, Stuart K. Nibbelink, Richard E. Parizek, Lonny E. Vlieger.
Application Number | 20100111997 12/684335 |
Document ID | / |
Family ID | 23633975 |
Filed Date | 2010-05-06 |
United States Patent
Application |
20100111997 |
Kind Code |
A1 |
Parizek; Richard E. ; et
al. |
May 6, 2010 |
Multi component vaccine containing clostridial and non-clostridial
organisms in a low dose
Abstract
Disclosed herein is a multicomponent low dose vaccine comprising
a safe and immunogenically effective combination of a protective
antigen component or components of clostridial organism, a
protective antigen component of a non-clostridial organism and an
adjuvant.
Inventors: |
Parizek; Richard E.;
(Lenexa, KS) ; Vlieger; Lonny E.; (Peculliar,
MD) ; Bryant; Sharon A.; (Shawnee, KS) ;
Nibbelink; Stuart K.; (Overland Park, KS) ; McGinley;
Michael J.; (Lenexa, KS) |
Correspondence
Address: |
Intervet/Schering-Plough Animal Health
Patent Dept. K-6-1, 1990, 2000 Galloping Hill Road
Kenilworth
NJ
07033-0530
US
|
Assignee: |
Bayer Corporation
Intervet International B.V.
|
Family ID: |
23633975 |
Appl. No.: |
12/684335 |
Filed: |
January 8, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10748524 |
Dec 29, 2003 |
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12684335 |
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08412676 |
Mar 29, 1995 |
6743430 |
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10748524 |
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Current U.S.
Class: |
424/203.1 |
Current CPC
Class: |
A61K 39/08 20130101;
A61K 39/102 20130101; A61P 33/02 20180101; Y02A 50/489 20180101;
Y02A 50/30 20180101; A61K 39/08 20130101; A61K 2300/00 20130101;
A61K 39/102 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/203.1 |
International
Class: |
A61K 39/116 20060101
A61K039/116 |
Claims
1-45. (canceled)
46. A method for reducing injection site lesion formation by at
least 41% when immunizing cattle using an encapsulating polymer
adjuvant in a multicomponent vaccine comprising an immunogenically
effective combination of protective antigen components from at
least six clostridial organisms, a protective antigen component
from Moraxella Bovis (M.Bovis), and an encapsulating polymer
adjuvant, comprising administering the multicomponent vaccine in a
dose size of about 2 ml., thereby reducing injection site lesion
formation by at least 41% compared with conventional injection of 5
ml of vaccine and accomplishing effective immunization.
47. A method for reducing injection site lesion formation by at
least 41% when immunizing cattle using an encapsulating polymer
adjuvant in a multicomponent vaccine comprising an immunogenically
effective combination of protective antigen components from seven
clostridial organisms, a protective antigen component from M.Bovis,
and an encapsulating polymer adjuvant, comprising administering the
multicomponent vaccine in a dose size of about 2 ml., thereby
reducing injection site lesion formation by at least 41% compared
with conventional injection of 5 ml of said vaccine and
accomplishing effective immunization.
48. (canceled)
49. The method of claim 46, wherein the protective antigen
components from clostridial organisms are selected from the group
consisting of Cl. chauvoei, Cl. septicum, Cl novyi, Cl. perfringens
type C, Cl. perfringens type D, Cl. sordellii, Cl. tetani and Cl.
haemolyticum.
50. The method of claim 46, wherein each of the protective antigen
components is adjuvanated separately, thereby encapsulating each
component separately and causing each to be released slowly over a
period of several weeks after administration.
51. The method of claim 46, wherein the multicomponent vaccine is
administered intramuscularly.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to low dose multicomponent
vaccines. More specifically, the invention relates to low dose
multicomponent vaccines comprising a safe and immunogenically
effective combination of: at least one protective antigen component
from clostridial organisms, at least one protective antigen
component from a non-clostridial organism and an adjuvant.
[0003] 2. Brief Description of the Prior Art
[0004] Preparation and formulation of multicomponent vaccines have
historically been complicated by physical and technological
hurdles. Multicomponent vaccines of interest are those vaccines
that contain as essential antigen components: one or more
protective antigens from one or more organisms and an adjuvant. The
protective antigen component can be in the form of a whole
bacterial culture, a whole virus culture, a cell-free toxoid, a
purified toxoid, or a subunit.
[0005] When one combines whole cultures of organisms (viruses or
bacteria) in a formulation of multicomponent vaccines, the
formulation would contain numerous antigens (hundreds to
thousands). Some of these are protective antigens as mentioned
above. Some of these antigens are detrimental to protection of the
animals or cause reaction in the animals ("detrimental antigens").
The detrimental antigens can interfere with the protective antigens
by either physically or chemically blocking the active sites of the
protective antigens. The interference prevents the protective
antigens from protecting animals. Also, the detrimental antigens
can produce negative responses such as local reactions, systemic
reactions, anaphylaxis and/or immunosuppression in the animals.
Therefore, the use of combinations of whole culture organisms can
cause problems with efficacy or with animal reactivity. Animal
reactivity produces localized reactions resulting in swellings or
abscesses at the injection sites or a systemic response such as
anaphylaxis that can result in death of the animal.
[0006] Aggravating the animal reactivity is the administration of
multi-component vaccines to large animals (e.g., cattle) in high
doses. The dose range has historically been from about 5 mL to 10
mL to allow incorporation of all of the protective antigens into
one formulation. Illustratively, up to seven clostridial whole
cultures or toxoids can be combined into a 5.0 mL dose of vaccine
for administration to cattle. See, for instance, pages 319, 320,
321, 322, and 432 of the Compendium of Veterinary Products, Third
Edition, 1995-1996). Also, 6 Clostridial whole cultures or toxoids
have been combined with Hemophilus somnus in a 5.0 mL dose
vaccines. See pages 191, 192, 319, 433, 490, and 1013 of the
Compendium of Veterinary Products, Third Edition, 1995-1996).
Reportedly, such vaccines demonstrate significant animal
reactivity.
[0007] Animal reactivity that produces localized reactions (often
called injection site lesions or blemishes) have become a matter of
significant concern for the beef industry. Many scientific and lay
articles since 1991 have addressed the concern with injection site
lesions. See Stokka et al, J. Am. Vet. Med. Assoc., 1994, Feb. 1,
204(3): 415-9, Effertz, Beef Today, March 1991 and Beef Today,
September 1992, Dittmer, CALF News Cattle Feeder, September 1992;
Smith, FEEDSTUFFS, August 24, 1992, and Hrehocik et al, dvm,
September 1992. During the past several years, many scientific and
lay articles have reported that injection site lesions are
deleterious to the quality of beef. The injection site lesions must
be cut out of the meat and discarded. This causes significant
monetary loses to retailers, beef packers and feedlots. It has been
estimated that 12-15% of prime beef cuts have some type of
injection site lesion that must be trimmed away (Effertz, Beef
Today, March 1991). This article attributes the main cause of the
injection site lesions to 7-way clostridial vaccines. Additionally,
there have been reports that up to 90% of cattle have injection
site lesions in their carcass. Injection site lesions have been
associated with: (1) the presence of many detrimental antigens or
contaminants which are present in whole culture vaccines, (2) the
adjuvants incorporated into such vaccines, (3) the method of
administration of such vaccines (4) the large dose size of some of
the multicomponent vaccines (5.0-10.0 mL), and (5) animal the
reactivity of the protective antigen components of the
vaccines.
[0008] Typically, clostridial vaccines are not highly purified
because purification can be cost prohibitive. As one would realize,
animal vaccine production must be necessarily economically
effective if the vaccines are to enjoy widespread use. Therefore,
highly purified animal vaccines are virtually cost prohibitive.
[0009] Somewhat related prior art involves two vaccines containing
six clostridial whole cultures or toxoids administered in a 2.0 mL
dose volume. See Compendium of Veterinary Products, Third Edition,
1995-1996, pages 133, 1183, 1184 and 1185 and the advertising
brochure entitled "ALPHA-7.TM.-JUST ONCE". However, these vaccines
do not include any additional component such as: additional
clostridial component(s) or one or more non-clostridial
component(s).
[0010] Antigenic components of clostridial vaccines were typically
obtained by concentrating whole cultures of the bacteria.
Concentration was accomplished by precipitating whole cultures with
ammonium salts such as ammonium sulfate or concentrating such whole
cultures via ultrafiltration. Both procedures are costly.
Additionally, these procedures produce massive amounts of cells
resulting in a high antigen mass that remains as an antigenic mass
of solids in the product. Such a high antigenic mass would induce
animal reactivity, particularly injection site lesions.
[0011] An even greater problem exists when one combines clostridial
organisms with non-clostridial organisms such as Gram-negative
bacteria, e.g., H. somnus and M. bovis and the Pasteurella spp.
Many of these organisms are, in themselves, highly reactive and
contain high levels of endotoxin that produce anaphylaxis. Also,
their antigenic components supposedly cause interference. The high
dose of the art-known combination of H. somnus and six clostridial
components, i.e., a 5.0 mL dose volume can be the source of animal
reactivity. In the case of non-clostridial viral formulations, the
addition of clostridial components to these formulations can
adversely affect viral epitopes. Consequently the viral components
of the formulation may become non-efficacious.
[0012] Because of the severity of the Clostridial diseases and
other disease complexes described herein, it is increasingly
important that calves and young cattle entering feedlots as well as
pregnant cows are properly vaccinated. The vaccines must contain
protective antigens described herein. While one could administer
each of the protective antigens in a monovalent vaccine, this mode
of administration would require several vaccinations for each
animal. This is impractical in a, because: 1) handling animals for
repeated vaccinations can result in undue stress and consequential
diseases; 2) labor for performing such vaccinations is expensive
compared to the profit obtained from each animal; 3) the more
injection sites on an animal, the more potential for injection site
reactions.
[0013] There is, therefore, a clear need for multicomponent
vaccines containing many protective antigens that do not contain
detrimental antigens and do not produce animal reactivity. By this
invention, there are provided low dose multicomponent vaccines
containing: protective antigen components of a clostridial
organism(s) and at least one non-clostridial protective antigen
component and an adjuvant, and the processes for making and using
the vaccines.
SUMMARY OF THE INVENTION
[0014] This invention relates to a multicomponent vaccine
comprising: a safe and immunogenically effective combination of
protective antigen components from at least one clostridial
organism, a protective antigen component from a non-clostridial
organism and an adjuvant, wherein the vaccine is in a low dose
volume. By "low dose" is meant dose volumes, including the adjuvant
which are less than 5.0 mL and which do not adversely affect the
protective antigen components or the animal post vaccination.
Generally, an antigen is that which produces an antibody response
against the antigen, which response is not necessarily protective.
By the term "protective antigen" is meant an antigen that produces
an immune response and imparts protection to the animal. A vaccine
containing such a protective antigen is characterized as
"immunogenically effective."
