U.S. patent application number 11/637541 was filed with the patent office on 2007-05-10 for equine protozoal myeloencephalitis vaccine.
This patent application is currently assigned to Wyeth. Invention is credited to Rocky Barry Bigbie, Terry Kaleung Ng, Joseph Wilson JR. Whalen.
Application Number | 20070104739 11/637541 |
Document ID | / |
Family ID | 26894769 |
Filed Date | 2007-05-10 |
United States Patent
Application |
20070104739 |
Kind Code |
A1 |
Bigbie; Rocky Barry ; et
al. |
May 10, 2007 |
Equine protozoal myeloencephalitis vaccine
Abstract
The present invention provides an immunogenically active
component comprising inactivated Sarcocystis neurona cells and/or
inactivated Neospora hughesi cells; antigens derived therefrom; DNA
derived therefrom; or a mixture; or in combination with other
vaccine components thereof. Further provided are vaccine
compositions useful for preventing or ameliorating equine protozoal
myeloencephalitis infection and disease and a method for the cell
culture propagation of protozoan parasites.
Inventors: |
Bigbie; Rocky Barry; (Spring
Hill, KS) ; Ng; Terry Kaleung; (Fort Dodge, IA)
; Whalen; Joseph Wilson JR.; (Fort Dodge, IA) |
Correspondence
Address: |
WYETH;PATENT LAW GROUP
5 GIRALDA FARMS
MADISON
NJ
07940
US
|
Assignee: |
Wyeth
|
Family ID: |
26894769 |
Appl. No.: |
11/637541 |
Filed: |
December 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09840485 |
Apr 23, 2001 |
7169398 |
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11637541 |
Dec 12, 2006 |
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60278695 |
Mar 26, 2001 |
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60199435 |
Apr 25, 2000 |
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Current U.S.
Class: |
424/269.1 |
Current CPC
Class: |
A61K 2039/555 20130101;
C07K 16/20 20130101; A61K 39/002 20130101; Y10S 435/947 20130101;
A61K 2039/505 20130101; A61P 33/02 20180101; A61P 33/00 20180101;
A61K 2039/541 20130101 |
Class at
Publication: |
424/269.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Claims
1. An immunogenically active component for preventing or
ameliorating equine protozoal myoencephalitis infection or disease
which comprises: tachyzoite antibody-inducing inactivated Neospora
hughesi cells, a mixture of merozoite antibody-inducing inactivated
Sarcocystis neurona cells and tachyzoite antibody-inducing
inactivated Neospora hughesi cells, a merozoite antibody-inducing
antigen derived from Sarcocystis neurona cells, a tachyzoite
antibody-inducing antigen derived from Neospora hughesi cells, DNA
derived from Sarcocystis neurona cells, DNA derived from Neospora
hughesi cells, or a mixture thereof.
2. The component according to claim 1 which comprises the antigen
derived from Sarcocystis neurona cells, DNA derived from said cells
or the mixture thereof.
3. The component according to claim 1 which comprises inactivated
Neospora hughesi cells, the antigen derived from said cells, DNA
derived from said cells or the mixture thereof.
4. The component according to claim 1 wherein said active component
is present in sufficient quantity to provide at least
1.times.10.sup.4 inactivated cells per dosage unit form.
5. A vaccine composition which comprises an effective immunizing
amount of the immunogenically active component of claim 1, a
pharmacologically acceptable carrier; and optionally an
immunogenically stimulating adjuvant.
6. The vaccine composition according to claim 5 wherein said active
component is present in sufficient quantity to provide at least
1.times.10.sup.4 inactivated cells per dosage unit form.
7. The vaccine composition according to claim 5 wherein said active
component is present in sufficient quantity to provide at least
1.times.10.sup.6 inactivated cells per dosage unit reform.
8. The vaccine composition according to claim 5 wherein said active
component comprises the antigen derived from Sarcocystis neurona
cells, DNA derived from said cells or the mixture thereof and the
active component is present in an amount sufficient to produce a
merozoite inducing serum neutralizing antibody response which has a
neutralizing effect on Sarcocystis neurona merozoites.
9. The vaccine composition according to claim 5 wherein said active
component comprises inactivated Neospora hughesi cells, the antigen
derived from said cells, DNA derived from said cells or the mixture
thereof and the active component is present in an amount sufficient
to produce a tachyzoite inducing serum neutralizing antibody
response which has a neutralizing effect on Neospora hughesi
tachyzoites.
10. The vaccine composition according to claim 5 wherein the
immunogenically stimulating adjuvant is present at about 1% to 50%
by weight.
11. The vaccine composition according to claim 10 wherein said
adjuvant is present at about 5% to 20% by weight.
12. (canceled)
13. The vaccine composition according to claim 5 wherein said
adjuvant is a metabolizable oil.
14. The vaccine composition according to claim 13 wherein the
pharmacologically acceptable carrier is a balanced salt
solution.
