U.S. patent application number 10/115478 was filed with the patent office on 2002-10-10 for neospora vaccines.
Invention is credited to Brown, Karen K., Choromanski, Leszek J..
Application Number | 20020146436 10/115478 |
Document ID | / |
Family ID | 25495565 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020146436 |
Kind Code |
A1 |
Choromanski, Leszek J. ; et
al. |
October 10, 2002 |
Neospora vaccines
Abstract
A Neospora caninum vaccine comprising tissue culture grown
Neospora and methods of making and using said vaccines. Neospora
caninum vaccines described include those containing whole Neospora
tachyzoites, extracts of Neospora tachyzoites and protective
antigen subunits of Neospora tachyzoites. The vaccines of this
invention may be inactivated or modified live and contain adjuvants
and/or stabilizers. The vaccines of this invention may be in a
liquid or lyophilized form.
Inventors: |
Choromanski, Leszek J.;
(Lenexa, KS) ; Brown, Karen K.; (Parkville,
MD) |
Correspondence
Address: |
INTERVET INC
405 STATE STREET
PO BOX 318
MILLSBORO
DE
19966
US
|
Family ID: |
25495565 |
Appl. No.: |
10/115478 |
Filed: |
April 2, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10115478 |
Apr 2, 2002 |
|
|
|
08954531 |
Oct 20, 1997 |
|
|
|
Current U.S.
Class: |
424/269.1 ;
435/258.1 |
Current CPC
Class: |
A61P 33/02 20180101;
C12N 1/105 20210501; C12R 2001/90 20210501; A61K 39/002
20130101 |
Class at
Publication: |
424/269.1 ;
435/258.1 |
International
Class: |
A61K 039/002; C12N
001/10 |
Claims
What is claimed is:
1. A Neospora caninum vaccine comprising tissue culture grown
Neospora.
2. The Neospora caninum vaccine according to claim 1 wherein the
tissue culture grown Neospora comprises an antigen selected from
the group consisting of a whole culture of Neospora tachyzoites, an
inactivated tissue culture of Neospora tachyzoites, a modified live
tissue culture Neospora tachyzoites, an extract from Neospora
tachyzoites and one or more subunits obtained from Neospora
tachyzoites.
3. The Neospora caninum vaccine according to claim 1 further
comprising an inactivating agent and an adjuvant.
4. The Neospora caninum vaccine according to claim 3 wherein the
inactivating agent is selected from the group consisting of
formalin, beta-propiolactone, heat, binary ethylenimine, detergents
and means for freezing/thawing.
5. The Neospora caninum vaccine according to claim 3 wherein the
adjuvant is selected from the group consisting of polymers, oil in
water, water-in-oil-in-water, lipids, aluminum hydroxide, aluminum
phosphate, aluminum sulfate, immunomodulators and combinations
thereof.
6. The Neospora caninum vaccine according to claim 5 wherein the
polymer adjuvant is selected from the group consisting of Carbopol,
HAVLOGEN.RTM. and POLYGEN.RTM..
7. The Neospora caninum vaccine according to claim 5 wherein the
oil-in-water adjuvant is selected from the group consisting of
EMULSIGEN.RTM. and EMULSIGEN PLUS.RTM..
8. The Neospora vaccine according to claim 1 wherein the Neospora
is modified live.
9. A method for growing a Neospora caninum in a susceptible tissue
culture to an amount sufficient to protect mammals against
infection or abortion caused by said Neospora caninum, comprising
inoculating the Neospora caninum onto said tissue culture and
harvesting the replicated Neospora caninum.
10. A method of producing a Neospora vaccine comprising the steps
of: a. growing Neospora caninum in a susceptible tissue culture
until a cytopathic effect is produced; b. harvesting said tissue
culture grown Neospora caninum; and c. formulating said harvest
into a vaccine.
11. A method of producing a Neospora vaccine comprising the steps
of: a. growing Neospora caninum in a susceptible tissue culture
until a cytopathic effect is produced; b. harvesting said tissue
culture grown Neospora caninum; c. inactivating said harvested
tissue culture grown Neospora caninum; and d. adjuvanting the
inactivated harvested tissue culture grown Neospora caninum to
produce a vaccine.
12. A method of producing a Neospora subunit vaccine comprising the
steps of: a. growing Neospora caninum in a susceptible tissue
culture until a cytopathic effect is produced; b. harvesting said
tissue culture grown Neospora caninum; c. extracting one or more
protective antigens from the harvested tissue culture grown
Neospora caninum to produce a subunit; d. inactivating the
subunit(s), optionally; and e. adjuvanting the subunit(s) to
produce a vaccine.
13. A method of producing a Neospora subunit vaccine comprising the
steps of: a. growing Neospora caninum in a susceptible tissue
culture until a cytopathic effect is produced; b. harvesting said
tissue culture grown Neospora caninum; c. inactivating the Neospora
caninum harvest; d. extracting one or more protective antigens from
the harvested, inactivated tissue culture grown Neospora caninum to
produce a subunit(s); and e. adjuvanting the subunit(s) to produce
a vaccine.
14. A method of protecting mammals from disease caused by Neospora
caninum comprising, administering to said mammals the vaccine
according to claims 1-8.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a vaccine for protection of
mammals from disease caused by Neospora caninum. More specifically,
the invention relates to safe and immunogenically effective
vaccines for protection of bovines and canines from abortion caused
by Neospora caninum.