[0015] Also, encompassed by the invention is a multicomponent
vaccine for ruminants comprising: a safe and immunogenically
effective combination of a protective antigen component from at
least two and preferably six to seven clostridial organisms; a
protective antigen component from a non-clostridial organism and an
adjuvant, wherein the vaccine is in a low dose volume.
[0016] In the present embodiment of the invention, the
multicomponent vaccine comprises a safe and immunogenically
effective combination of an antigen component from one or more
clostridial organisms; an antigen component from an organism
selected from the group consisting of a Gram negative organism, a
Gram positive organism, a virus, a parasite and a rickettsia and an
adjuvant wherein the vaccine is in a dose size of 3.0 mL or
less.
[0017] In a preferred embodiment of the invention, the
multicomponent vaccine for ruminants comprises a safe and
immunogenically effective combination of an antigenic component
from six clostridial organisms, which are Clostridium chauvoei,
Clostridium septicum, Clostridium novyi, Clostridium perfringens
type C, Clostridium perfringens type D and Clostridium sordellii,
an antigen component from H. somnus or M. bovis and an adjuvant,
wherein the vaccine is in a dose size of 3.0 mL or less.
[0018] In another preferred embodiment of this invention, the
multi-component vaccine for ruminants comprises: a safe and
immunogenically effective combination of a protective antigen
component from seven clostridial organisms which are Cl. chauvoei,
Cl. septicum, Cl. novyi, Cl. perfringens type C, Cl. perfringens,
type D, Cl. sordellii, and Cl. haemolyticum; an antigen component
from Haemophilus somnus or Moraxella bovis and an adjuvant, wherein
the vaccine is in a dose size of 3.0 mL or less.
[0019] In another preferred embodiment of this invention, the
multi-component vaccine for ruminants comprises: a safe and
immunogenically effective combination of an antigen component from
at least two clostridial organisms such as Cl. perfringens type C
and Cl. perfringens type D; an antigen component from a virus such
as an infectious bovine rhinotracheitis virus (IBRV) and an
adjuvant, wherein the vaccine is in a dose size of 3.0 mL or
less.
[0020] A particularly preferred embodiment of this invention
includes a multicomponent vaccine for ruminants comprising: a safe
and immunogenically effective combination of a protective antigen
component from more than two clostridial organisms selected from
the group consisting of Cl. chauvoei, Cl. septicum, Cl. novyi, Cl.
perfringens type C, Cl. perfringens type D, Cl sordellii, and Cl.
haemolyticum; protective antigen components from viruses which are
selected from the group consisting of an infectious bovine
rhinotracheitis virus (IBRV), a parainfluenza type 3 virus
(Pl.sub.3V), a bovine virus diarrhea virus (BVDV) and a bovine
respiratory syncytial virus (BRSV) and an adjuvant, wherein the
vaccine is in a dose size of 3.0 mL or less.
[0021] In another particularly preferred embodiment of the
invention the multicomponent vaccine comprises: a safe and
immunogenically effective combination of a protective antigen
component from at least six clostridial organisms; a protective
antigen component from a plurality of viruses and an adjuvant,
wherein the vaccine is in a dose size of 3.0 mL or less.
[0022] The most preferred embodiment of the invention is a
multi-component vaccine comprising: a safe and immunogenically
effective combination of a protective antigen component from at
least seven clostridial organisms; protective antigen components
from at least four viruses and an adjuvant, wherein the vaccine is
in a dose size of 3.0 mL or less.
[0023] Further encompassed by the invention is a method for
producing a multicomponent vaccine comprising a safe and
immunogenically effective combination of protective antigen
components from clostridial organisms and a protective antigen
component from a non-clostridial organism and an adjuvant wherein
the vaccine is in a dose size of 3.0 mL or less, said method
comprising: 1) identifying the protective antigen component of each
organism by in vivo or in vitro methods; 2) quantitating the
protective antigen components using antigen quantitation assays to
provide the protective antigen component in an amount sufficient to
produce a protective vaccine with the least antigenic mass; 3)
identifying components of the organisms containing detrimental
antigens by using the antigen quantitation assays and animal
reactivity testing; 4) purifying the protective antigen components
which contain detrimental antigens to remove the detrimental
antigens; 5) selecting for each organism requiring inactivation, an
effective inactivating agent which kills the organism without
denaturing the protective antigen component; 6) selecting an
effective adjuvant which produces enhancement of immune response
without causing unacceptable animal reactivity for each component;
7) adjuvanting the protective antigen components sensitive to the
effects of detrimental antigens organisms individually; 8) pooling
all protective antigen components.
[0024] Also, encompassed by the invention is a process for
administering the vaccines of the invention to ruminants.
[0025] By the present invention, it has been demonstrated that
there is a significant difference in the size of injection site
lesions in cattle vaccinated with: (1) a conventional 5.0 mL dose
multicomponent clostridial product and (2) the low dose (2.0 mL)
multicomponent vaccine of this invention. The area of the injection
site lesion produced by the low dose vaccine is significantly
smaller, post injection than the lesion produced by the
conventional 5.0 mL dose vaccine. The low dose multicomponent
vaccine produced injection site lesions in an insignificant number
of cattle as compared with the conventional vaccine.
DETAILED DESCRIPTION OF THE INVENTION
[0026] In accordance with the invention it has been discovered that
in the preparation of multicomponent vaccines such as those
containing seven clostridial organisms, one can: identify and
reduce the required antigenic mass and combine it with a compatible
adjuvant to produce a low dose, safe and immunogenically effective
vaccine. This discovery is the basis of the inventive concept
described herein. According to this inventive concept, the skilled
artisan can combine: protective antigen components from the
clostridial organisms and non-clostridial organisms, and an
adjuvant in a low dose volume, and safely administer it to
ruminants to protect them against diseases described more fully
hereunder.
[0027] More specifically, the invention relates to a multicomponent
vaccine comprising a safe and immunogenically effective combination
of: an antigen component from one or more clostridial organisms; an
antigen component from a non-clostridial organism selected from the
group consisting of a Gram negative organism, a Gram positive
organism, a virus, a parasite and a rickettsia and an adjuvant,
wherein the vaccine is in a dose size of 3.0 mL or less.
Non-limiting examples of the clostridial organisms and diseases in
ruminants are as follows:
[0028] Clostridium chauvoei causes the disease blackleg. This
organism, like all Clostridial organisms, produces spores that can
survive in soil for years and, during this time, can infect
susceptible animals (cattle and sheep) which ingest them. Blackleg
is an acute, infectious but noncontagious, disease of cattle and
sheep characterized by gaseous tissue swelling, usually in the
heavy muscles. The organism enters cattle or sheep via feed or cuts
or by shearing, docking, or castration. The onset of the disease is
quite sudden. Body temperature rises rapidly and muscular
stiffness, depression and reluctance to move are prominent. When
infection is extensive, death often occurs within 16-72 hours.
Treatment of sick animals is futile since there is often permanent
damage done to their meat. [0029] Clostridium septicum causes the
disease of malignant edema, or gas gangrene, a rapidly extending
edematous swelling, in subcutaneous tissues of cattle. The disease
is characterized by gangrene and gaseous swelling surrounding a
wound. Incidence of the disease often follows castration,
dehorning, accidental puncture wounds and lacerations, abortions,
and vaccination with unclean needles. The incubation period is
short and death occurs within 12 to 48 hours. Death is primarily
caused by toxins released by multiplying organisms after infection
occurs. As with Cl. chauvoei, it is impractical to treat the
animals. [0030] Clostridium novyi causes the condition of black
disease or infectious necrotic hepatitis which is an acute
infectious disease of cattle and sheep. The causative spore-forming
organism may enter cattle through the digestive tract, lungs or
wounds. In areas where liver flukes are endemic, Cl. novyi is
especially dangerous because the organism will multiply in damaged
areas resulting from the migration of liver flukes. The organism
multiplies rapidly and produces a highly fatal exotoxin causing
toxemia and death. Death is usually sudden with no well-defined
signs. Because of the rapidity of death, treatment is not
practical. [0031] Clostridium sordellii causes a disease similar to
Cl. novyi and Cl. septicum. The organism is an inhabitant of the
soil and of the animal intestine. Most infections by the organisms
are associated with wounds or liver flukes. Lesions at the site of
the infection progress rapidly, followed by fever, depression and
edema that is similar to that produced in Cl. novyi infections. A
rank odor is detected in diseased tissues. Death is also sudden
indicating that treatment is not practical. [0032] Clostridium
perfringens types B, C, and D are found as spores in the soil but
are also parts of the normal intestinal flora of healthy animals.
Under favorable conditions, such as when animals are being fed high
protein diets in feedlots, the organisms multiply rapidly in the
intestines. They produce lethal toxins which kill infected animals.
Cl. perfringens type B causes sudden death in cattle and lambs. Cl.
perfringens type C produces an acute hemorrhagic enteritis in
calves, lambs, piglets and older cattle and sheep on high-energy
feeds. Cl. perfringens type D causes overeating disease in feedlot
cattle unaccustomed to high-energy concentration rations. All of
the syndromes produced by the various types of Cl. perfringens have
rapid onset and result in death before the animals can be
effectively treated. [0033] Clostridium tetani causes tetanus that
can afflict all mammals. The disease results from organisms
entering their body via puncture wounds. As the organisms multiply,
toxins which affect the central nervous system are produced.
Infected animals become stiff, have difficulty swallowing and
breathing, and are afflicted with spasmodic contractions of the
musculature. While treatment with antitoxin is viable, it is
extremely expensive and cost inefficient.
[0034] As set forth above, the non-clostridial organism can be
selected from the group consisting of: a Gram negative organism, a
Gram positive organism, a virus, a parasite and a rickettsia. The
following is a non-limiting illustration of the Gram negative
organisms. [0035] Haemophilus somnus (H. somnus) is an organism
that causes a complex of disease conditions found mainly in feedlot
cattle The disease is, also, found in dairy and pasture cattle.
This organism can cause a thromboembolic meningoencephalitis
(TEME), a respiratory tract disease, reproductive diseases and a
general septicemia. It is a non-motile, rod-shaped bacterium which
is difficult to isolate and is most likely spread by respiratory
secretions and discharges. Its incubation period is two to seven
days. Infected animals can be treated successfully with antibiotics
if they are treated early enough in the course of the disease.
Unfortunately, once the infection becomes systemic, antibiotic
effectiveness is decreased. Vaccination is the best method for
protecting a herd of cattle from these H. somnus-induced diseases.
The fact that H. somnus is a Gram-negative organism, and therefore
contains endotoxin, renders the formulation of a non-reactive
vaccine difficult. [0036] Moraxella bovis (M. bovis) is a
Gram-negative organism that causes pink-eye in cattle. This disease
is often chronic in herds of cattle and causes cattle to develop
keratoconjunctivitis, with blindness a sequelae, after a period of
time. Treatment is expensive as it must be continued for long
periods of time. [0037] M. bovis has the potential to cause
anaphylaxis and/or severe local reactions. [0038] Campylobacter
fetus is a Gram-negative organism that causes a venereal disease
transmitted during breeding. Although the disease is often
subclinical, it causes temporary infertility, irregular estrous
cycles, delayed conception and, occasionally, abortion in cows.