15. A vaccine composition for the prevention or amelioration of
equine protozoal myoencephalitis disease in equines comprising: a
first immunogenically active component comprising merozoite
antibody-inducing inactivated Sarcocystis neurona cells, a
merozoite antibody-inducing antigen derived from said cells, DNA
derived from said cells capable of inducing a merozoite antibody
immune response or a mixture thereof, a second immunogenically
active component comprising tachyzoite antibody-inducing
inactivated Neospora hughesi cells, a tachyzoite antibody-inducing
antigen derived from said cells, DNA derived from said cells
capable of inducing a tachyzoite antibody immune response, or a
mixture thereof, a pharmacologically acceptable carrier and
optionally an immunogenically stimulating adjuvant.
16. The vaccine composition according to claim 15 wherein said
first immunologically active component comprises the inactivated
Sarcocystis neurona cells and said second immunologically effective
component comprises the inactivated Neospora hughesi cells.
17. The vaccine composition according to claim 15 wherein said
first immunologically active component is present in an amount
sufficient to produce a merozoite inducing serum neutralizing
antibody response which has a neutralizing effect on Sarcocystis
neurona merozoites, and wherein said second immunologically active
component is present in an amount sufficient to produce a
tachyzoite inducing serum neutralizing antibody response which has
a neutralizing effect on Neospora hughesi tachyzoites.
18. A method for the prevention or amelioration of equine protozoal
myoencephalitis disease in equines which comprises administering to
said equine the immunogenically active component of claim 1.
19. A method for the prevention or amelioration of equine protozoal
myoencephalitis disease in equines which comprises administering to
said equine a therapeutically effective amount of the vaccine
composition of claim 5 comprises.
20. A method for the prevention or amelioration of equine protozoal
myoencephalitis disease in equines which comprises administering to
said equine the vaccine composition of claim 15.
21. The method according to claim 19 wherein said vaccine is
administered parenterally.
22. The method according to claim 19 wherein said vaccine is
administered intramuscularly.
23. A method for the cell culture propagation of Sarcocystis
neurona or Neospora hughesi protozoan parasite which comprises: a)
growing a monolayer of cells having a confluency of 80%-100%; b)
refeeding said cells with supplemented growth media; c) inoculating
said cells with merozoites or tachyzoites; d) holding the
inoculated cells for 4-12 days; e) decanting the supplemented
growth media from the inoculated cells; and f) refeeding said cells
a second time with supplemented growth media.
24. The method according to claim 23 wherein the cells are selected
from the group consisting of Equine Dermal cells; Maiden Darby
Bovine Kidney cells; African Green Monkey Kidney cells; Canine
Monocyte cells; Mouse Monocyte cells; Fetal Rhesus Monkey Kidney
cells; Feline Kidney cells, Maiden Darby Canine Kidney cells; and
Baby Hamster Kidney cells.
25. The method according to claim 23 wherein the cells are Equine
Dermal cells or African Green Monkey Kidney cells.
Description
BACKGROUND OF THE INVENTION
[0001] Equine protozoal myeloencephalitis (EPM) is a debilitating
neurologic disease of equines which can affect the brain, the brain
stem, spinal cord or any combination of these three areas of the
equine's central nervous system. EPM is caused by the protozoan
parasites Sarcocystis neurona or Neospora hughesi.
[0002] A horse of any age, breed or gender may be affected by EPM.
The disease has been reported in two-month olds, as well as
thirty-year olds. In fact, any horse demonstrating neurologic
abnormalities may be infected. Clinical signs of a condition depend
upon the location of the organism within the central nervous
system. These signs include weakness, malposition of a limb, muscle
atrophy, spinal ataxia or the like. A severely EPM-affected horse
may go down and be unable to rise. Lameness not traceable to
orthopedic disease or any combination of the aforementioned signs
may occur in early or less severe infections.
[0003] Initially EPM was thought to only be caused by Sarcocystis
neurona. The opossum (Didelphis virginiana) has been identified as
the definitive host for this agents. The intermediate host for this
organism is still unknown. The horse ingests feed which has been
contaminated with opossum fecal material containing Sarcocystis
neurona sporocysts. These sporocysts then excyst in the intestinal
epithelium of the intermediate and incidental hosts. In the case of
the intermediate host, the merezoites would encyst in the tissues
of the host forming sarcocysts. In the case of the aberrant host,
the Sarcocystis neurona multiply in the Central Nervous System
(spinal cord) and fail to encyst. In horses, the only observed
forms of Sarcocystis neurona have been the meront or merozoite.
[0004] Recently Neospora hughesi has been identified as a second
organism which will cause the EPM clinical disease. Neospora
hughesi will not only infect the spinal cord as Sarcocystis neurona
does, but will also colonize the brain. At this point in time the
definitive and intermediate hosts for Neospora hughesi remain
unknown. It is believed that fecal contamination of horse feed or
water with sporulated oocysts is the route of horse infection. The
oocysts will release tachyzoites which will infect cells as do the
merozoites of Sarcocystis neurona.