[0003] 2. Brief Description of the Prior Art
[0004] Neospora caninum was first reported by Dubey et al (JAVMA,
Vol. 192, No. 9, May 1, 1988) as a Toxoplasmosis-like illness
affecting dogs. Neospora caninum was found to be structurally
distinct from Toxoplasma gondii and did not react with anti-T.
gondii antiserum in an immunoperoxidase test. Dubey et al described
the major lesions associated with the organism as
meningoencephalomyelitis and myositis. Within the past few years,
neosporosis has become recognized as a major reproductive disease
in cattle (Anderson et al., 1994, Food Animal Practice, 10:
439-461) with cases reported in North and South America, Europe,
Africa, the Pacific-rim countries as well as in the United States.
The major clinical manifestation of bovine neosporosis is fetal
abortion, with focal nonsuppurative necrotizing encephalitis,
nonsuppurative myocarditis, and myositis in the fetus (Anderson et
al., 1991, Journal of the American Veterinary Medical Association,
198: 241-244). According to Anderson et al., 1997 (Journal of the
Veterinary Medical Association, 210: 1169-1172), retrospective
studies of cattle in California indicate that neosporosis has been
endemic since at least 1985. These authors state that 18 to 19% of
all aborted bovine fetuses submitted to the California Veterinary
Diagnostic Laboratory System have Neospora sp infection. In a
prospective survey of selected dairies in California, the number of
abortions attributed to Neospora sp infections was even greater
(42.5%).
[0005] Ho et al (J. Parasitol., 1997, 83(3)) have recently reported
the successful reproduction of bovine abortion and fetal infection
by infecting pregnant cows with tachyzoites of Neospora caninum.
This publication suggests that there may be a correlation between
serological titer as measured by indirect fluorescent antibody
(IFA) testing and protection from abortion caused by Neospora
caninum in cows. Cows with IFA titers of 320 and 640 did not abort
after infection with tachyzoites of this organism.
[0006] As mentioned previously, neosporosis has also been reported
in puppies and in dogs as old as 15 years of age. The percentage of
infected dogs that show clinical signs is unknown. In dogs,
Neospora caninum can infect any tissue, although it is most
commonly found in the central nervous system and spinal nerve
roots. The most severe infections are seen in puppies that were
infected in utero. These puppies exhibit ascending paralysis.
Abortion can be reproduced in experimental infection of pregnant
bitches during the early stage of gestation. Sulfonamides,
pyrimethamine and clindamycin have been used to treat neosporosis
in dogs.
[0007] Neospora caninum can also produce a fatal infection in
experimentally inoculated cats. However, the disease has not yet
been reported to occur naturally in cats.
[0008] Neosporosis has been observed to cause abortion in sheep and
goats but to a lesser extent than is found in cattle. Experimental
infection is readily induced in sheep and goats by subcutaneous
injection of tachyzoites.
[0009] Although neosporosis, especially in cattle, appears to pose
an increasingly serious problem and there is certainly a long felt
need to solve this problem by protecting mammals using a vaccine,
there are no descriptions of vaccines, vaccine development nor
suggestions of methods of preparing vaccines to protect cattle and
other animals from disease caused by Neospora caninum.
SUMMARY OF THE INVENTION
[0010] It is a focus of this invention to describe a vaccine
composition for protection of mammals from disease caused by
Neospora caninum comprising tissue culture grown Neospora caninum
tachyzoites as a whole culture or in an extract form or as subunit
antigens obtained therefrom. In addition, it is a focus of this
invention to describe a method of producing a vaccine for
protection of mammals from disease caused by Neospora caninum
comprising the steps of: growing Neospora caninum in a susceptible
tissue culture until a cytopathic effect (CPE) is produced,
harvesting said tissue culture grown Neospora caninum and
formulating said harvest into a vaccine. A modified live vaccine
produced in this manner can be administered to mammals without
inactivating the Neospora sp. However, said non-inactivated
Neospora sp would need to be attenuated by art-known techniques
prior to growing it in tissue culture. Another method of producing
a vaccine for protection of mammals from disease caused by Neospora
caninum comprises the steps of: 1) growing Neospora caninum in a
susceptible tissue culture until a CPE is produced; 2) harvesting
said tissue culture grown Neospora caninum; 3) inactivating said
harvested tissue culture grown Neospora caninum; and 4) adjuvanting
the inactivated harvested tissue culture grown Neospora caninum to
produce a vaccine. Still another method of producing a vaccine for
protection of mammals from disease caused by Neospora caninum
comprises the steps of: 1) growing Neospora caninum in a
susceptible tissue culture until a CPE is produced; 2) harvesting
said tissue culture grown Neospora caninum; 3) extracting
protective antigens from the harvested tissue culture grown
Neospora caninum to produce subunits; 4) inactivating the subunits
if necessary; and 5) adjuvanting the subunits to produce a vaccine.