[0039] Leptospira spp. infect and localize in the kidneys and are
shed in the urine. Infection with Leptospira spp. can cause anemia,
bloody urine, fever, loss of appetite and prostration in calves.
Infection is usually subclinical in adult cattle. Infected pregnant
cows, however, often abort, and dairy cows may exhibit a marked
decrease in milk production. There are at least six major serovars
in the species L. interrogans (L. pomona, L. canicola, L.
grippotyphosa, L. icterohaemorrhaqiae, L. hardjo, and L.
bratislava), [0040] Pasteurella haemolytica and Pasteurella
multocida are causative agents of bovine pneumonia in feedlot
cattle and young calves. They are the most significant components
of the shipping fever complex and induce clinical pneumonia in
cattle which are predisposed to infections with: infectious bovine
rhinotracheitis, parainfluenza type 3 virus, bovine respiratory
syncytial virus or bovine virus diarrhea virus. [0041] Infectious
bovine rhinotracheitis virus causes a severe respiratory infection
of cattle, specifically in feedlot conditions. The disease is
characterized by: high temperature, excessive nasal discharge,
conjunctivitis and ocular discharge, inflamed nasal mucosa,
increased rate of respiration, coughing, loss of appetite,
depression and/or reproductive failure in cattle. Infection with
this virus often predisposes cattle to bacterial infections that
cause death. [0042] Parainfluenza type 3 virus (Pl.sub.3) usually
causes a localized upper respiratory tract infection, producing
elevated temperatures and moderate nasal and ocular discharge.
Although clinical signs of Pl.sub.3 are typically mild, this
infection weakens the respiratory defenses and allows replication
of other pathogens, particularly Pasteurella spp. [0043] Bovine
virus diarrhea (BVD) is a major cause of abortion, fetal resorption
or congenital fetal malformation. If susceptible cows are infected
with non cytopathic BVD virus during the first trimester of
pregnancy, their calves may be born persistently infected with the
virus. Exposure of those calves to certain virulent cytopathic BVD
virus strains may precipitate BVD-mucosal disease. Clinical signs
of this disease include loss of appetite, ulcerations in the mouth,
profuse salivation, elevated temperature, diarrhea, dehydration and
lameness. The disease usually affects feedlot cattle. [0044] Bovine
respiratory syncytial virus (BRSV) infects cattle of all ages and
causes: rapid breathing, coughing, loss of appetite, discharge from
the nose and eyes, fever and swelling in the cervical area. In an
acute outbreak, death may follow 48 hours after the onset of
signs.
[0045] The following is a non-limiting illustration of the
parasites that are employed herein. [0046] Neospora spp. have been
recently isolated form aborted fetuses. These organisms are
parasites which have been proposed as a major cause of abortion in
pregnant cows throughout the world. If this proves to be correct, a
vaccine for protection of pregnant cattle against Neospora spp.
could be a requirement in the future.
[0047] In accordance with the invention, clostridial organisms can
be selected from the group consisting of: Cl. chauvoei, Cl.
septicum, Cl. novyi, Cl. perfringens type C, Cl. perfringens type
D, Cl sordellii, and Cl. haemolyticum. Preferably, the protective
antigen of the clostridial component is derived from six to seven
clostridial organisms.
[0048] The non-clostridial protective antigen component can be
selected from the group consisting of Gram negative bacteria, Gram
positive bacteria, viruses, parasites, rickettsia and a combination
thereof. Non-limiting examples of the Gram negative organisms can
be selected from the group consisting of: H. somnus, M. bovis, E.
coli, Salmonella typhimurium, Pasteurella hemolytica, Pasteurella
multocida, Campylobacter fetus, Leptospira spp and a combination
thereof. Preferred herein are H. somnus and M. bovis.
[0049] Non-limiting examples of the Gram positive organisms are
Clostridium tetani, Bacillus anthracis, Listeria monocytogenes,
Actinomyces pyogenes and a combination thereof.
[0050] Non-limiting examples of the virus can be selected from the
group consisting of: infectious bovine rhinotracheitis (IBRV),
parainfluenza virus type 3 (Pl.sub.3V), bovine virus diarrhea virus
(BVDV) bovine respiratory syncytial virus (BRSV) and a combination
thereof.
[0051] Non-limiting examples of the parasites are Neospora spp.,
Tritrichimonas foetus, Cryptosporidia spp. and a combination
thereof.
[0052] A non-limiting example of the rickettsia is Ehrlichia
bovis.
[0053] In accordance with the invention, the clostridial and
non-clostridial protective antigen components can be in the form
of: inactivated or modified live whole cultures, toxoids, cell-free
toxoids, purified toxoids, subunits or combinations thereof.
[0054] Adjuvants useful herein are by definition chemical compounds
added to vaccines to enhance the production of an immune response
by the animal receiving the vaccine. Most adjuvants function by:
(1) producing an irritation at the site of injection causing
leukocytes (immune cells) to infiltrate the area, and/or (2) by
producing a depot effect--holding the antigen(s) at the injection
site for as long as possible. If infiltration of leukocytes to the
injection site is extensive, swelling and injection-site lesions
will occur. Such leukocytes carry the antigens from the vaccine to
cells within the immune system (of the vaccinated animal) which can
produce a protective response. Some newer polymer adjuvants
function by encapsulating antigens and releasing them slowly over a
period of weeks or months. These newer adjuvants can help in
protecting antigens from interference and are generally less likely
to cause extensive infiltration of leukocytes to the injection
site. In accordance with the invention, the adjuvants can be
selected from the group consisting of: oil-in-water, water-in-oil,
Al(OH).sub.3, Al.sub.2(SO.sub.4).sub.3, AlPO.sub.4, extracts of
bacterial cell walls (Mycobacterium, Propionibacterium, etc.),
extracts of plants (acemannan, saponin or Quil A), polymers,
including block copolymers, liposomes and combinations thereof.
Preferred herein are adjuvants that function by encapsulating
antigens and releasing them slowly over a period of weeks or months
Preferably, the adjuvants are polymers, including block copolymers
(alternately referred to herein as polymer adjuvants. A specific
example of the preferred adjuvant is carbopol. Generally, the more
effective the adjuvant is, the more irritating it is and the more
likely it is to cause an animal reaction. It is a distinct feature
of the invention that effective adjuvants can be formulated with
the protective antigens to produce vaccines that are safe and
effective.
[0055] It is also a distinct feature of the invention that a
multicomponent vaccine for ruminants would include all the required
protective antigen components and adjuvant, in a low dose. In
essence, fewer than five protective antigens from each organism
would be required to make a vaccine immunogenically effective.
However, a vaccine containing only the protective antigens would be
essentially a very pure vaccine. Because of the high purity of the
antigens, it would be difficult adjuvant them with commonly used
adjuvants. The pure antigen would require adjuvants that are
different from the typical adjuvants. Therefore, a commercial scale
production of clostridial vaccines containing very pure protective
antigen components would be technically difficult. At any rate, the
preparation of a very pure animal vaccine on a commercial scale is
prohibitive because of the cost of purification.
[0056] In accordance with the invention, individual components of
the multicomponent vaccines described herein can be formulated with
protective antigens derived from: whole culture bacteria, whole
culture viruses, cell-free toxoids, purified toxoids and/or
subunits. Whole cultures contain numerous antigens. Some of the
antigens impart protection (protective antigens), some produce
negative response (detrimental antigens) and some are essentially
neutral (neutral antigens). Subunits can be obtained from the
organisms themselves by conventional methods such as:
centrifugation, ultrafiltration, and extraction with detergents or
organic solvents. Alternately, the subunits can be produced by
recombinant technology and expressed in live vectors or other
organisms and isolated and purified. It would be understood that
protective antigen components may contain few to many antigens at
least one of which is protective or immunogenically effective.
[0057] In the preparation of the vaccine of the invention, one can
incorporate protective antigen components from a plurality of
organisms. This occasions the likelihood of one protective antigen
component interfering with another. This is particularly the case
if the protective antigens are derived from clostridial organisms.
The interference may result from: (1) physical masking or hiding of
an active site of one protective antigen component by another, (2)
aggregation or agglomeration of one or more protective antigen
components so that one or more active sites are hidden from the
immune system, (3) chemical interaction wherein there is a change
in the active site of one protective antigen component by another.
The latter change can result from a toxic effect, chemical binding
or a conformational change in a critical portion of an active
site.
[0058] It is a distinct feature of the invention that the effects
of the detrimental antigens can be avoided by the process of the
invention. The process comprises: using specialized procedures for
identifying the protective antigen components; quantitating the
protective antigen components; identifying those protective antigen
components that contain detrimental antigens; purifying those
protective antigen components that contain detrimental antigens to
remove such detrimental antigens; selecting adjuvants that produce
the necessary enhancement of the immune response without causing
unacceptable reactivity and protect against interference
;individually adjuvanting the protective antigen components that
are sensitive to the effects of detrimental antigens; pooling the
various protective antigen components into a low dose volume
vaccine.
[0059] In the preparation of the multicomponent vaccines, the
inventors employ adjuvant that protect the active sites of the
various protective antigen components. In effect, the adjuvants
interact with targeted protective antigens, and not other antigens.
As would be realized, the selection of an adjuvant is critical. The
adjuvant must be one that is potent enough to produce significant
enhancement of the immune response without producing unacceptable
local or systemic reactions. The term "produce significant
enhancement of the immune response" refers to stimulation of the
immune system such that protection of the host animal results from
vaccination. Additionally, the adjuvant must reduce or prevent the
interference with the protective antigens. An adjuvant that
encapsulates antigens is preferred. This characteristic is usually
associated with polymer- or block copolymer-type adjuvants. The
preferred adjuvant for this invention is one containing "carbopol"
or the equivalent thereof.
[0060] An integral part of the invention is the use of a specified
test method for antigen quantitation of the protective antigen
components. Illustratively, the test method for quantitation of a
clostridial protective antigen component involves injection of mice
with combinations of antigen and specific antisera. The test method
is referred to herein as "a combining power test". The resultant
measurement of antigen is designated as "combining power unit"
(CPU). The CPU test, developed in accordance with the invention, is
an integral part of the formulation of combination clostridial
products. The test comprises adding varying volumes of test
material to a series of tubes. The total volume of test material in
each tube is brought to 1.0 mL using Peptone Sodium Chloride
Diluent [8.5 gm Sodium Chloride and 10 gm Bactone Peptone/liter
(PND)]. One half milliliter of PND containing one International
Unit of antitoxin, obtained from the clostridial organism being
tested, plus enough excess antitoxin to neutralize approximately
100 MLD of toxin, is added to each tube. The tubes are mixed and 18
to 20 gm mice are inoculated intravenously with 0.5 mL from each
tube. The mice are observed for 48 hours and death is recorded. The
CPU of the test material is calculated as follows:
CPU / mL = Reciprocal of the dilution of the toxoid .times. 2
Smallest volume of the above dilution which kills 100 % of
inoculated mice ##EQU00001##
Other test methods that produce substantially the same results as
described herein are encompassed by the claimed invention.