[0005] In both cases the horse is an aberrant dead-end host and
infectious forms of the parasite are not passed from horse to horse
or from an infected horse to a definitive or true intermediate
host.
[0006] There is currently no vaccine or approved animal drug
product available for the effective treatment of EPM. The currently
available treatments are expensive, of limited efficacy and may
include adverse side effects such as anemia, abortion, diarrhea,
low white blood cell counts or the like. There remains an
unfulfilled need for treatment for EPM-afflicted equines,
particularly horses, which is effective, convenient to administer
and useful for the reduction of resistant strains.
[0007] Therefore, it is an object of this invention to provide an
immunogenically active component useful for the prevention or
amelioration of EPM.
[0008] It is another object of this invention to provide a vaccine
composition suitable for use in equines against infection and
disease caused by the protozoan parasites Sarcocystis neurona
and/or Neospora hughesi.
[0009] It is a further object of this invention to provide a method
for the prevention or amelioration of EPM disease in equines that
need such protection. Other objects and features of the invention
will become apparent from the detailed description set forth herein
below.
SUMMARY OF THE INVENTION
[0010] The present invention provides an immunogenically active
component which comprises inactivated Sarcocystis neurona cells or
inactivated Neospora hughesi cells; DNA derived therefrom; or a
mixture; or in combination with other vaccine components.
[0011] The present invention further provides an immunogenically
active component which comprises a member selected from the group
consisting of merozoite antibody inducing, inactivated Sarcocystis
neurona cells; tachyzoite antibody inducing, inactivated Neospora
hughesi cells; a merozoite or tachyzoite antibody inducing antigen
derived or extracted from said cells; DNA derived from said cells
capable of inducing a merozoite or tachyzoite antibody immune
response; and a mixture thereof.
[0012] Further provided is a vaccine composition which comprises an
effective immunizing amount of at least one of the above said
immunogenically active components and a pharmacologically
acceptable carrier.
[0013] Still further provided is a vaccine composition which
comprises a) an effective amount of one immunologically active
component selected from merozoite antibody inducing, inactivated
Sarcocystis neurona cells; a merozoite antibody inducing antigen
derived or extracted from said cells; DNA derived from said cells
capable of inducing a merozoite antibody immune response, and a
mixture thereof; b) an effective amount of a second immunologically
active component selected from tachyzoite antibody inducing,
inactivated Neospora hughesi cells; a tachyzoite antibody inducing
antigen derived or extracted from said cells; DNA derived from said
cells capable of inducing a tachyzoite antibody immune response;
and a mixture thereof; and c) a pharmacologically acceptable
carrier.
[0014] The present invention also provides a method for the
prevention or amelioration of infection or disease caused by
Sarcocystis neurona protozoa in equines that need such protection.
The method for the prevention or amelioration of EPM infection or
disease in equines comprises administering to said equine an
immunogenically active component which comprises a member selected
from the group consisting of merozoite antibody inducing,
inactivated Sarcocystis neurona cells; tachyzoite antibody
inducing, inactivated Neospora hughesi cells; a merozoite or
tachyzoite antibody inducing antigen derived from said cells; DNA
derived from said cells capable of inducing a merozoite or
tachyzoite antibody immune response; or a mixture thereof; and,
optionally, a pharmacologically acceptable carrier.
[0015] Also provided is a method for the cell culture propagation
of protozoan parasites, including Sarcocystis spp. and Neospora
spp.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Sarcocystis neurona or Neospora hughesi protozoa are the
causative agents of equine protozoal myeloencephalitis (EPM)
disease, which is a serious, and sometimes fatal, neurological
disease in equines, particularly horses. EPM symptoms include
hypermetria, decreased proprioception, weakness, cranial nerve
deficits, general ataxia or the like. The opossum has been
identified as the definitive host for these organisms. However an
intermediate host is, as yet, unknown. Equines are the aberrant
host and apparently become infected when ingesting feed which has
been contaminated with the Sarcocystis neurona or Neospora hughesi
protozoans via opossum fecal contamination. EPM disease when
untreated will progress from initial numbness of limbs to final
central nervous system destruction, resulting in death. Heretofore,
there were no known vaccination or immunization treatments
available against EPM.