It is within the scope of this invention to inactivate the Neospora
caninum prior to extraction of the protective antigen subunits
DETAILED DESCRIPTION OF INVENTION
[0011] As set forth above, the present invention is directed to
vaccine compositions comprising a modified live Neospora caninum
grown in a susceptible tissue culture or an inactivated, adjuvanted
Neospora caninum grown in a susceptible tissue culture or subunits
derived from Neospora caninum. The method of producing the above
vaccine compositions comprises growing Neospora caninum under
artificial conditions, in tissue culture, for the purpose of
obtaining parasite antigens for use in vaccines. The Neospora
caninum can be obtained from any source. It is preferred that a
vaccine for bovines contain a Neospora caninum isolated from an
aborted bovine fetus. Additionally, it is preferred that a vaccine
intended for canines contain a Neospora caninum isolated from a
canine. Illustratively, the brain of an infected fetus is
harvested, homogenized in a growth medium such as Minimal Essential
Media (MEM) or in a diluent such as phosphate buffered saline (PBS)
supplemented with antibiotics to minimize the potential for
contamination. Such a homogenate is centrifuged to remove the large
particulate matter and the supernate is inoculated onto various
tissue cultures and passaged in tissue cultures, if necessary,
until a cytopathic effect (CPE) is produced on at least one tissue
culture. The tissue culture is preferably a cell line in which the
parasite grows to a high titer so that a Master Seed can be
prepared. A high titer means that the parasite tachyzoites grow to
produce a count, as visualized under a microscope, or a titer when
placed back into tissue culture of at least 1.times.10.sup.4 tissue
culture infective dose.sub.50/mL (TCID.sub.50/mL). Preferably,
1.times.10.sup.5 TCID.sub.50/mL are produced and more preferably,
1.times.10.sup.6 TCID.sub.50/mL are produced. A Master Seed means
that the tissue culture grown Neospora sp is grown to a high titer,
aliquoted into equivalent volumes in freezing vials and frozen,
after which it is tested for freedom from contaminants (bacteria,
fungi and viruses) and then used to prepare Working Seeds and
Production Seeds. Working Seeds and Production Seeds mean further
passage of the Master Seed in a susceptible tissue culture,
aliquoting, freezing and repeat testing so that vaccines can be
produced from the Production Seeds instead of using the Master Seed
and all vaccine is prepared from the same origin material. A
susceptible tissue culture means a tissue culture that, when
inoculated with Neospora sp, is able to grow the parasite
tachyzoites and produce CPE.
[0012] At least three types of vaccines can be made according to
this invention, a modified live vaccine, an inactivated vaccine or
a subunit vaccine. If a modified live vaccine is to be made, the
Neospora sp must be mutated or genetically modified so that the
parasite loses its virulence, by art-known techniques including but
not limited to chemical mutagenesis and genetic engineering, prior
to making the Master Seed. Once the non-virulent (avirulent) mutant
is prepared, a Master Seed is made by growing the mutated Neospora
sp in a susceptible tissue culture and frozen as described above.
Preparation of a modified live vaccine comprises the steps of
growing the mutated Neospora caninum in a susceptible tissue
culture until CPE is produced, harvesting the tissue culture grown
Neospora sp and formulating said harvest into a vaccine.
Formulation may include addition of stabilizers and/or adjuvants or
immunomodulators. The vaccine may remain in a liquid form or may be
lyophilized.
[0013] The method for preparation of an inactivated Neospora
caninum vaccine requires that the organism be grown to higher titer
and comprises the steps of growing Neospora caninum in a
susceptible tissue culture until a CPE is produced, harvesting said
tissue culture grown Neospora caninum, inactivating said harvested
tissue culture grown Neospora caninum; and adjuvanting the
inactivated harvested tissue culture grown Neospora caninum to
produce a vaccine.
[0014] The method for preparation of a subunit Neospora caninum
vaccine comprises the steps of growing Neospora caninum in a
susceptible tissue culture until a CPE is produced, harvesting said
tissue culture grown Neospora caninum, extracting protective
antigens from the harvested tissue culture grown Neospora caninum
to produce protective antigen subunits, inactivating the subunits
if necessary; and adjuvanting the subunits to produce a vaccine. It
is within the scope of this invention to inactivate the Neospora
caninum prior to extraction of the protective antigen subunits in
order to prepare a subunit vaccine.
[0015] Inactivating agents may be selected from the group
consisting of formalin, beta-propiolactone (BPL), heat, binary
ethylenimine (BEI), detergents and freeze/thaw with the preferred
inactivating agents being BEI and BPL.
[0016] Adjuvants used to increase the immunogenicity of the
Neospora vaccines of this invention may be selected from the group
consisting of polymers such as Carbopol, HAVLOGEN.RTM. and
POLYGEN.RTM., oil in water such as EMULSIGEN.RTM. and EMULSIGEN
PLUS.RTM., water-in-oil-in-water, aluminum hydroxide, aluminum
phosphate, aluminum sulfate, immuno-modulators such as
BAYPAMUN.RTM., lipid based adjuvants such as Bay R-1005 and
liposomes and combinations thereof.
[0017] The inactivated Neospora vaccines of this invention may
include stabilizers which are added before or after adjuvanting in
order to maintain the antigen content over long periods of time and
under adverse conditions of high or low temperatures. Stabilizers
are selected from the group consisting of protease inhibitors,
sugars such as sucrose and glycerol, encapsulating polymers,
chelating agents such as ethylene-diaminetetracetic acid (EDTA),
proteins and polypeptides such as gelatin and polyglycine and
combinations thereof.