Non-limiting examples of other test methods can be ELISA assays and
liquid chromatography, which quantitate antigens directly in
vaccines. In accordance with the foregoing, the skilled artisan can
employ the required CPU/mL or the equivalent Elisa antigen
quantitation unit to ascertain the value of the amounts of the
antigenic components that are useful in making and using the
vaccines of the invention.
[0061] The inventors have unexpectedly found that multicomponent
vaccines containing a plurality of clostridial protective antigen
components plus at least one non-clostridial protective antigen
component and an adjuvant in a low dose volume can be produced by:
identifying the protective antigen component of each organism by in
vivo or in vitro methods; quantifying the protective antigen
components during formulation and manufacture of the vaccine, using
antigen quantitation assays described above to provide the
protective antigen component in an amount sufficient to produce a
protective vaccine with the least antigenic mass; identifying the
antigenic components of the organisms which contain detrimental
antigens by using the antigen quantitation assays and animal
reactivity testing; purifying the protective antigen components
which contain detrimental antigens to remove such antigens;
selecting the inactivating agent for each organism requiring
inactivation such that the organism is killed without denaturing
the protective antigen component; selecting an adjuvant for each
protective antigen component that requires an adjuvant by
evaluating the adjuvant's ability to enhance the immune response to
the specific protective antigen component without causing
unacceptable animal reactivity; adjuvanting, individually, the
protective antigen components that require such adjuvanting;
pooling the protective antigenic components into a low dose vaccine
that imparts protection to animals to which the vaccine is
administered. By this method, one can produces a
commercially-viable, cost effective safe, immunogenically effective
multicomponent vaccine. The multicomponent vaccine contains a
combination of: one or more clostridial protective antigen
components with one or more non-clostridial protective antigen
components and an adjuvant within a low dose volume of 3.0 mL or
less. The use of multicomponent vaccines, i.e., commercial scale
vaccines of this infection, do not produce significant
injection-site lesions upon subcutaneous or intramuscular
administration.
[0062] The following is a specific description of the invention
that is intended to assist those skilled in the practice of the
invention. More specifically, the description relates to the
characterization of the antigenic components and the manner in
which they are formulated, including inactivation and
adjuvanting.
[0063] Cl. chauvoei protective antigens have been found by the
inventors to be associated with cells These protective antigens are
not found in proteinaceous material excreted into the culture
supernatant while the organism is being grown in fermenters. It has
also been found that the Cl. chauvoei protective antigen component
does not interfere with other protective antigen components in the
multicomponent clostridial vaccine. Therefore, a whole cell
bacterin or a cell extract can be used. The whole cell bacterin or
cell extract may be inactivated with formaldehyde (0.05-1.5%),
Betapropriolactone (BPL) at 0.05 to 0.3% or Binary ethyleneimine
(BEI) at 0.05 to 0.3%. After inactivation, this component must be
adjuvanted separately. If BPL or BEI are used for inactivation they
must be neutralized prior to adjuvanting. Adjuvants which enhance
this protective antigen component are Al(OH).sub.3, oils, saponin,
Quil A, block co-polymers and polymers such as "carbopol". Oil
adjuvants can be used as co-adjuvants with polymers. Carbopol is
more preferred and is added to the inactivated whole culture at a
low pH. The pH is then adjusted upward to approximately 7.0 with,
say, sodium hydroxide (NaOH). This pH adjustment step allows for
the protective antigen components of the Cl. chauvoei to become
encapsulated in the polymer adjuvant. Without being bound to any
particular theory of the invention, it is believed the Cl. chauvoei
antigens are released over a period of several weeks. Because of
the slow release, these antigens do not cause the typical animal
reaction. The long-term release causes an enhanced immune response
by the vaccinated animal.
[0064] The protective antigen component of Cl. septicum is
associated both with the cell and with a toxin. The toxin is
secreted into a supernatant while the organism is growing.
Therefore, this protective antigen component is derived from the
cell and supernatant. Apparently, Cl. septicum does not interfere
with other protective antigen components in multicomponent
clostridial vaccines containing non-clostridial protective antigen
components. The whole cell bacterin or cell extract can be
inactivated with formaldehyde (0.05-1.5%), BPL (0.05-0.3%) or BEI
(0.05-0.3%). After inactivation, this protective antigen component
must be adjuvanted separately. When BPL or BEI are used for
inactivation, they must be neutralized before adjuvanting.
Adjuvants that enhance this protective antigen component can be:
Al(OH).sub.3, oils, saponin, Quil A, block co-polymers and polymers
such as carbopol. Oil adjuvants can be used if combined as
co-adjuvants with polymers. The preferred adjuvant are the polymer
adjuvant. Preferably, the adjuvant is added to the inactivated
whole culture at a low pH. Then the pH is adjusted upward to
approximately 7.0 with NaOH. This pH adjustment step increases the
pH from approximately 5.0 to 7.0 during which the antigens of the
Cl. septicum become encapsulated in the carbopol. The resulting
vaccine does not cause the typical animal reactivity but releases
the Cl. septicum antigens over a period of several weeks. This mode
of release causes an enhanced immune response by the vaccinated
animal.
[0065] The protective antigen component of Cl. novyi, is believed
by the inventors to be associated with a cell protein, and a toxin
that is excreted into a supernatant. Therefore, this protective
antigen component is derived from both the cell and supernatant, in
either concentrated or non-concentrated form. Apparently, the
protective antigen of the Cl. novyi does not interfere with other
protective antigen components in multicomponent clostridial
vaccines when combined with non-clostridial protective antigen
components. The whole cell bacterin or cell extract may be
inactivated with formaldehyde (0.05-1.5%), BPL (0.05-0.3%) or BEI
(0.05-0.3%) and must be adjuvanted separately. If BPL or BEI is
used, it must be neutralized before adjuvanting. Adjuvants that
enhance this protective antigen component are Al(OH).sub.3, oils,
saponin, Quil A, block co-polymers and polymers such as carbopol.
Oil adjuvants can be used if combined as co-adjuvants with
polymers. The carbopol polymer adjuvants are preferred. The polymer
adjuvant is added to the inactivated whole culture at a low pH.
Then the pH is adjusted upward to approximately 7.0 with NaOH. This
pH adjustment step increases the pH from approximately 5.0 to 7.0
during which the antigens of the Cl. novyi become encapsulated in
polymer. The resulting vaccine does not cause the typical animal
reactivity but releases the Cl. novyi antigens over a period of
several weeks. The long-term release causes an enhanced immune
response by the vaccinated animal.
[0066] The protective antigen component of Cl. sordellii is
believed to be associated with a toxin that is secreted into the
supernatant as the culture is growing. Therefore, this protective
antigen component is derived from the supernatant. This protective
antigen component is typically concentrated via ultrafiltration
through a 10,000 dalton molecular weight (MW) cartridge before
adjuvanting. The Cl. sordellii toxin may be inactivated with
formaldehyde (0.05-1.5%), BPL (0.05-0.3%) or BEI (0.05-0.3%) prior
to adjuvanting, and must be adjuvanted separately. If BPL or BEI is
used for inactivation, it must be neutralized before adjuvanting.
Adjuvants that enhance this protective antigen component are
Al(OH).sub.3, oils, saponin, block co-polymers and polymers such as
carbopol. Oil adjuvants can be used if combined as co-adjuvants
with polymers. The polymer adjuvant is are preferred. The carbopol
polymer adjuvant is added to the inactivated whole culture at a low
pH. Then the pH is adjusted upward to approximately 7.0 with NaOH.
This pH adjustment step increases the pH from approximately 5.0 to
7.0 during which the antigens encapsulated in polymer adjuvant. The
resulting vaccine does not cause the typical animal reactivity but
releases the Cl. sordellii antigens over a period of several weeks.
The long-term release causes an enhanced immune response by the
vaccinated animal.
[0067] The protective antigen components of Cl. perfringens types C
and D are known to be toxoids that are excreted by the cells.
Because they cross-protect against Cl perfringens type B, these
protective antigen components only need to contain cell-free
supernatant containing inactivated toxin (toxoid). These two
components are considered to represent 3 components (B, C, and D).
In formulations of a multicomponent clostridial vaccine, one may
use Cl. perfringens types C and D protective antigen components
that contain cells or have the cells removed therefrom (cell free
toxoid). Before the removal of the cells, the whole culture is
harvested from the fermenter and inactivated with formaldehyde
(0.5-1.5%), BPL (0.05-0.5%) or BEI (0.05-0.5%) and before
adjuvanting. The cells can be removed by, say, filtration or
centrifugation, In either case, the respective antigens must be
adjuvanted separately. If BPL or BEI is used for inactivation, it
must be neutralized before cell removal. Adjuvants which enhance
this protective antigen component are Al(OH).sub.3, oils, saponin,
Quil A, block co-polymers and polymers such as carbopol. Oil
adjuvants can be used if combined as co-adjuvants with polymers.
Preferred here is the polymer adjuvant. The carbopol adjuvant is
added to the inactivated whole culture at a low pH. Then the pH is
adjusted upward to approximately 7.0 with NaOH. This pH adjustment
step increases the pH from approximately 5.0 to 7.0. During this
increase the protective antigen components of the Cl. perfringens
become encapsulated in the polymer adjuvant.
[0068] The protective antigen component of Cl. haemolyticum is
believed to be both cell-associated and excreted as a toxin into
the supernatant. Therefore, this protective antigen component
contains antigens from the cells and supernatant. Because of its
high cell mass, this protective antigen component can cause
interference with other protective antigen components of a
multicomponent clostridial vaccine. Typically, this protective
antigen is concentrated by, say, ultrafiltration with a 10,000
molecular weight cartridge before adjuvanting. The Cl. haemolyticum
whole culture can be inactivated with formaldehyde (0.05-t5%), BPL
(0.05-0.3%) or BEI (0.05-0.3%) before concentration. The
inactivated, concentrated material must be adjuvanted separately.
If BPL or BEI are used for inactivation, it must be neutralized
prior to adjuvanting. Adjuvants which enhance this protective
antigen component are Al(OH).sub.3, oils, saponin, Quil A, block
co-polymers and polymers such as carbopol. Oil adjuvants can be
used if combined as co-adjuvants with polymers. Preferred herein is
the polymer adjuvant. The carbopol adjuvant is added to the
inactivated whole culture at a low pH. Then the pH is adjusted
upward to approximately 7.0 with NaOH. This pH adjustment step
increases the pH from approximately 5.0 to 7.0. During the
increase, the protective antigen components of the Cl. haemolyticum
become encapsulated in polymer adjuvant. The resulting vaccine does
not cause the typical animal reactivity but releases the Cl.
haemolyticum antigens over a period of several weeks. The long-term
release causes an enhanced immune response by the vaccinated
animal.