[0017] Surprisingly, it has now been found that an immunogenically
active component which comprises inactivated Sarcocystis neurona
cells or antigens, subunit proteins or plasmid DNA; inactivated
Neospora hughesi cells or antigens, subunit proteins or plasmid
DNA; or mixtures thereof may be administered in the form of a
vaccine composition to prevent or ameliorate EPM disease in
equines, particularly horses. Antigens derived from Sarcocystis
neurona or Neospora hughesi may be obtained using conventional
procedures such as outer membrane extraction. Plasmid DNA derived
from Sarcocystis neurona or Neospora hughesi may be obtained via
isolation from sources such as the fluids or tissues of equine
mammalians diagnosed to have EPM. Such sources include cerebral
spinal fluid or sections of spinal cord or brain. Alternatively,
the precursor of the infectious stage in horses (sporocyst or cyst)
may be obtained from feces or intestinal scrapings of opossums or
other wild life present in endemic locales. Sarcocystis Spp. or
Neospora SPP. cells, thus obtained, may be maintained in the
infected equine or in suitable tissue culture media, such as RPMI
1640 medium or in cells known in the art such as African green
monkey kidney (Vero) cells or equine dermal (E. Derm) cells. The
Sarcocystis Spp. or Neospora Spp. protozoa may then be separated
from the tissue culture of cell media using conventional techniques
such as centrifugation, filtration, or the like. A useful starting
isolate for the vaccines of the invention include, for example, for
Sarcocystis neurona, the isolate designated SN3; other such
isolates are those known as SN1, SN2, SN4, SN5, SN6, UCD-1, UCD-2
and UCD-3 and are variously available from the University of
Kentucky, Dr. J. P Dubey at the USDA, U. of California--Davis,
Oregon State University, the University of Missouri and others. A
culture of one such Sarcocystis neurona isolate designated SNg,
originally isolated from the intestinal scrapings of the opossum
and confirmed to be a representative Sarcocystis neurona by PCR,
was deposited with the ATCC on Jan. 25, 2001, and given ATCC
Accession No. PTA-2972. A useful starting isolate for the vaccines
of the invention include, for example, for Neospora hughesi, the
isolate designated NEQ1; another such isolate is that known as NE1,
which has been described by Antoinette Marsh et al, Journal of
Parasitology, 84 (5), 1998, pp 983-991. A culture of one such
Neospora hughesi isolate has been deposited with the ATCC and given
ATCC Accession No. 209622 (NE1) as disclosed in U.S. Pat. No.
6,071,737. Surprisingly, it has now been found that protozoan
parasites such as Sarcocystis spp. or Neospora Spp. may be
propagated in increased yield and increased active viability via
cell culture propagation by growing suitable cells to a monolayer
having a confluency of about 80%-100% in a growth media; decanting
the growth media; refeeding the cells with fresh growth media;
inoculating the cells with merozoites or tachyzoites; after 4-12
days, decanting the growth media; and refeeding the inoculated
cells a second time with growth media. Cells suitable for use in
the method of the invention include cells such as E. Derm cells,
Vero cells, Maiden Darby Bovine Kidney (MDBK) cells, Canine
Monocyte (DH82) cells, Mouse Monocyte (P388) cells, Fetal Rhesus
Monkey Kidney cells, Feline Kidney (FKCU) cells, Maiden Darby
Canine Kidney (MDCK) cells, Baby Hamster Kidney (BHK21) cells, or
the like, preferably E. Derm or Vero cells, more preferably E. Derm
cells.
[0018] In actual practice, the cells are grown to a monolayer
having at least 80%, preferably 90%-100%, confluency in a growth
media such as MEM with 0.05% lacalbumin hydrosylate (LAH) or
Optimem (LTI, Gaithersburg, Md.) supplemented with 10% fetal bovine
serum, iron fortified fetal calf serum or donor serum. When the
cell monolayer has been formed, the culture is decanted to remove
the original growth media, the cells are refed with a growth media
such as RPMI 1640 with no antibiotics and 25 M .mu.hepes buffer
supplemented with 1% sodium pyruvate/2-mercaptoethanol solution
having a pH of 6.8-7.8, preferably 7.2-7.4, and up to 10% fetal
bovine serum. The refed cells are then inoculated with merozoites
or tachyzoites, held for 4 to 12 days and decanted to remove the
growth media. The culture is then refed a second time with growth
media as described above and monitored for disease progression.
When a level of cytopathology of >60% is obtained, the culture
may be harvested.
[0019] The thus obtained whole cell isolates of Sarcocystis Spp. or
Neospora Spp. protozoa may be inactivated by conventional
inactivating means, for example chemical inactivation using
chemical inactivating agents such as binary ethyleneimine,
beta-propiolactone, formalin, merthiolate, gluteraldehyde, sodium
dodecyl sulfate, or the like or a mixture thereof, preferably
formalin. Said whole cell isolates may also be inactivated by heat
or psoralen in the presence of ultraviolet light.
[0020] As used herein the term "immunogenically active" designates
the ability to stimulate an immune response, i.e., to stimulate the
production of antibodies, particularly humoral antibodies, or to
stimulate a cell-mediated response. For example, the ability to
stimulate the production of circulating or secretory antibodies or
the production of a cell-mediated response in local mucosal
regions, i.e., intestinal mucosa, peripheral blood, cerebral spinal
fluid or the like.