[0018] The examples to follow represent compositions of Neospora
caninum vaccines and describe their methods of production including
growing the tachyzoites of this organism in such diverse cell lines
as an Equine Dermal cell line (ATCC No.CCL-57), a Vero cell line
and an African Green Monkey kidney cell line (BIOWHITTAKER No.
75-104) which was cloned at Bayer Corporation to produce a cell
line designated MA 104 Clone B as well as describing their use in
vaccinating bovines to produce protective indirect fluorescent
antibody (IFA) titers.
[0019] The invention is further illustrated but is not intended to
be limited by the following examples in which all parts and
percentages are by weight unless otherwise specified.
EXAMPLES
Example 1
[0020] In order to determine whether Neospora caninum vaccines can
produce protection against abortion in pregnant cows in a model
known in the art (Ho et al., 1997), the inventors produced Neospora
caninum vaccines by growing the Neospora caninum on a Vero cell
line in 850 cm.sup.2 roller bottles. A vial of Working Cells of the
Vero cell line was removed from liquid nitrogen storage, thawed
rapidly, diluted and placed into 850 cm.sup.2 roller bottles
containing 250 mL of DMEM (high glucose), hereinafter designated
DMEMH, at a rate of 4.times.10.sup.7 cells per roller. The medium
was supplemented with Neomycin Sulfate at 1 mL/L and Horse Serum at
5% v/v. Cells were incubated at 36 to 38.degree. C. for 5 to 7 days
until the cells were between 95 and 100% confluent. The Working
Cells were removed from the roller bottles by rinsing the cell
sheet with Phosphate Buffered Saline (PBS) and then adding 10 mL of
a Trypsin-Ethylene-diaminetetracetic acid disodium salt (EDTA)
solution (2.5 g/L of Trypsin+1 g/L EDTA) to each roller bottle,
agitating the bottles gently for at least 10 minutes until the
cells slough from the surface and then rinsing the bottle surface
with DMEMH and pooling the contents of all of the bottles. The
cells from these bottles (Production Cells) were re-inoculated into
new 850cm.sup.2 roller bottles at 4.5.times.10.sup.7 cells per
roller bottle. The Production Cells were incubated for 24 hours at
36 to 38.degree. C. after which they were infected with
freshly-passaged Neospora caninum tachyzoites of Strain BPA-1
(3.times.10.sup.8 to 4.5.times.10.sup.8/850 cm.sup.2 roller
bottle). At the time of infection, the production cells were at
least 50% confluent. Infected roller bottles were incubated at 36
to 38.degree. C. for 120 to 168 hours on rotating roller racks set
at between 0.2 and 0.4 rpm. At that time, the cell sheet was
displaying typical CPE affecting at least 80% of the cell sheet. At
the end of the incubation period the Neospora fluids were harvested
by pooling the contents from all of the roller bottles into a
sterile vessel and a sample was removed for live Neospora
titration. Acceptable harvest fluids must have a titer of at least
3.times.10.sup.5/mL. The harvest titer for the present batch was
3.times.10.sup.50/mL. The harvest fluids were frozen and thawed
twice by holding the harvest fluids at -70.degree. C. and thawing
them rapidly at temperatures no higher than 37.degree. C. After
this treatment, the harvest fluids were inactivated for a period of
48 hours at 4.degree. C. with 0.2 M Binary Ethylenimine (BEI).
After inactivation, the BEI was neutralized with 3.16 M sodium
thiosulfate. The inactivated harvest fluids were concentrated by
centrifugation at 3500 rpm for 15 minutes and the pellet was
re-suspended in PBS to a concentration of 3.0.times.10.sup.7 based
on a microscopic count. Aliquots of these inactivated and
concentrated harvest fluids were adjuvanted with two different
types of adjuvants in order to prepare two different vaccine
formulations. One half of the inactivated and concentrated harvest
fluids was adjuvanted with 10% (v/v) HAVLOGEN.RTM. while the
remainder of the inactivated concentrated harvest fluid was
adjuvanted with 15% (v/v) of EMULSIGEN.RTM.. HAVLOGEN.RTM. is a
polymer based adjuvant containing Carbopol while EMULSIGEN.RTM. is
an oil-in-water based adjuvant.
[0021] The two vaccine formulations were used to vaccinate heifers
ranging in age from two to two and one-half years of age. All
heifers were bred and when pregnancy was confirmed at 30.+-.5 days,
these animals were divided into four groups which were treated as
follows:
[0022] Group 1 heifers (No. 21, 30, 39 and 20) were injected
subcutaneously two times at 4 week intervals with a Neospora
vaccine containing 10% HAVLOGEN.RTM. adjuvant.
[0023] Group 2 heifers (No. 18, 37, 40 and 431) were injected
subcutaneously two times at 4 week intervals with a Neospora
vaccine containing 15% EMULSIGEN.RTM..
[0024] Group 3 heifers (No. 429, 25, 28 and 2) served as controls
and were injected subcutaneously two times at 4 week intervals with
a control preparation containing only uninfected Vero cell cultures
containing 15% EMULSIGEN.RTM..
[0025] Group 4 heifers (No. 19, 10, 4, and 14) served as contact
controls and were neither vaccinated nor challenged.