[0069] With the foregoing description and the examples to follow,
it would be within the purview of the skilled artisan to make and
use the low dose, multicomponent vaccines of the invention. In the
practice of the invention, the multicomponent, low-dose vaccines
can be administered subcutaneously or intramuscularly to protect
animals without causing significant injection-site lesions.
[0070] This and other aspects of the invention are further
illustrated by the following non-limiting examples.
EXAMPLES
Example 1A
[0071] This example illustrates the embodiment of this invention
comprising a combination of protective antigen components from at
least 6 clostridial organisms with protective antigen components
from at least 1 non-clostridial component such as a Gram-negative
organism. First a multi-component bacterin was formulated with a
combination of protective antigen components derived from: Cl.
chauvoei, Cl. septicum, Cl. novyi, Cl. sordellii, Cl. perfringens
types C and D; a protective antigen component from H. somnus and a
carbopol adjuvant. The H. somnus protective antigen component was
purified enough to prevent animal reactivity but not so much as to
make it non-cost effective. Two isolates of H. somnus were used in
the experiments. One isolate was designated 8025T and the other was
designated 14767. Each isolate was grown separately in 160 L of
media containing the following components: Pancreatic Digest of
Casein, Yeast Extract, Proteose Peptone, NaCl, and
Na.sub.2HPO.sub.4. The growth medium was supplemented with 0.5%
dextrose and 10% horse serum. Dissolved oxygen was controlled
during the fermentation cycle at approximately 10% (between 5% and
20%). Fermenters were inoculated with either 3.5% seed (isolate
14767) or 5% seed (isolate 8025T). Cultures were incubated at
37.degree. C., with pH control between 7.1 and 7.3 and allowed to
grow until optical densities (absorbance at 540 nm) reached
approximately 1.20 (5-24 hours) at which time cultures were
inactivated with 0.3% formalin. Inactivation of the H. somnus was
done with formaldehyde (0.05-1.5%), BPL (0.05-0.5%) or BEI
(0.05-0.5%) prior to concentration and adjuvanting. In the use of
BPL and BEI, they were neutralized before being used for
inactivation. Carbopol was added to the inactivated whole culture
at a low pH. Then the pH was adjusted up to 7.0 with NaOH.
[0072] Following inactivation, the whole bacterial cultures were
concentrated 10.times. using a 0.1 micron ultrafiltration
cartridge, followed by diafiltration with 11 volumes of Phosphate
Buffered Saline (PBS). The washed concentrates were then
centrifuged at 7000 RPM using a Sorvall RC5B refrigerated
centrifuge and the pellets were resuspended in 100 mL of chilled
PBS. Centrifuged concentrates were adjusted to either 10.times. or
20.times. concentration (based on initial who culture volume) and
adjuvanted with 10% v/v 10.times. modified carbopol adjuvant. This
adjuvant was comprised of: up to 0.25% Carbopol, 934P, Tween 80,
Span 20 and Cotton Seed Oil. For further experimentation, a
1.times. dose of H. somnus 8025T consisted of either 0.061 mL of
adjuvanted H. somnus 8025T 20.times. concentrate or 0.122 mL of
adjuvanted 10.times. concentrate. Likewise, a dose of H. somnus
14767 consisted of either 0.061 mL of adjuvanted 20.times.
concentrate or 0.122 mL of adjuvanted 10.times. concentrate. These
volumes corresponded to the amount of antigen contained in 1.0 mL
of 14767 or 8025T whole culture, each having an optical density of
1.3 at 540 nm.
[0073] Relative purity of the above-described H. somnus
preparations was demonstrated by comparing their endotoxin levels
after the various purification steps. The preparations were
compared to whole culture H. somnus. Samples of H. somnus 8025T and
14767 10.times. concentrates were removed at various stages in the
purification process and diluted to 1.times. with PBS.
[0074] Endotoxin assays were run on the samples using an automated
BioWhitaker apparatus and results were normalized against an E.
coli LPS standard prepared to contain one million endotoxin units
per mL. Results are shown in TABLE 1. Results show that the H.
somnus cultures can be purified using centrifugation or a
combination of ultrafiltration and diafiltration. The resultant
cultures had endotoxin levels that were less than 10% of those seen
in original inactivated whole cultures. This level of endotoxin
reduction is adequate to eliminate significant animal reactivity
and is cost effective.
TABLE-US-00001 TABLE 1 ENDOTOXIN LEVELS OF PURIFIED H. somnus
CONCENTRATES ENDOTOXIN UNITS/mL (.times.1000) MATERIAL TESTED
ISOLATE 14767 ISOLATE 8025T INACTIVATED 1X WHOLE 5266 8705 CULTURE
10X CONCENTRATE, 681 1332 DIAFILTERED WITH 11 VOLUMES PBS, RECON.
TO 1X 10X CONCENTRATE, 422 397 DIAFILTERED WITH 11 VOLUMES PBS,
CENT., RECON. TO 1X 10X CONCENTRATE, CENT., 408 397 RECONSTITUTED
TO 1X CENTRIFUGED WHOLE 431 256 CULTURE, RECON. TO 1X
Example 1B
[0075] This example illustrates that immunogenicity is maintained
when only the cells were used to produce the protective antigen
components. After the purification of H. somnus as described in
Example 1A, the washed-cell preparations thereof were formulated at
various antigen concentrations with a plurality of clostridial
protective antigen components and tested as either a 2.0 mL dose or
a 5.0 mL dose (positive control) in a mouse vaccination/challenge
test [approved by the U.S. Animal Plant Health Inspection Service
(APHIS)]. The test was conducted by vaccinating mice with a
fractional dose of the test product, boostering such mice with the
same dose at 14 days post vaccination and challenging such mice
with a virulent H. somnus culture at 10-14 days post booster. The
challenge culture was mixed with an equal volume of 7% gastric
mucin prior to injection. The resulting mixture was strong enough
to kill 80% of the control mice (16 of 20). For a satisfactory
test, at least 14 of 20 vaccinated mice must survive. The
clostridial fractions were produced as follows:
[0076] Although any commercial Cl. chauvoei whole bacterial culture
could be used as the protective antigen component, for purposes of
this experiment the Cl. chauvoei was grown under strict anaerobic
conditions in large-scale fermenters under pH control conditions
between 6.5 and 7.6; inactivated with 0.5% formaldehyde and
adjuvanted with the modified carbopol adjuvant as a separate
non-concentrated whole bacterial culture. The modified carbopol
adjuvant was the same as that described in Example 1A. The adjuvant
was added in a 10% v/v ratio to the Cl. chauvoei whole bacterial
culture, mixed to allow complete contact with adjuvant while at a
low pH, and then pH adjusted to approximately 7.0 with 5 or 10N
NaOH.
[0077] Although it is expected that any commercial Cl. septicum
whole culture bacterial culture could be used as the protective
antigen component, for purposes of this experiment the Cl. septicum
was grown under strict anaerobic conditions in large-scale
fermenters with pH control between 6.5 and 7.6; inactivated with
0.5% formaldehyde, concentrated minimally using a 10,000 dalton MW
ultrafiltration system and adjuvanted with the modified carbopol
adjuvant by adding the adjuvant directly to the concentrated whole
bacterial culture Cl. septicum. The modified carbopol adjuvant is
the same as that described previously. The adjuvant was added in a
10% v/v ratio to the Cl. septicum concentrate, mixed to allow
complete contact with adjuvant at the low pH, and then pH adjusted
to approximately 7.0 with 5 or 10N NaOH.
[0078] Cl. novyi was grown under strict anaerobic conditions in
large-scale fermenters with pH control between 6.5 and 7.6,
inactivated with 0.5% formaldehyde and adjuvanted as a
non-concentrated whole bacterial culture with the modified carbopol
adjuvant as described previously. The adjuvant was added in a 10%
v/v ratio to the Cl. novyi whole bacterial culture, mixed to allow
complete contact with adjuvant at low pH, and then pH adjusted to
approximately 7.0 with 5 or 10N NaOH. Combining Power Unit (CPU)
was measured, as described above, in the culture post inactivation
and post adjuvanting. The CPU of the final protective antigen
component was adjusted to 10 CPU/mL with adjuvanted PBS.
[0079] Cl. sordellii was grown under strict anaerobic, conditions
in large-scale fermenters with pH control between 6.5 and 7.6. At
the end of the growth phase, the culture was maintained at a pH of
approximately 8.0 for 8-10 hours to facilitate cell lysis. The
lysed culture was then inactivated with 0.5% formaldehyde (lysed
toxoid), concentrated using a 10,000 dalton MW ultrafiltration
cartridge and adjuvanted with the modified carbopol adjuvant
described previously. The adjuvant was added in a 10% v/v ratio to
the Cl. sordellii lysed toxoid, mixed to allow complete contact
with adjuvant at the low pH, and then pH adjusted to approximately
7.0 with 5 or 10N NaOH. After adjuvanting, the combining power was
measured and the protective antigen component was adjusted to 100
CPU/mL by dilution with adjuvanted PBS.
[0080] Clostridium perfringens types C and D were grown under
strict anaerobic conditions in large-scale fermenters with pH
control between 7.3 and 7.5 for 4-8 hours. The whole bacterial
cultures were inactivated with 0.5% formaldehyde. For purposes of
this experiment, cells were removed by centrifugation in a Sorvall
centrifuge at 7000 RPM. The remaining supernatants contained Cl.
perfringens C or D toxoids. The toxoids were individually
concentrated by ultrafiltration through a 10,000 dalton MW
cartridge and the concentrates were assayed for their quantity of
protective antigen component by the previously-described combining
power test. After adjustment of the antigen concentration (CPU),
each protective antigen component was individually adjuvanted using
the modified carbopol adjuvant described previously. The adjuvant
was added in a 10% v/v ratio to the individual Cl. perfringens
toxoids (C or D), mixed to allow complete contact with adjuvant at
the low pH, and then pH adjusted to approximately 7.0 with 5 or 10N
NaOH.
[0081] Cl. haemolyticum was grown under strict anaerobic conditions
in large-scale fermenters with pH control between 6.8 and 7.3. The
culture was harvested and inactivated with 0.5% formaldehyde prior
to concentration. A 10,000 dalton MW ultrafiltration cartridge was
used to concentrate the whole culture which was then adjuvanted
with the modified carbopol adjuvant described in Example 1A. The
adjuvant was added in a 10% v/v ratio to the Cl. haemolyticum
culture concentrate, mixed to allow complete contact with adjuvant
at low pH, and then pH adjusted to approximately 7.0 with 5 or 10N
NaOH.