[0021] The immunogenically active component of the invention may be
incorporated into liposomes using known technology such as that
described in Nature, 1974, 252, 252-254 or Journal of Immunology,
1978, 120, 1109-13. Further, the immunogenically active component
of the invention may be conjugated to suitable biological compounds
such as polysaccharides, peptides, proteins, or the like, or a
combination thereof.
[0022] Advantageously, the immunogenically active component of the
invention may be formulated as a vaccine composition in dosage unit
form to facilitate administration and insure uniformity of dosage.
The vaccine composition of the invention comprises an effective
immunizing amount of the immunogenically active component described
hereinabove, a pharmacologically acceptable carrier and optionally
an immunogenically stimulating adjuvant. The effective immunizing
amount of the immunogenically active component may vary and may be
any amount sufficient to evoke an immune response. Amounts wherein
the dosage unit comprises at least about 1.times.10.sup.4
inactivated Sarcocystis Spp. cells or Neospora Spp. cells or a
mixture thereof, preferably at least about 1.times.10.sup.6 cells,
are suitable.
[0023] As used in the specification and claims, the term
"immunogenically stimulating adjuvant" designates a compound which
is capable of potentiating or stimulating the immune response in a
subject animal when administered in combination with the
immunogenically active component of the invention. Examples of an
immunogenically stimulating adjuvant suitable for use in the
vaccine composition of the invention include: surfactants such as
hexadecylamine, octadecylamine, lysolecithin, dimethyl dioctadicyl
ammonium bromide, N,N-dioctadecyl-N'-N-bis(2-hydroxyethyl-propane
diamine), methoxyhexadecylglycerol, PLURONIC polyols, saponin,
Quil.RTM. A, or the like; polyanions such as pyran, dextran
sulfate, polynucleotide complex of polyinosinicpolycytidylic acid,
polyacrylic acid, carboxypolymethylenes and carboxyvinyl polymers
such as CARBOPOL.RTM., aluminum hydroxide, aluminum phosphate, or
the like; peptides such as muramyl dipeptide, dimethyl glycine,
tuftsin or the like; oil emulsions; immunomodulators such as
interleukin-1, interleukin-2, interleukin-12, GM-CSF or the like;
or a combination thereof. A preferred immunogenically stimulating
adjuvant suitable for use in the vaccine composition of the
invention is a mixture of squalane and a
polyoxyethylene-polyoxypropylene block copolymer (e.g.,
Pluronic.RTM. L121, BASF, Parsippany, N.J.) capable of forming
small liposomes. The immunogenically stimulating adjuvant may be
present in the vaccine composition of the invention in wt/wt
amounts of about 1% to 50%, preferably about 5% to 20%.
[0024] Pharmacologically acceptable carriers suitable for use in
the vaccine composition of the invention may be any conventional
liquid carrier suitable for veterinary pharmaceutical compositions,
preferably a balanced salt solution suitable for use in tissue
culture media.
[0025] In addition to the immunogenically active component as
active ingredient, it is contemplated the vaccine composition of
the invention may also contain other active components such as an
antipathogenic component directed against rabies virus, Eastern
equine encephalitis virus, Western equine encephalitis virus,
Venezuelan equine encephalitis virus, equine herpes virus such as
EHV-1 or EHV-4, Ehrlichia risticii, Streptococcus equi, tetanus
toxoid, or the like or a combination thereof.
[0026] The inventive vaccine composition may be administered
parenterally, for example, intramuscularly, subcutaneously,
intraperitoneally, intradermally or the like, preferably
intramuscularly; or said composition may be administered orally or
intranasally.
[0027] The vaccine composition of the invention is useful for the
prevention or amelioration of EPM infections in equine that need
such protection. In actual practice, the vaccine composition of the
invention is administered parenterally, orally, or intranasally,
preferable parenterally, more preferably intramuscularly, in
effective amounts according to a schedule determined by the time of
potential exposure to infective Sarcocystis Spp. or Neospora Spp.
sporocysts. In this way, the treated animal may have time to build
immunity prior to natural exposure. For example, a typical
treatment schedule may include parenteral administration,
preferably intramuscular injection, at least 5-8 weeks prior to
potential exposure. At least two administrations are preferred, for
example one at about 8 weeks and a second at about 3 weeks prior to
potential exposure of the treated animal.
[0028] For a more clear understanding of the invention, the
following examples are set forth below. These examples are merely
illustrative and are not understood to limit the scope or
underlying principles of the invention in any way. Indeed, various
modifications of the invention, in addition to those shown and
described herein, will become apparent to those skilled in the art
from the following examples and the foregoing description. Such
modifications are also intended to fall with the scope of the
appended claims.
[0029] Unless otherwise noted, all parts are parts by weight.