[0026] Serum samples from all heifers were taken at least one week
prior to vaccination (P.V.), on the day of first vaccination (day
0) and on Weeks 5, 6 and 7 post vaccination, on the day of booster
(boost), the day of challenge (between Week 11 and 12, and weekly
thereafter through Week 16 post vaccination. All heifers started
the study as seronegative. Only the titers measured on the day of
challenge are listed in Table 1 as these are the most titers for
this study.
[0027] Heifers from Groups 1-3 were challenged with
8.times.10.sup.7 virulent Neospora caninum tachyzoites of strain
BPA-1 grown in Vero cells. Challenge occurred at 85.+-.5 days of
gestation. Fetuses were removed by caesarian section from the
heifers at 40.+-.6 days (114 to 120 days) of gestation and
evaluated by gross examination. The presence of dead fetuses were
interpreted to indicate that the vaccine did not protect the
fetuses and would have resulted in abortion of the fetuses. The
presence of live fetuses was interpreted as demonstrating
protection of the fetuses and that abortion should not have
occurred.
[0028] Table 1 shows the results of the fetal evaluation and lists
the serological titers of the heifers on the day of challenge. The
results shown in this table indicate that the heifers in Group 1
contained two live fetuses and two dead fetuses suggesting that the
HAVLOGEN.RTM. adjuvanted Neospora vaccine produced 50% protection
from abortion. It should be noted that the two protected heifers
had titers at challenge of 320 and 640 respectively. The heifers in
Group 2 vaccinated with EMULSIGEN.RTM. adjuvanted Neospora vaccine
contained one live fetus from a heifer with a serological titer of
320. The remaining heifers in this vaccine group had dead fetuses
and titers lower than 320 at challenge. The first two heifers in
Group 3 (Control Group receiving adjuvanted Vero cells without
Neospora) had dead fetuses and titers <80. The remaining two
heifers in this group were challenged at a later time than all of
the other heifers and it is proposed that they did not receive a
high enough challenge dose and, therefore, had live fetuses. Their
titers were <80 on the day of challenge and at a later
histological examination it was shown that these heifers were not
infected. Group 4 heifers did not develop antibody titers during
the study indicating that the other groups did not shed Neospora
organisms. This latter group was not challenged since they only
served as contact controls.
[0029] This experiment supports the inventor's interpretation of
the Ho et al data wherein the inventors proposed that a 320 IFA
titer might be indicative of protection from fetal abortion.
1TABLE 1 Results of Fetal Evaluation Post Challenge with Virulent
Neospora BPA-1 Gestational Age at Appearance Titer of Removal of
Fetus Heifer at Group Heifer No. of Fetus at Removal Challenge 1
HAV 21 119 days LIVE 320 1 HAV 30 119 days DEAD 160 1 HAV 39 114
days LIVE 640 1 HAV 20 120 days DEAD 160 2 EMUL 18 119 days DEAD
160 2 EMUL 37 119 days DEAD 160 2 EMUL 40 118 days LIVE 320 2 EMUL
431 111 days DEAD 160 Controls 429 118 days DEAD <80 25 114 days
DEAD <80 Controls 28 120 days LIVE/NI* <80 2 120 days
LIVE/NI* <80 Contact 19 121 days LIVE <80 Controls 10 121
days LIVE <80 Contact 4 120 days LIVE <80 Controls 14 120
days LIVE <80 HAV = HAVLOGEN .RTM. EMUL = EMULSIGEN .RTM. *NI =
Not Infected as determined by later histopathology
Example 2
[0030] This experiment was conducted in order to determine whether
a Neospora caninum organism could be grown in another tissue
culture cell line, inactivated and formulated to prepare a vaccine
which could produce antibody titers in cattle which would be
similar to those observed in EXAMPLE 1 with Neospora vaccines
produced on a Vero cell line.
[0031] An Equine Dermal Cell Line, Master Cell Passage 11, derived
from ATCC No. CCL-57 was diluted to a cell count of
2.times.10.sup.7 cells per roller bottle in a Dulbecco's Modified
Eagles Medium (DMEM) containing 10% Horse Serum and inoculated into
850 cm.sup.2 roller bottles at a volume of 250 mL per roller
bottle. The cells were grown to confluency after which they were
infected with 2.4.times.10.sup.7 Neospora caninum tachyzoites in
14.1 mL of DMEM. Each roller bottle contained 264 mL of DMEM plus
10% Horse Serum. The neospora-infected tissue cultures were
incubated at 37.degree. C. until at least 50% of the cells
demonstrated CPE (approximately 7 to 9 days). Fluids were harvested
and tachyzoites were centrifuged for 30 minutes at 3500 rpm in
order to concentrate the harvested antigen. The pelleted
concentrated Neospora caninum antigen was re-suspended in 200 mL of
decanted DMEM supernatant from the centrifuged tachyzoites. This
concentrated preparation containing 8.times.10.sup.6 tachyzoites
per mL was frozen for 16 hours at -70.degree. C. and then thawed at
room temperature. The preparation was then inactivated using 0.05 M
binary ethylenimine (BEI) incubated at 4.degree. C. for 48 hours.