[0082] H. somnus was prepared according to the description in
Example 1A. The pre-adjuvanted clostridial components, as
afore-described, were formulated into one pool as shown in TABLE 2.
To this pool was added the adjuvanted H. somnus component and
adjuvanted PBS to equal the dose size being tested.
[0083] Experimental serials were made with varying amounts of H.
somnus washed cell suspension, as described in Example 1A, in
combination with 6 or 7 clostridial protective antigen components,
in order to determine whether the potency of this component was
adversely affected by the purification process or by the mixture of
the more purified H. somnus with the clostridia! components.
Serials of product containing 6 clostridial protective antigen
components plus H. somnus or 7 clostridial protective antigen
components+H. somnus were prepared as shown in Table 2 and tested
for potency of the H. somnus protective antigen component according
to the mouse test described in Example 1A. Host animal doses of 5.0
mL and 2.0 mL were tested. The results of these tests are shown in
TABLE 3 along with a listing of dose size tested and the amounts of
H. somnus per dose.
[0084] This experiment demonstrates that the protective antigens of
H. somnus are associated with the cells and not with the
supernatant which contains the endotoxins. Additionally, the washed
cell suspension did not appear to be adversely affected by the 6
clostridial protective antigen components. The H. somnus protective
antigen component was still potent when the washed cells were
resuspended to a concentration equal to one-half the concentration
of the original whole culture and mixed with 6 clostridial
protective antigen components. When Cl. haemolyticum was added to
the 6 original clostridial protective antigen components it
appeared to adversely affect the H. somnus protective antigen
component only slightly--not enough to require a dose size greater
than 2.0 mL. Therefore, it is commercially feasible to produce a
vaccine with protective antigen components from 7 clostridial
organisms in combination with a protective antigen component from a
Gram-negative organism such as H. somnus.
TABLE-US-00002 TABLE 2 GENERAL COMPONENT FORMULATIONS -
PREADJUVANTED MINIMUM ACTUAL AMOUNT OF VOLUME OF COMPONENT/
COMPONENT/ DESCRIPTION ORGANISM DOSE DOSE OF ANTIGEN Cl. chauvoei
>0.2 mL of 0.2 mL WC Nonconc. WC* Cl. septicum 0.11 mL of 0.11
mL WC 7.4X WC* Concentrate Cl. novyi 2.0 CPU 0.2 mL @ 2.0 CPU 10
CPU/mL Toxoid + WC Cl. sordellii 27.0 CPU 0.27 mL @ 27.0 CPU 100
CPU/mL Toxoid + WC Cl. 20 mL 0.28 mL 7.2X Conc. hemolyticum
equivalents of Toxoid + WC whole culture Cl. perfringens 600
CPU/dose 0.375 mL of WC Nonconc. type C whole culture = Purified
600 CPU/dose Cl. perfringens 359 CPU/dose 0.39 mL of WC Nonconc.
type D whole culture = Purified 350 CPU/dose Adjuvanted N/A Amt.
needed to N/A PBS bring total dose to volume required *WC = Whole
Culture
TABLE-US-00003 TABLE 3 POTENCY TESTING OF THE PURIFIED H. somnus
COMPONENT WHEN COMBINED WITH CLOSTRIDIAL COMPONENTS POTENCY TEST
RESULT AMOUNT OF H. Somnus PER (PROTECTED TYPE OF DOSE DOSE*
MICE/TOTAL SERIAL NUMBER PRODUCT SIZE (mL) ISOLATE 8025T ISOLATE
14767 INFECTED) 1093-1 6-WAY + H. somnus 5.0 0.183 0.183 20/20 1.5X
1.5X 1093-2 6-WAY + H. somnus 5.0 0.122 0.122 20/20 1.0X 1.0X
1093-3 6-WAY + H. somnus 5.0 0.061 0.061 20/20 0.5 1.0X 1093-4
6-WAY + H. somnus 2.0 0.183 0.183 20/20 1.5X 1.5X 1093-5 6-WAY + H.
somnus 2.0 0.122 0.122 20/20 1.0X 1.0X 1093-6 6-WAY + H. somnus 2.0
0.061 0.061 19/20 0.5X 0.5X 1093-7 1093-5 DILUTED 2.0 0.5X 0.5X
19/20 1:2 1093-8 7-WAY + H. somnus 2.0 0.061 0.061 20/20 1.0X 1.0X
1093-9 6-WAY + H. somnus 2.0 0.244 NONE 20/20 1.5X 1093-10 6-WAY +
H. somnus 2.0 NONE 0.244 18/20 1.0X 1093-11 H. somnus ONLY 2.0
0.122 0.122 20/20 1.0X 1.0X *The amount as designated by X
indicates the concentration as relative to the original whole
culture. 6-WAY components = Cl. chauvoei, Cl. novyi, Cl. septicum,
Cl. sordellii, Cl. perfringens types C and D 7-WAY components = Cl.
chauvoei, Cl. novyi, Cl. septicum, Cl. sordellii, Cl. perfringens
types C and D, Cl. haemolyticum Cl. perfringens type C contained
600 CPU per dose Cl. perfringens type D contained 350 CPU per
dose
Example 3
[0085] This example shows the effect of detrimental antigens on
relatively weak protective antigen components such as C.
perfringens types C and D. The effect of the detrimental antigens
were evaluated in a multi-component vaccine containing protective
antigen components from 6 clostridial organisms and one protective
antigen component from one non-clostridial. Clostridial protective
antigen components were produced as described in Example 1 B.
Serials were formulated with varying levels of Cl. perfringens type
C and D toxoids. CPU levels for type C were adjusted to 600, 900,
1200 or 1800 per dose whereas CPU levels of type D toxoid were
adjusted to 350, 500, 700 or 1000 per dose.
[0086] Six clostridial protective antigen components were combined
with two protective antigen components from H. somnus in various
formulations containing differing concentrations of the two Cl.
perfringens protective antigen components. TABLE 4 illustrates the
amounts of each protective antigen component added to the
formulations excluding the Cl. perfringens types C and D.
TABLE-US-00004 TABLE 4 GENERAL PROTECTIVE ANTIGEN COMPONENT
FORMULATIONS - PREADJUVANTED MINIMUM ACTUAL AMOUNT OF VOLUME OF
COMPONENT/ COMPONENT/ DESCRIPTION ORGANISM DOSE DOSE OF ANTIGEN Cl.
chauvoei >0.2 mL of WC* 0.2 mL WC Nonconc. Cl. septicum 0.8 mL
of WC* 0.11 mL WC 7.4X Concentrate Cl. novyi 2.0 CPU 0.2 mL @ 2.0
CPU 10 CPU/mL Toxoid + WC Cl. sordellii 27.0 CPU 0.27 mL @ 27.0 CPU
100 CPU/mL Toxoid + WC H. somnus Conc. equivalent 0.122 mL 10X
Conc. 8025T to 1.0 mL of Toxoid + WC WC* at harvest H somnus Conc.
equivalent 0.122 mL 10X Conc. 14767 to 1.0 mL of Washed cells WC*
at harvest Cl. 2.0 mL 0.28 mL 7.2X Conc. haemolyticum equivalents
of Washed cells whole culture Adjuvanted N/A Amt. needed to N/A PBS
bring total dose to 2.0 mL *WC = Whole Culture
[0087] Because Cl. perfringens types C and D were more purified
toxoids in this experimental preparation, it was important to
determine whether these protective antigen components would be
adversely affected by the other clostridial protective antigen
components or by a non-clostridial protective antigen component
such as H. somnus. Therefore, this experiment involved preparation
of a clostridial vaccine combined with H. somnus in a 2.0 mL dose
size and included varying the amounts of the Cl. perfringens types
C and D components. CPU levels of types C & D ranged from 600
to 1800 CPU per dose for type C and from 350 to 1000 CPU per dose
for type D. TABLE 5 shows the Cl. perfringens types C & D
components along with the test results after injection of
animals.
[0088] The five multicomponent clostridial vaccines and one vaccine
containing a plurality of clostridial protective antigen components
combined with H. somnus were tested according to procedures
required by the U.S. government Animal Plant Health Inspection
Service (APHIS). Guinea pigs, rabbits or mice were used for the
testing. For the clostridial components, guinea pigs or rabbits
were vaccinated respectively with a dose equivalent to 1/5 or 1/2
the field dose. These animals were boostered 10 to 14 days later
with the same dose of vaccine. Guinea pigs were challenged with
live organisms of either Cl. chauvoei or Cl. haemolyticum. To
correlate with protection in cattle, at least 80% of the guinea
pigs must survive these challenges. Mice were vaccinated, boostered
and challenged to demonstrate that a vaccine was protective against
H. somnus. The challenge was a live culture of H. somnus which must
kill at least 80% of the non-vaccinated control mice. An acceptable
vaccine must protect 14 of 20 vaccinated mice. Rabbits were
vaccinated, boostered and bled to test for antibody titers against
Cl. septicum, Cl. sordellii, Cl. novyi, and Cl. perfringens types C
and D. Antibody quantitation was conducted according to prescribed
APHIS testing against known standard toxins and antitoxins.
[0089] Animal test results [comparing Cl. perfringens types C and
D, Cl. novyi and Cl. sordellii antitoxin responses obtained with
five multi-component vaccines containing protective antigen
components from 7 clostridial organisms (7-way) and one
multicomponent vaccine containing protective antigen components
from 7 clostridial organisms and one Gram-negative organism (H.
somnus)] indicate that as little as 600 CPU of Cl. perfringens type
C and 350 CPU of Cl. perfringens type D are necessary to protect
animals in a vaccine containing 7 clostridial protective antigen
components. Three-fold increases in the amounts of these toxoids
did not interfere with other protective antigen components of these
multicomponent vaccines. When H. somnus was added to the 7
clostridial protective antigen components, there appeared to be a
slight depression of response to the Cl. perfringens types C &
D. Therefore, the amounts of these protective antigen components
would be increased in order to assure host animal protection in a
multicomponent vaccine containing at least one non-clostridial
antigen. TABLE 5 (below) shows that CPU levels of 1200 for Cl.
perfringens type C and 700 for Cl. perfringens type D compensate
for the affect of H. somnus. Apparently, the amounts of Cl.
sordellii and Cl. novyi, can be decreased since the amounts thereof
appear to be significantly greater than necessary to protect
animals.