EXAMPLE 1
A--Vaccine Preparation
[0030] An equine spinal cord isolate of Sarcocystis neurona is
obtained from a horse which has been diagnosed to have EPM. The
isolate is cultivated in multiple cultures of E. Derm cells in RPMI
tissue culture medium at 37.degree. C. These merozoite harvests are
counted at the time of harvest and then inactivated by means of
addition of a 10% formalin solution to a final concentration of
0.05%. This is allowed to inactivate at 37.degree. C. for a period
of no less than 48 hours.
[0031] To remove unnecessary serum proteins associated with tissue
culture the harvests are pooled and diafiltrated/concentrated
against 0.01M phosphate buffered saline to a level of
3.14.times.10.sup.7 merozoites per mL.
[0032] The vaccines are formulated by suspending the appropriate
volume of inactivated cells in an adjuvant containing 1-20% by
volume of a metabolizable oil adjuvant per 1 mL dose, e.g., 5% of
the stock adjuvant described herinbelow.
B--Formulation of A Preferred Stock Adjuvant
[0033] A preferred adjuvant for use in the present invention was
prepared according to the following formulation: TABLE-US-00001
Polyoxyethylene-polyoxypropylene block copolymer 20 ml (e.g.,
Pluronic .RTM. L121, BASF, Parsippany, NJ) Squalane (e.g. Kodak,
Rochester, NY) 40 ml Polyoxyethylenesorbitan monooleate 3.2 ml
(e.g., Tween .RTM. 80, Sigma Chemical, St. Louis, MO) buffered salt
solution 936.8 ml (e.g., D-V PAS Solution, Ca, Mg free)
The ingredients are mixed and homogenized until a stable mass or
emulsion is formed. Prior to homogenization, the ingredients or
mixture can be autoclaved. The emulsion may be further sterilized
by filtration. Formalin may be added up to a final concentration of
0.2%. Thimerosal may be added to a final dilution of 1:10,000.
EXAMPLE 2
[0034] Antibody response to intramuscular injection of vaccine In
this evaluation, horses that are found to be naive to Sarcocystis
neurona merozoite antigen by means of Indirect Fluorescent Antibody
(IFA) testing are employed. Horses are randomly divided into four
groups: one group of nine horses are administered vaccine at the
level of 1.times.10.sup.5 merozoites per dose; a second group of
twenty-one horses are administered vaccine blended at
1.times.10.sup.6 merozoites per dose; a third group of ten horses
are administered vaccine at 1.times.10.sup.7 merozoites per dose;
and a fourth of group of ten horses are maintained as
non-vaccinated environmental controls. Treated horses are given a
first dose of vaccine according to the group to which they are
assigned. At twenty-one days following administration of the first
dose, a second dose of the same vaccine is administered. All horses
are bled for serum at the time of administration of the first and
second dose and at weekly intervals through 28 days post second
dose administration.
[0035] In this evaluation, the vaccine compositions contain
formalin-inactivated, E. Derm cell line-grown Sarcocystis neurona
merozoites with an adjuvant system. The method of serologic
measurement of antibodies is conducted by IFA. The IFA is run using
Vero cell line-grown Sarcocystis neurona merozoites to eliminate
anti-E. Derm antibody titers.
[0036] The serological data is shown in Table I below, wherein: 0
DPV 1 designates day zero, pre vaccination; 0 DPV 2 designates day
zero, post vaccination; 7 DPV 2 designates day 7, post vaccination;
and 14 DPV 2 designates day 14, post vaccination.
[0037] As can be seen from the data on Table I, treated horses from
all groups showed significant increases in antibodies to
Sarcocystis neurona merozoites while the control horses maintained
a low to non-existent antibody level. The level of response in the
horses that received vaccine was dependent upon the level of
antigen in the vaccine that they received. TABLE-US-00002 TABLE I
EPM (Sarcocystis neurona) Dose Titration IFA Serology 0 DPV 7 DPV
14 DPV No. Vaccine Antigen Load 0 DPV 1 2 2 2 1 1 1 .times.
10{circumflex over ( )}5 mer. <1:10 1:80 1:640 1:800 2 1 1
.times. 10{circumflex over ( )}5 mer. <1:10 1:480 1:480 1:3200 3
1 1 .times. 10{circumflex over ( )}5 mer. <1:10 1:40 1:320 1:800
4 1 1 .times. 10{circumflex over ( )}5 mer. <1:10 1:160 1:320
1:3200 5 1 1 .times. 10{circumflex over ( )}5 mer. <1:10 1:320
1:480 1:3200 6 1 1 .times. 10{circumflex over ( )}5 mer. <1:10
1:320 1:160 1:1600 7 1 1 .times. 10{circumflex over ( )}5 mer.