The inactivated preparation was neutralized using 3.16 M sodium
thiosulfate. Two equal aliquots of the inactivated, neutralized
Neospora caninum antigen preparation were then adjuvanted with
different adjuvants as in EXAMPLE 1. One half of the preparation
was adjuvanted with HAVLOGEN.RTM., a Carbopol-based polymer
adjuvant, by adding adjuvant to a 10% concentration (v/v). The
other half of the preparation was adjuvanted with EMULSIGEN.RTM.,
an oil-based adjuvant, by adding adjuvant to a 15% concentration
(v/v).
[0032] The adjuvanted Neospora caninum vaccines produced on Equine
Dermal Cells were injected subcutaneously into calves ranging in
age from 9 to 12 months. One calf (#954) received a 5.0 mL dose of
the HAVLOGEN.RTM. adjuvanted vaccine while a second calf (#955)
received a 5.0 mL dose of EMULSIGEN.RTM. adjuvanted vaccine. Each
calf was boostered with the homologous vaccine 10 days later.
Calves were bled at each vaccination and 10 days post booster
vaccination. Serum was analyzed for titer using an indirect
fluorescent antibody (IFA) test. Serological titers of these calves
are shown in Table 2. These results indicate that the
EMULSIGEN.RTM. adjuvanted Neospora vaccine produced protective
titers while the HAVLOGEN.RTM. adjuvanted Neospora vaccine produced
a lower titer which was close to protective.
2TABLE 2 Antibody Titers of Calves Vaccinated with inactivated
adjuvanted Neospora caninum vaccines grown in Equine Dermal Cells
VACCINE SERUM ANTIBODY TITER (IFA) ADJUVANT DAY 0 DAY 14 DAY 24
HAVLOGEN .RTM. <80 80 160 EMULSIGEN .RTM. <80 80 2580
Example 3
[0033] After noting from EXAMPLES 1 and 2 that a Neospora caninum
vaccine produced in a continuous cell line could produce protective
antibody titers in cattle which correlated to protection from
abortion, it was the object of this experiment to evaluate the
effect of growing the Neospora caninum in a totally different
cloned cell line derived from African Green Monkey Kidneys (MA-104
Clone B) and evaluating the effects of several different types of
adjuvants on production of antibody titers in cattle.
[0034] A Neospora caninum vaccine was produced as follows. A vial
of Working Cells (MA-104 Clone B horse serum) was removed from
liquid nitrogen storage, thawed rapidly, diluted and inoculated
into 850 cm.sup.2 roller bottles containing 250 mL of DMEM (high
glucose), hereinafter designated DMEMH, at a concentration of
4.times.10.sup.7 cells per roller. The medium was supplemented with
Neomycin Sulfate at 1 mL/L and Horse Serum at 5% v/v. Cells were
incubated at 36 to 38.degree. C. for 5 to 7 days until the cells
were between 95 and 100% confluent. The Working Cells were removed
from the roller bottles by rinsing the cell sheet with Phosphate
Buffered Saline (PBS) and then adding 10 mL of a Trypsin-EDTA)
solution (2.5 g/L of Trypsin+1 g/L EDTA) to each roller bottle,
agitating the bottles gently for at least 10 minutes until the
cells slough from the surface and then rinsing the bottle surface
with DMEMH and pooling the contents of all of the bottles. The
cells from these bottles (Production Cells) were re-inoculated into
new 850 cm.sup.2 roller bottles at 4.5.times.10.sup.7 cells per
roller bottle. The Production Cells were incubated for 24 hours at
36 to 38.degree. C. after which they were infected with
freshly-passaged Neospora caninum tachyzoites
(1.2.times.10.sup.7/850 cm.sup.2 roller bottle). At the time of
infection, the production cells were at least 50% confluent.
Infected roller bottles were incubated at 36-38.degree. C. for 120
to 168 hours on rotating roller racks set at between 0.2 and 0.4
rpm. At that time, the cell sheet was displaying typical CPE
affecting at least 50% of the cell sheet. At the end of the
incubation period, the Neospora fluids were harvested by pooling
the contents from all of the roller bottles into a sterile vessel
from which a sample was removed for live Neospora tachyzoite
titration. Acceptable harvest fluids must have a titer of at least
3.times.10.sup.5/mL. The harvest titer for the present batch was
2.3.times.10.sup.6. In this case the harvest fluids were
concentrated by centrifugation in order to obtain
2.4.times.10.sup.7 tachyzoites/mL. Other concentration methods
include but are not limited to ultrafiltration and column
chromatography. The harvest fluids were inactivated by addition of
0.2 M binary ethylenimine (BEI) to a final concentration of 0.01 M
and incubation at 2 to 7.degree. C. for at least 96 hours. After
this incubation, the BEI was neutralized by addition of 3.16 M
sodium thiosulfate.
[0035] After inactivation and neutralization, the fluids were
divided into four aliquots. Each aliquot was adjuvanted with a
different adjuvant as follows:
[0036] Formula A: 1.0 mL of inactivated harvest fluids plus 3.5 mL
of PBS plus 0.5 mL of a Carbopol-based polymer adjuvant designated
HAVLOGEN.RTM..
[0037] Formula B: 1.0 mL of inactivated harvest fluids plus 3.25 mL
PBS plus 0.75 mL of a polymer-based adjuvant designated
POLYGEN.RTM..