TABLE-US-00005 TABLE 5 CRITICAL POTENCY RESULTS OF 7-WAY AND 7-WAY
+ H. somnus ANTITOX UNITS* CL. PERFRINGENS Cl. Cl. Cl. SERIAL CPUs
Cl. perf. C perf. D novyi sordellii 3X1094-A C = 600 CPU >10.0
2.0 4-5 >8 7-WAY D = 350 CPU 3X1094-B C = 900 CPU 10.0 2.0 NT NT
7-WAY D = 500 CPU 3X1094-C C = 1200 CPU 20.0 3.0 3.0 >8 7-WAY D
= 700 CPU 3X1094-D C = 1800 CPU 10.0 3.0 NT NT 7-WAY D = 1000 CPU
3X1094-E C = 1200 CPU 15.0 2.0 3.0 5-7 7-WAY + D = 700 CPU H.
somnus 3X1094-F C = 1200 CPU 20.0 2.0 NT NT 7-WAY (pH adj. to 6.0)
D = 700 CPU *Necessary for Host Animal Protection: Cl. perf. C = 10
au; Cl. perf. D = 2 au; Cl. novyi = 0.5 au; Cl. sordellii = 1.0 au
**NT = Not Tested
Example 4
[0090] This example shows the incorporation of the protective
antigen components from the clostridial organisms and H. somnus in
a commercial size serial of a vaccine, and the test for potency of
the components. A 160 L batch of 6-way clostridial product
containing Cl. chauvoei, Cl. septicum, Cl. novyi, Cl. sordellii,
Cl. perfringens types C and D was prepared in the proportions as
listed in TABLE 4 and formulated as in Example 2 with H. somnus
isolates 8025T and 14767 at a 1.times. concentration as described
in Example 1A. This serial was tested for potency according to the
previously-described APHIS requirements. The results of the tests
are shown in TABLE 6. All protective antigen components of the
6-WAY clostridial plus H. somnus multicomponent vaccine showed
potency results which exceed the minimum requirements for
protection of animals as determined by APHIS.
TABLE-US-00006 TABLE 6 ANIMAL TEST RESULTS OF 6-WAY CLOSTRIDIAL +
H. somnus REQUIREMENT TEST ANIMAL FOR POTENCY TYPE OF SATISFACTORY
RESULT ORGANISM TEST POTENCY (live/total) Cl. chauvoei Guinea Pig
7/8 guinea pigs 8/8 Challenge must survive challenge Cl. septicum
Rabbit 7/8 rabbits must 8/8 Challenge survive challenge Cl. novyi
Rabbit Serology 0.5 antitoxin 4.0 au units in the rabbit serum Cl.
sordellii Rabbit Serology 1.0 antitoxin >10.0 au units in the
rabbit serum Cl. perfringens Rabbit Serology 10.0 antitoxin 25.0 au
Type C units in the rabbit serum Cl. perfringens Rabbit Serology
2.0 antitoxin 3.0 au Type D units in the rabbit serum H. somnus
Mouse 15 of 20 mice 20/20 Challenge must survive the challenge
Example 5
[0091] Seven clostridial protective antigen components were
combined with the protective antigen component from H. somnus
according to the procedures described in Example 2 and tested in
APHIS-required potency tests (as described previously) as a 2.0 mL
dose. The actual formulation specifications are listed in TABLE 7.
Results of the APHIS-required animal testing are shown in TABLE 8.
All the protective antigen components passed the testing. These
data demonstrate that 7 clostridial protective antigen components
can be combined with a protective antigen component from H. somnus
or some other non-clostridial organism to produce a vaccine which
is immunogenically effective. In fact, there is little difference
between the animal test results produced by the 6-way plus H.
somnus and those produced by the 7-way plus H. somnus (compare
results in TABLES 6 and 8).
TABLE-US-00007 TABLE 7 FORMULATION OF PROTECTIVE ANTIGEN COMPONENTS
OF 7-WAY + H. somnus SERIAL 102994 AMOUNT LOT PER 2.0 mL ORGANISM
STRAIN NUMBER CONC. DOSE Cl. chauvoei 5677-2 264 NONE 0.400 mL Cl.
septicum 6750-2 296 6.6X 0.121 mL Cl. novyi 3047 165 NONE 0.167 mL
Cl. sordellii 4513 227 NONE 0.090 mL Cl. 5982 194 7.15X 0.280 mL
haemolyticum Cl. perfringens 3602 540 NONE 0.400 mL type C/B Cl.
perfringens 455E 155 NONE 0.364 mL type D/B H. somnus 8025T N/A 20X
0.061 mL H. somnus 14767 N/A 20X 0.061 mL Adjuvanted N/A N/A N/A
0.056 mL PBS
TABLE-US-00008 TABLE 8 ANIMAL TEST RESULTS PRODUCED BY 7-WAY + H.
somnus REQUIREMENT POTENCY TEST ANIMAL FOR RESULT TYPE OF
SATISFACTORY 7-WAY + ORGANISM TEST POTENCY H. somnus Cl. chauvoei
Guinea Pig 7/8 guinea pigs 8/8 Challenge must survive Live/Total
challenge Cl. septicum Rabbit 7/8 rabbits must 8/8 Challenge
survive challenge Live/Total Cl. novyi Rabbit 0.5 antitoxin units
>0.5 Serology in the rabbit Antitoxin Units serum Cl. sordellii
Rabbit 1.0 antitoxin units >1.0 Serology in the rabbit Antitoxin
Units serum Cl. Rabbit 10.0 antitoxin >10.0 perfringens Serology
units in the rabbits Antitoxin Units Type C serum Cl. Rabbit 2.0
antitoxin units >2.0 perfringens Serology in the rabbit
Antitoxin Units Type D serum Cl. Guinea Pig 7/8 guinea pigs 8/8
haemolyticum Challenge must survive Live/Total challenge H. somnus
Mouse 14 of 20 mice 16/20 Challenge must survive Live/Total
challenge
Example 6
[0092] This example illustrates vaccines wherein viruses are
combined with clostridial components. Modified live infectious
bovine rhinotracheitis virus (IBRV) was combined with a plurality
of clostridial protective antigen components (Cl. perfringens types
C and D).
[0093] The clostridial protective antigen components were prepared
and formulated according to methods discussed in Example 1B. The
IBRV utilized for this experiment was one which had been modified
such that it would a disease if the live virus is injected into
animals. Vaccines prepared from such viruses are called modified
live vaccines. Since modified live vaccines contain live viruses as
their protective antigen component, the efficacy of such vaccines
depends on the amount of live virus contained within them. It has
been determined by cattle vaccination/challenge studies that
infectious bovine rhinotracheitis virus when prepared in a
lyophilized vaccine protects cattle if the titer is at least
10.sup.4.2TCID.sub.50/mL. The reference IBRV used for this
experiment was grown in roller bottle culture on bovine kidney
cells after which the IBRV harvest fluids were lyophilized such
that the titer post lyophilization was 10.sup.7.0/mL.
[0094] To avoid loss of efficacy of the vaccine, the multicomponent
vaccine containing protective antigen components from Cl.
perfringens types C and D and from IBRV is formulated as a
two-container vaccine. One container will contain the lyophilized
modified live IBRV protective antigen component and the second
container will contain the inactivated, adjuvanted liquid Cl.
perfringens types C and D protective antigen components. In using
the vaccine, the liquid Cl. perfringens types C and D protective
antigen component is removed from its container with a syringe and
injected into the lyophilized modified live IBRV container causing
rehydration of the lyophilized IBRV. In order to determine whether
a modified live virus is negatively affected by the rehydration,
one retitrates the combined multicomponent vaccine. If there is a
detrimental effect (viricidal activity) of the rehydration of the
virus protective antigen component it will be apparent within the
first 2 hours after rehydration. Therefore, all such modified jive
vaccines which are combined with non-modified live components be
tested for and pass a virucidal activity test. APHIS defines
viricidal activity as the loss of more than 0.7 logs of virus titer
within 2 hours after rehydrating the virus component. Any
multicomponent vaccine in which the virus protective antigen
component loses more than 0.7 logs of virus titer within 2 hours
post rehydration by the diluent therefore would be considered to
have failed the viricidal activity test.
[0095] Several formulations of the 3-way multicomponent vaccine
containing Cl. perfringens types C and D and IBRV were prepared and
formulated. An APHIS-required viricidal activity test was conducted
on each of these formulations. The specifics of the formulation of
the combinations and results of the viricidal activity testing are
shown in TABLE 9. It is apparent that all formulations, even those
containing non-purified Cl. perfringens types C and D were
acceptable showing no viricidal activity. Therefore, it has been
demonstrated that a plurality of clostridial protective antigen
components can be added to virus protective antigen components
without causing a detrimental effect when prepared according to the
methods described herein. More specifically there were no contrary
indications that clostridial protective antigen components or
adjuvants or combinations thereof are virucidal, or that there was
an interference between the clostridial protective antigen
components and the virus protective antigen components.
TABLE-US-00009 TABLE 9 FORMULATION AND TESTING OF COMBINATION Cl.
perfringens types C and D + IBRV SERIAL NO. Cl. perfringens Cl.
perfringens TESTED AS Type C Type C IBRV TITER IBRV (LOG A 20. mL
Amount of Amount of POST CHANGE IN DOSE Purif. CPU Purif. CPU
REHYD. TITER) 12X894-A NONPURIF. 600 NONPURIF. 400 10.sup.7.5 +0.5
CELL-FREE CELL-FREE TOXOID TOXOID 12x894-B NONPURIF. 1200 NONPURIF.
700 10.sup.7.0 0.0 CELL-FREE CELL-FREE TOXOID TOXOID 12X894-C
PURIF. CELL- 600 PURIF. CELL- 400 10.sup.7.0 0.0 FREE FREE TOXOID
TOXOID 12X894-D PURIF. CELL- 900 PURIF. CELL- 550 10.sup.6.9 -0.1
FREE FREE TOXOID TOXOID 12X894-E PURIF. CELL- 1200 PURIF. CELL- 700
10.sup.6.7 -0.3 FREE FREE TOXOID TOXOID NOTE: The reference titer
for the IBRV rehydrated with sterile diluent was 10.sup.7.0.
[0096] The Cl. perfringens types C and D from the above
multicomponent vaccines were also tested for potency in order to
assure that the virus did not have a detrimental effect on the
clostridial protective antigen components. Results of the
clostridial testing are shown in TABLE 10. It was found that the
clostridial protective antigen components were not detrimentally
affected by the virus component. Apparently, the purification
improved the potency of the clostridial protective antigen
components, as does addition of antigen. This was evidenced by
higher CPUs producing higher rabbit antitoxin units. This example
shows that clostridial protective antigen components and virus
protective antigen components can be successfully combined to
produce effective multicomponent vaccines.