<1:10 1:40 1:80 1:400 9 1 1 .times. 10{circumflex over ( )}5
mer. <1:10 1:320 1:160 1:800 10 1 1 .times. 10{circumflex over (
)}5 mer. <1:10 1:480 1:640 1:4800 GMT <1:10 1:211 1:300
1:1550 11 2 1 .times. 10{circumflex over ( )}6 mer. <1:10 1:640
1:960 1:4800 12 2 1 .times. 10{circumflex over ( )}6 mer. <1:10
1:960 1:1920 1:4800 13 2 1 .times. 10{circumflex over ( )}6 mer.
<1:10 1:160 1:240 1:800 14 2 1 .times. 10{circumflex over ( )}6
mer. <1:10 1:640 1:1280 1:4800 15 2 1 .times. 10{circumflex over
( )}6 mer. <1:10 1:1280 1:2560 1:12800 16 2 1 .times.
10{circumflex over ( )}6 mer. <1:10 1:320 1:1280 1:4800 17 2 1
.times. 10{circumflex over ( )}6 mer. <1:10 1:320 1:640 1:3200
18 2 1 .times. 10{circumflex over ( )}6 mer. <1:10 1:1280 1:960
1:3200 19 2 1 .times. 10{circumflex over ( )}6 mer. <1:10 1:160
1:960 1:4800 20 2 1 .times. 10{circumflex over ( )}6 mer. <1:10
1:320 1:640 1:800 21 2 1 .times. 10{circumflex over ( )}6 mer.
<1:10 1:320 1:640 1:4800 22 2 1 .times. 10{circumflex over ( )}6
mer. <1:10 1:960 1:1920 1:4800 23 2 1 .times. 10{circumflex over
( )}6 mer. <1:10 1:640 1:1280 1:4800 24 2 1 .times.
10{circumflex over ( )}6 mer. <1:10 1:1280 1:2560 1:3200 25 2 1
.times. 10{circumflex over ( )}6 mer. <1:10 1:640 1:1280 1:4800
26 2 1 .times. 10{circumflex over ( )}6 mer. <1:10 1:1280 1:960
1:3200 27 2 1 .times. 10{circumflex over ( )}6 mer. <1:10 1:1280
1:1280 1:3200 28 2 1 .times. 10{circumflex over ( )}6 mer. <1:10
1:1280 1:1280 1:4800 29 2 1 .times. 10{circumflex over ( )}6 mer.
<1:10 1:2560 1:960 1:4800 30 2 1 .times. 10{circumflex over (
)}6 mer. <1:10 1:2560 1:2560 1:3200 31 2 1 .times. 10{circumflex
over ( )}6 mer. <1:10 1:1280 1:1280 1:3200 GMT <1:10 1:734
1:1147 1:3704 32 3 1 .times. 10{circumflex over ( )}7 mer. <1:10
1:1280 1:1920 1:3200 33 3 1 .times. 10{circumflex over ( )}7 mer.
<1:10 1:640 1:1280 1:4800 34 3 1 .times. 10{circumflex over (
)}7 mer. <1:10 1:640 1:1280 1:1600 35 3 1 .times. 10{circumflex
over ( )}7 mer. <1:10 1:2560 1:2560 1:4800 36 3 1 .times.
10{circumflex over ( )}7 mer. <1:10 1:2560 1:5120 1:4800 37 3 1
.times. 10{circumflex over ( )}7 mer. <1:10 1:2560 1:5120 1:4800
38 3 1 .times. 10{circumflex over ( )}7 mer. <1:10 1:1280 1:1280
1:12800 39 3 1 .times. 10{circumflex over ( )}7 mer. <1:10
1:2560 1:7680 1:19200 40 3 1 .times. 10{circumflex over ( )}7 mer.
<1:10 1:1920 1:2560 1:19200 41 3 1 .times. 10{circumflex over (
)}7 mer. <1:10 1:1280 NS 1:12800 GMT <1:10 1:1429 1:2296
1:6630 42 4 Control <1:10 <1:10 <1:10 <1:10 43 4
Control <1:10 <1:10 <1:10 <1:10 44 4 Control <1:10
<1:10 <1:10 <1:10 45 4 Control <1:10 <1:10 <1:10
<1:10 46 4 Control <1:10 <1:10 <1:10 <1:10 47 4
Control <1:10 <1:10 <1:10 <1:10 48 4 Control <1:10
<1:10 <1:10 <1:10 49 4 Control <1:10 <1:10 <1:10
<1:10 50 4 Control <1:10 <1:10 <1:10 <1:10 51 4
Control <1:10 <1:10 <1:10 <1:10 GMT <1:10 <1:10
<1:10 <1:10 Sample Type Material IFA Titer MSU1 Positive
Control Sera 1:800 MSU2 Positive Control Sera 1:800 Blakely
Positive Control Sera 1:400 Sport Positive Control Sera 1:160
EXAMPLE 3
Plaque Reduction Effect as Determined by Serum of Vaccinated
Horses
[0038] In this evaluation, an assay is performed to determine if
the Sarcocystis neurona antibody found by IFA in the serum of EPM
vaccinated horses would have a neutralizing effect on Sarcocystis
neurona merozoites at varying levels of the organism.