[0038] Formula C: 1.0 mL of inactivated harvest fluids plus 0.5 mL
of HAVLOGEN.RTM. plus 3.5 mL of lipid-based adjuvant designated Bay
R-1005.
[0039] Formula D: 1.0 mL of inactivated harvest fluids plus 0.5 mL
of PBS plus 3.5 mL of MONTANIDE.RTM. 773.
[0040] Eighteen heifers ranging in age from 1.5 to 2.0 years of age
were randomly separated into six groups. Group 1 heifers (No. U148,
S85 and A184) did not receive a Neospora caninum vaccine. They
served as contact controls and received uninfected MA104 Clone B
cells. Group 2 heifers (No. A29, 13 and Z55) served as positive
controls and received live Neospora tachyzoites (3.times.10.sup.7
intravenously and 8.times.10.sup.7 intramuscularly). Group 3
heifers (No. 40, 1851, and A71) were vaccinated with three 5.0 mL
doses of Formula A, administered subcutaneously at 4 week
intervals. Group 4 heifers (No. 237, Y21, and U93) were vaccinated
with three 5.0 mL doses of Formula B, administered subcutaneously
at 4 week intervals. Group 5 heifers (No. Y6, X7, and 800) were
vaccinated with three 5.0 mL doses of Formula C, administered
subcutaneously at 4 week intervals. Group 6 heifers (No. A144, S74,
and 5212) were vaccinated with three 5.0 mL doses of Formula D
administered subcutaneously at 4 week intervals. All animals were
bled at day 0 and bi-weekly thereafter.
[0041] Serum samples were analyzed for conversion to Neospora
specific titers by use of an indirect fluorescent antibody (IFA)
test. Table 3 shows the serological results. All of the vaccine
preparations produced protective titer levels (>320) in the
heifers. However, the polymer-based adjuvants appear to produce a
better titer response than the oil-based adjuvant formulations.
Since the contact control cattle remained serologically negative
(within the test variation) for the duration of the experiment, it
is clear that the titers produced in the vaccinated animals were
not produced by shedding from the heifers injected with live
Neospora tachyzoites but were a result of the vaccination.
3TABLE 3 IFA Titers of heifers vaccinated with Neospora caninum
Vaccine Containing Four Different Adjuvants Weeks Post Vaccination
Week 0 Week 4 Week 8 Treatment (Vacc) Week 2 (Vacc) Week 6 (Vacc)
Week 10 Week 12 Formula A <80 320 160 160 160 320 320 <80 160
80 160 160 640 320 <80 320 80 160 320 640 160 GMT <80 254 101
160 202 508 254 Formula B <80 320 320 2560 1280 1280 1280 <80
160 80 640 640 1280 640 <80 320 160 640 640 1280 640 GMT <80
254 160 1016 806 1280 806 Formula C 80 640 160 1280 2560 2560 1280
80 320 160 1280 1280 2560 2560 <80 160 160 2560 1280 1280 1280
GMT 19 320 160 1613 1613 2032 1613 Formula D <80 80 80 160 160
640 1280 <80 80 160 160 320 1280 2560 <80 80 160 640 640 1280
1280 GMT <80 80 127 254 320 1016 1613 Live Tachys <80 2560
5120 10240 5120 2560 1280 <80 1280 20480 20480 20480 20480 20480
<80 20480 20480 20480 20480 20480 5120 GMT <80 4064 12902
16255 12902 10240 5120 Contact <80 <80 <80 <80 <80
<80 <80 Controls <80 <80 <80 <80 <80 <80
<80 <80 80 80 160 <80 80 <80 GMT <80 4 4 5 <80 4
<80 GMT = Geometric Mean Titer Vacc = Vaccination
Example 4
[0042] This experiment was conducted in order to determine the
impact of Neospora caninum antigen quantity in the vaccines, and to
evaluate a Neospora vaccine comprising subunit antigens. Also
incorporated in this vaccine production process was the use of a
"soft kill" technique which is defined as an inactivation process
utilizing reduced quantities of inactivating agents and lower
incubation temperatures and shorter inactivation times. For this
experiment, the Neospora caninum was grown and processed in a
manner similar to that described in EXAMPLE 3. The inactivation
process was modified as follows. Binary ethylenimine was added to
the harvested Neospora caninum to a final concentration of 0.01 M
but was incubated at room temperature for only 24 hours after which
it was neutralized by addition of sodium thiosulfate to a final
concentration of 0.01 M. Subunits were obtained by removing
aliquots of the inactivated tachyzoite fluids, centrifuging them at
3500 rpm for 15 minutes and decanting off the supernatant fluids.
The Neospora tachyzoite pellets were re-suspended in Dulbecco's
Phosphate Buffered Saline (DPBS) to produce a subunit vaccine
containing only the tachyzoite antigens and not the exoantigens
which are excreted by the tachyzoites into the medium. A second
Neospora vaccine was prepared by re-suspending the Neospora
tachyzoite pellet in the supernatant fluids which had been removed
and saved. Three batches of subunit DPBS re-suspended Neospora
caninum were formulated to contain 1.2.times.10.sup.7,
2.4.times.10.sup.7 and 3.6.times.10.sup.7 tachyzoites per dose,
respectively. Three batches of supernatant re-suspended Neospora
caninum were formulated to contain equivalent numbers of
tachyzoites (1.2.times.10.sup.7, 2.4.times.10.sup.7 and
3.6.times.10.sup.7) per dose. All formulations were adjuvanted with
HAVLOGEN.RTM. and brought to a final 5.0 mL/dose concentration by
addition of DPBS (to subunit vaccine) or supernatant fluid
respectively.