TABLE-US-00010 TABLE 10 POTENCY RESULTS OF THE Cl. Perfringens
types C and D FROM THE COMBINATION CLOSTRIDIAL VACCINE CONTAINING
IBRV Rabbit Cl. Units Antitox perf. Cl. perf. Cl. SERIAL Type C
Type D perf. Cl. perf. NO. DESCRIPTION CPU CPU type C type D
12X894-A NON-PURIFIED 600 400 20-30 3-4 CELL-FREE TOXOID 12X894-B
NON-PURIFIED 1200 700 20-30 4-5 CELL-FREE TOXOID 12X894-C PURIFIED
600 400 30-40 >5 CELL-FREE TOXOID 12X894-D PURIFIED 900 550
40-60 5-6 CELL-FREE TOXOID 12X894-E PURIFIED 1200 700 30-40 >6
CELL-FREE TOXOID
Example 7
[0097] This example shows that a larger combination of virus
protective antigen components and clostridial protective antigen
components could be successfully prepared in a low dose
formulation. Several preparations of Cl. perfringens types C and D
protective antigen components were prepared as described in Example
1B and combined with modified live IBRV, modified live bovine virus
diarrhea virus (BVDV), modified live parainfluenza type 3 virus
(Pl.sub.3) and modified live bovine respiratory syncytial virus
(BRSV). The four modified live virus protective antigen components
were prepared by art-known techniques. As part of the preparation,
the detrimental effect of the clostridial protective antigen
components on any of the modified live virus protective antigen
components was determined. Therefore, the APHIS-required viricidal
activity test was conducted on the various multicomponent vaccines.
Since clostridial vaccines historically contain residual
formaldehyde as a preservative and since it is known that
formaldehyde can have a detrimental effect on modified live
viruses, part of this experiment involved adding known amounts of
formaldehyde to the formulations to determine maximum allowable
amounts of this preservative. TABLE 11 lists the formulation
differences and the results of the viricidal activity testing for
the four virus protective antigen components. The results indicate
that the clostridial protective antigen components are somewhat
viricidal especially to IBRV and BVDV. Additionally, higher
concentrations of formaldehyde significantly reduce the titers of
these two virus whereas BRSV and Pl.sub.3V are only adversely
affected by the highest level of formaldehyde. However, it is
apparent that such a combination of clostridial protective antigen
components and modified live virus protective antigen components
would be commercially viable. This experiment also demonstrates
that purification of the clostridial protective antigen components
may not be required.
TABLE-US-00011 TABLE 11 RESULTS OF THE VIRICIDAL ACTIVITY TESTING
FOR THE COMBINATION CONTAINING MULTIPLE CLOSTRIDIAL AND VIRAL
PROTECTIVE ANTIGEN COMPONENTS BRSV IBRV TITER/ BVDV TITER/ PI.sub.3
TITER/LOG TITER/LOG LOG CHANGE LOG CHANGE CHANGE IN CHANGE IN
SERIAL DESCRIPTION IN TITER IN TITER TITER TITER 4X1594-A
NON-PURIF. Cl. 10.sup.7.6 10.sup.6.6 10.sup.7.0 10.sup.5.9
perfringens -0.1* -0.8 -0.3* -0.0* types C and D, 0.1% Form.
4x1594-B NON-PURIF. Cl. 10.sup.7.1 10.sup.6.9 10.sup.7.3 10.sup.5.8
perfringens types -0.6* -0.5* -0.0* -0.1* C and D, 0.1% Form.
4X1594-C NON-PURIF. Cl. 10.sup.6.9 10.sup.6.0 10.sup.6.9 10.sup.5.7
perfringens types -0.8 -1.4 -0.4* -0.2* C and D, 0.17% Form.
4X1594-D NON-PURIF. Cl. 10.sup.6.6 10.sup.5.7 10.sup.6.9 10.sup.5.7
perfringens types -1.1 -1.7 -0.4* -0.2* C and D, 0.25% Form.
4X1594-E NON-PURIF. Cl. 10.sup.6.0 10.sup.5.9 10.sup.6.3 10.sup.5.2
perfringens types -1.7 -1.5 -1.0 -0.7* C and D, 0.32% Form.
4X1594-F NON-PURIF. 10.sup.7.0 10.sup.6.8 10.sup.7.5 10.sup.5.7
CELL-FREE -0.7* -0.6* +0.2* -0.2* Cl. perfringens types C and D,
0.05% Form. Reference Virus titers 10.sup.7.7 10.sup.7.4 10.sup.7.3
10.sup.5.9 FORM. = Formaldehyde
Example 8
[0098] This example illustrates the safety of the vaccines of the
invention. In order to show that the described low dose,
multicomponent vaccines are actually safer for animals and would
not cause significant animal reactivity, including injection site
lesions (as routinely noted with the current 5.0 mL dose
clostridial combination products on the market) several field
safety studies were conducted. The first study involved a
comparison of injection sites from cattle injected subcutaneously
with either a 5.0 mL dose, 6-way conventional clostridial product
or a 2.0 mL dose multicomponent vaccine comprising protective
antigen components from 6 clostridial organisms (6-way clostridial
vaccine) prepared according to the methods described herein.
[0099] Two sources of yearling cattle were randomly allocated to
treatment groups of 54 head each. Two-milliliter dose 6-way
clostridial vaccine (formulated as in Example 2) was given
subcutaneously to one group and 5.0 mL dose, 6-way vaccines
formulated via conventional methods but containing the modified
carbopol adjuvant was administered subcutaneously to the other
group. The cattle were commingled throughout the trial. Evaluations
of the injection sites were made on days 7, 21, 49 and 95 days post
injection. Results are shown in FIGS. 1 and 2. On day 7, all
animals had a palpable injection site response in both groups. The
animals receiving the 2.0 mL dose multicomponent vaccine had
significantly smaller lesions than the animals receiving the 5.0 mL
dose conventional product (p=<0.0001). This difference continued
on days 21, 49 and 95. At slaughter (95 days) there were
significantly fewer (p=<0.001) 2.0 mL dose vaccinates with
lesions (3.5%) as compared to the 5.0 mL dose vaccinates with
lesions (30%). Additionally, the 2.0 mL dose vaccinates had
consistently smaller lesions at the injection sites.
[0100] In the second field safety study, calves with a known
injection history were used to evaluate the incidence and duration
of injection site lesions in carcasses from animals injected
intramuscularly. The calves were at branding and weaning age.
Forty-two steer calves and 42 heifer calves, of known history,
located at Colorado State University, were selected for the study.
These calves had received no injections prior to the beginning of
the trial and were individually identified using plastic ear tags
and assigned randomly to a product treatment group. A 5.0 mL dose
conventional 6-way clostridial product or a 2.0 mL dose 6-way
clostridial multicomponent vaccine prepared by the methods of this
invention were administered in the semimembranosus muscle (inside
round steak location) at branding using an 18 gauge, 1-inch needle.
Animals were vaccinated with the same vaccines at weaning. However,
injections were administered in the biceps femoris (top and gluteus
medium muscles (top sirloin butt location) using a 16 gauge, 1.5
inch needle. Calves were managed from birth to slaughter. Following
weaning, animals were fed a typical finishing diet. Calves were
branded at approximately 1.5 months of age, weaned at 6.5 months of
age and slaughtered at 14 months of age. At slaughter, 82.7% of the
cattle graded choice or better. Upon completion of the finishing
phase, steers were slaughtered/dressed using conventional
procedures. Following the slaughter process, the top sirloin butt
and inside round subprimal cuts were collected. From a total of 84
head, 160 inside rounds and 159 top sirloin butts were collected
after slaughter and fabrication at the packing plant. Cuts were
subjected to evaluation, dissection into one-inch strips and
observation for the presence of injection-site lesions. Results
showing the incidence of lesions, the distribution of lesions by
score and the quantity of trim required to remove the lesions are
presented in TABLES 12, 13 and 14.
TABLE-US-00012 TABLE 12 INCIDENCE OF INJECTION-SITE LESIONS AFTER
INJECTING 5.0 mL DOSE OR 2.0 mL DOSE 6-WAY CLOSTRIDIAL VACCINES
6-WAY VACCINE INCIDENCE OF LESIONS DOSE NUMBER BRANDING NUMBER
WEANING 5.0 mL 38 OF 41 92.7% 31 OF 39 79.5% 2.0 mL 29 OF 40 72.5%
19 OF 41 46.3%
TABLE-US-00013 TABLE 13 LESION CLASSIFICATION BY INJECTION TIME AND
VACCINE INJECTED 5.0 mL Dose 2.0 mL Dose 6-WAY Clostridial 6-WAY
Clostridial TYPE OF VACC. AT VACC. AT VACC. AT VACC. AT LESION
BRANDING WEANING BRANDING WEANING CALLOUSED 33 27 22 19 LESION
CLEAR 5 4 7 0 LESION MINER- 0 0 0 0 ALIZED LESION LESION 0 0 0 0
WITH NODULES LESIONS 0 0 0 0 WITH FLUID VACC = VACCINATION
TABLE-US-00014 TABLE 14 QUANTITY OF TRIM (IN GRAMS) TO REMOVE
INJECTION SITE LESIONS AFTER INJECTING 5.0 mL DOSE OR 2.0 mL DOSE
6-WAY CLOSTRIDIAL VACCINES INTRAMUSCULARLY INTO CALVES AT BRANDING
OR WEANING QUANTITY OF TRIM TO REMOVE LESION LESIONS LESIONS 6-WAY
NUMBER WHEN NUMBER WHEN VACCINE OF VACC. AT OF VACC. AT DOSE CALVES
BRANDING CALVES WEANING 5.0 mL 38 86.0 31 69.4 Conventional 2.0 mL
29 48.8 19 30.3
[0101] These results indicate that a 2.0 mL dose 6-way
multicomponent clostridial vaccine of the invention was less
reactive in calves than a 5.0 mL dose conventional technology 6-way
clostridial product. The incidence of lesions was significantly
lower (p=<0.05) for the 2.0 mL group than for the 5.0 mL group
when administration occurred at both branding and weaning times.
The blemishes resulting from use of the 5.0 mL clostridial also
necessitated more trim (p=<0.05) to remove the lesions than was
the case for those in the 2.0 mL group.
[0102] In the final field safety trial, a 2.0 mL dose vaccine
containing 6 clostridial protective antigen components combined
with protective antigen components from H. somnus was prepared
according to the methods described in Example 2 and administered to
1,528 calves by six veterinarians in five states. The field trial
was conducted from November 1994 through January 1995. Vaccine was
administered by the normal routes of administration for the herd
and included both intramuscular and subcutaneous routes.
Veterinarians were requested to observe the calves for injection
site reactions and/or lesions. At the end of the trial, no
significant unfavorable local or systemic reactions were noted by
any of the participating veterinarians.
[0103] As a result of these field safety studies, especially the
final study which involved a true field evaluation of a
commercial-size production serial, it has been demonstrated that a
multicomponent vaccine containing protective antigen components
from at least 6 clostridial organisms, protective antigen
components from at least one non-clostridial organism such as a
Gram-negative bacteria like H. somnus and an adjuvant such as
carbopol, can be produced commercially in a dose volume less than
3.0 mL and safely injected to protect animal under field
conditions.
[0104] Although the invention has been described in detail in the
foregoing for the purpose of illustration, it is to be understood
that such detail is solely for that purpose and that variations can
be made therein by those skilled in the art without departing from
the spirit and scope of the invention except as it may be limited
by the claims.
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