[0039] Horse serum samples are collected at 14 days post second
vaccination from the Example 2 study group 3, which received
vaccine containing 1.times.10.sup.7 merozoites per dose; and the
samples are pooled. Duplicate sets of this serum are diluted 1:2 to
a 1.0 mL volume and are mixed with 1.0 mL volumes of varying levels
of viable Sarcocystis neurona merozoites, resulting in a final
serum dilution of 1:4. The organism (merozoite) levels used 1:10
are 2.5.times.10.sup.5, 1:100 are 2.5.times.10.sup.4, and 1:1000
are 2.5.times.10.sup.3 merozoites per mL. Duplicate sets of
serum/organism tubes are set up using a serum pool from the group 4
non-vaccinated horses to stand as a negative control group for
comparison. The 2.0 mL organism/serum mixtures are incubated for 1
hour at 37.degree. C. and then added to 25 cm.sup.2 of E. Derm
cells with the appropriate media to support Sarcocystis neurona. At
14 days post inoculation all flasks are fixed using a 10%
formalin/crystal violet stain and are counted for the number of
plaques present in each flask.
[0040] There was a marked reduction in the number of plaques
observed in the flasks which had received the serum from the group
3 vaccinate horses which had been incubated with organism at all
organism dilutions when compared to similar flasks which had the
non-vaccinated control serum. This data is shown in Table II
below.
[0041] As can be seen from the data in Table II, the degree of
plaque reduction in every case of the vaccinated horse serum pools
exceeded 70%. TABLE-US-00003 TABLE II Sarcocystis neurona Plaque
Reduction Serology Organism Serum Plaques Average No. Percent
Dilution Sample Dilution Observed of Plaques Reduction.sup.2 1:10
Vaccine 3 1:4 87 97.0 89.22 1:10 Vaccine 3 1:4 107 1:100 Vaccine 3
1:4 16 14.0 73.33 1:100 Vaccine 3 1:4 12 1:1000 Vaccine 3 1:4 2 1.5
85.00 1:1000 Vaccine 3 1:4 1 1:10 Controls 1:4 TNTC.sup.1 TNTC NA
1:10 Controls 1:4 TNTC 1:100 Controls 1:4 55 52.5 NA 1:100 Controls
1:4 50 1:1000 Controls 1:4 12 10.0 NA 1:1000 Controls 1:4 8
.sup.1TNTC estimated to be 900-1000 plaques (900 used for
calculations). .sup.2Percent Reduction as compared to the number of
plaques in the corresponding control serum dilution plaque
count.
EXAMPLE 4
Vaccine Preparation
[0042] Neospora hughesi is obtained from the brain or spinal column
of a horse that has been diagnosed to have EPM. The isolate is
cultivated in multiple cultures of E. Derm or Vero cells in RPMI
tissue culture medium at 37.degree. C. The tachyzoites harvested
are counted at the time of harvest and then inactivated by means of
addition of a 10% formalin solution to a final concentration of
0.05%. This is allowed to inactivate at 37.degree. C. for a period
of no less than 48 hours.
[0043] To possibly remove unnecessary serum proteins associated
with tissue culture the harvests are pooled and may be
diafiltrated/concentrated against 0.01M phosphate buffered saline
to a suitable level of tachyzoites per mL for final vaccine
formulation.
[0044] The vaccine is formulated with antigen as in Example 1.
EXAMPLE 5
Cell Culture Propagation of Sarcocystis spp. and Neospora spp.
[0045] Equine dermal (E. derm) cells that have been grown to
achieve a monolayer of 90-100% confluency are decanted to remove
the original cell growth media (OptiMEM supplemented with 10% fetal
bovine serum). The E. derm cells are then refed with RPMI 1640
media supplemented with 1% sodium pyruvate/2-mercaptoethanol.sup.1
solution having a pH of 7.2-7.4 and 10% fetal bovine serum and
inoculated with viable merozoites or tachyzoites. After 4-12 days,
the resultant culture is decanted to remove the growth media and
then refed a second time with RPMI 1640 media supplemented with 1%
sodium pyruvate/2-mercaptoethanol solution.sup.1 having a pH of
7.2-7.4 and 2%-10% bovine fetal serum. The resultant culture is
then monitored for disease progression and when a level of greater
than 60% cytopathology is obtained the culture is harvested.
.sup.1The sodium pyruvate/2-mercaptoethanol solution consists of
0.175 mL 2-mercaptoethanol and 0.600 g sodium pyruvate in 500 mL of
RPMI 1640 media (pH 7.2-7.4) which has been sterile filtered.
* * * * *