[0043] These formulations were administered to Neospora
seronegative heifers between the ages of 7 and 9 months of age. Six
vaccine groups were comprised of five heifers each (n=5) and two
control groups were comprised of three heifers each (n=3). Heifers
in the experimental vaccine groups were each injected
subcutaneously (SC) with 5.0 mL of one of the Neospora tachyzoite
vaccine preparations and revaccinated four weeks later. Vaccine
groups received the following vaccines:
[0044] Group 1 Subunit Neospora vaccine containing
1.2.times.10.sup.7 Neospora tachyzoites with 10% HAVLOGEN.RTM..
[0045] Group 2 Subunit Neospora vaccine containing
2.4.times.10.sup.7 Neospora tachyzoites with 10% HAVLOGEN.RTM..
[0046] Group 3 Subunit Neospora vaccine containing
3.6.times.10.sup.7 Neospora tachyzoites with 10% HAVLOGEN.RTM..
[0047] Group 4 Neospora vaccine containing 1.2.times.10.sup.7
Neospora tachyzoites with 10% HAVLOGEN.RTM. and supernatant
diluent.
[0048] Group 5 Neospora vaccine containing 2.4.times.10.sup.7
Neospora tachyzoites with 10% HAVLOGEN.RTM. and supernatant
diluent.
[0049] Group 6 Neospora vaccine containing 3.6.times.10.sup.7
Neospora tachyzoites with 10% HAVLOGEN.RTM. and supernatant
diluent.
[0050] Group 7 Contact Controls--These heifers received no
vaccine.
[0051] Group 8 Positive Controls--These heifers received a
challenge containing 5.times.10.sup.6 live Neospora tachyzoites
administered intravenously in a 5.0 mL dose and 3.times.10.sup.6
live Neospora tachyzoites administered intramuscularly in a 5.0 mL
dose.
[0052] All heifers were housed in the same lot, bled weekly for 7
weeks and all serum samples were tested for antibody titer specific
for Neospora caninum using IFA. Additionally, the vaccines were
evaluated for local reactivity by observing the sites of
vaccination. Any local reactions were measured and recorded in
centimeters. The serological titer responses of the heifers are
shown in Table 4.
[0053] The results listed in Table 4 indicate that the vaccines
containing the supernatant fluids added back to the Neospora pellet
produce a slightly higher antibody response than the Neospora
subunit vaccines. The antibody responses produced by the Neospora
caninum vaccines containing the added-back supernatant also
produced antibody responses which appear to be somewhat dose
related. However, all of the vaccines were effective in producing
protective levels of antibody in cattle.
[0054] None of the vaccines produced significant local reactions
post vaccination. Therefore, all of the formulations could be
considered acceptable commercial vaccine candidates.
4TABLE 4 Geometric Mean IFA Titers of Heifers Vaccinated with
Different Types of Neospora Vaccines Containing Increasing
Concentrations of Neospora Tachyzoites Days Post Vaccination Group
0 7 14 21 28* 35 42 49 56 64 1 <40 <80 80 320 160 2560 2560
2560 2560 1280 <40 <80 320 320 320 2560 2560 2560 640 640
<40 320 640 640 320 1280 2560 640 320 1280 80 320 640 640 640
640 1280 640 640 640 40 <80 160 80 80 320 2560 640 320 320 GMT 5
10 279 320 243 1114 2229 1114 640 735 2 <40 <80 160 160 160
320 5120 1280 320 320 <40 80 80 640 320 1280 5120 2560 2560 640
80 160 640 320 320 640 640 320 320 320 40 80 160 320 320 1280 1280
1280 1280 640 <40 <80 320 160 160 320 320 320 160 320 GMT 5
16 211 279 243 640 1470 844 557 422 3 <40 160 320 320 160 1280
1280 640 640 640 <40 <80 640 160 160 640 1280 1280 320 1280
40 320 1280 1280 640 2560 1280 1280 1280 320 40 <80 <80 80 80
320 2560 640 <80 640 80 160 320 160 160 2560 5120 2560 640 320
GMT 11 24 153 243 184 114 1940 1114 176 557 4 40 320 1280 640 80
640 2560 2560 320 320 <40 160 320 640 160 2560 5120 5120 2560
640 80 160 640 320 80 1280 1280 1280 640 640 80 320 1280 1280 1280
2560 5120 2560 2560 1280 <40 <80 160 160 160 1280 5120 1280
640 320 GMT 12 77 557 485 184 1470 3379 2229 970 557 5 <40
<80 160 320 160 1280 2560 2560 1280 1280 <40 160 160 640 160
2560 5120 1280 320 1280 <40 <80 640 320 160 640 2560 1280 640
640 <40 160 640 160 320 2560 5120 1280 1280 1280 <40 <80
160 80 80 160 1280 1280 640 160 GMT <40 7 279 243 160 970 2941
931 735 735 GMT = Geometric Mean Titer Vacc = Vaccination
[0055] 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 claim.
* * * * *