U.S. patent application number 10/872949 was filed with the patent office on 2004-11-11 for neospora vaccine.
Invention is credited to Brake, David A., Campos, Manuel.
Application Number | 20040223982 10/872949 |
Document ID | / |
Family ID | 22007613 |
Filed Date | 2004-11-11 |
United States Patent
Application |
20040223982 |
Kind Code |
A1 |
Brake, David A. ; et
al. |
November 11, 2004 |
Neospora vaccine
Abstract
The present invention provides an homogenate prepared from cells
of Neospora, and vaccines against neosporosis prepared therefrom
which are useful in the prevention of clinical disease and abortion
in mammals.
Inventors: |
Brake, David A.; (East Lyme,
CT) ; Campos, Manuel; (Stonington, CT) |
Correspondence
Address: |
SCULLY SCOTT MURPHY & PRESSER, PC
400 GARDEN CITY PLAZA
GARDEN CITY
NY
11530
|
Family ID: |
22007613 |
Appl. No.: |
10/872949 |
Filed: |
June 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10872949 |
Jun 21, 2004 |
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10036351 |
Nov 9, 2001 |
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6787146 |
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10036351 |
Nov 9, 2001 |
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09138985 |
Aug 24, 1998 |
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60056956 |
Aug 26, 1997 |
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Current U.S.
Class: |
424/269.1 |
Current CPC
Class: |
Y02A 50/489 20180101;
C07K 16/20 20130101; A61P 31/00 20180101; A61K 39/002 20130101 |
Class at
Publication: |
424/269.1 |
International
Class: |
A61K 039/002; A61K
039/005; A61K 039/008 |
Claims
1. An homogenate prepared from cells of Neospora which is capable
of inducing a protective response against neosporosis in a
mammal.
2. The homogenate of claim i., wherein the species of Neospora from
which the homogenate is prepared is N. caninum.
3. The homogenate of claim 1 which is capable of inducing the
production of antibodies that recognize one or more antigenic
components present in an homogenate of cells of N. caninum strain
NC-1.
4. The homogenate of claim 3, wherein the species of Neospora from
which the homogenate is prepared is N. caninum.
5. The homogenate of claim 4, wherein the strain of N. caninum from
which the homogenate is prepared is NC-1.
6. The homogenate of claim 1 which is prepared from
tachyzoites.
7. A vaccine to protect a mammal against neosporosis, comprising an
immunologically effective amount of an homogenate prepared from
cells of Neospora, which homogenate is capable of inducing a
protective response against neosporosis in a mammal, and a
veterinarily acceptable carrier.
8. The vaccine of claim 7, wherein the species of Neospora from
which the homogenate is prepared is N. caninum.
9. The vaccine of claim 7, which is capable of inducing the
production of antibodies that recognize one or more antigenic
components present in an homogenate of cells of N. caninum strain
NC-1.
10. The vaccine of claim 9, wherein the species of Neospora from
which the homogenate of the vaccine is prepared is N. caninum.
11. The vaccine of claim 10, wherein the strain of N. caninum from
which the homogenate of the vaccine is prepared is NC-1.
12. The vaccine of claim 7, wherein the homogenate is prepared from
tachyzoites.
13. The vaccine of claim 7, further comprising one or more
additional immunomodulatory components
14. The vaccine of claim 13, in which the additional
immunomodulatory component is an adjuvant.
15. The vaccine of claim 14, in which the adjuvant is selected from
the group consisting of the RIBI adjuvant system (Ribi Inc.), alum,
aluminum hydroxide gel, an oil-inwater emulsion, a water-in-oil
emulsion, Block co polymer, QS-21, SAF-M, AMPHIGEN.RTM. adjuvant,
saponin, Quil A, monophosphoryl lipid A, and Avridine lipid-amine
adjuvant.
16. The vaccine of claim 15, in which the adjuvant is an
oil-in-water emulsion selected from the group consisting of SEAM62
and SEAM 1/2.
17. The vaccine of claim 13, in which the additional
immunomodulatory component is a cytokine.
18. The vaccine of claim 7, wherein the Neospora cells from which
the homogenate is prepared have been modified to delete the
expression of one or more antigenic components normally associated
with Neospora cells or a homogenate prepared therefrom.
19-38. (Canceled).
39. A combination vaccine for protecting a mammal against
neosporosis and, optionally, one or more other diseases or
pathological conditions that can afflict the mammal, which
combination vaccine comprises an immunologically effective amount
of a first composition comprising an homogenate prepared from cells
of Neospora, which homogenate is capable of inducing a protective
response against neosporosis in a mammal; an immunologically
effective amount of a second composition capable of inducing a
protective response against a disease or pathological condition
that can afflict the mammal; and a veterinarily acceptable
carrier.
40. The combination vaccine of claim 39, wherein the species of
Neospora from which the homogenate of the first composition is
prepared is N. caninum.
41. The combination vaccine of claim 39, which is capable of
inducing the production of antibodies that recognize one or more
antigenic components present in an homogenate of cells of N.
caninum strain NC-1.
42. The combination vaccine of claim 41, wherein the species of
Neospora from which the homogenate of the first composition is
prepared is N. caninum.
43. The combination vaccine of claim 42, wherein the strain of N.
caninum from which the homogenate of the first composition is
prepared is NC-1.
44. The combination vaccine of claim 39, wherein the homogenate of
the first composition is prepared from tachyzoites.
45. The combination vaccine of claim 39, wherein the second
composition is capable of inducing in the mammal a protective
response against a pathogen selected from the group consisting of
bovine herpes virus, bovine respiratory syncitial virus, bovine
viral diarrhea virus, parainfluenza virus types I, II, or III,
Leptospira spp., Campylobacter spp., Staphylococcus aureus,
Streptococcus agalactiae, Mycoplasma spp., Klebsiella spp.,
Salmonella spp., rotavirus, coronavirus, rabies, Pasteurella
haemolytica, Pasteurella multocida, Clostridia spp., Tetanus
toxoid, E. coli, Cryptosporidum spp., Eimeria spp. and Neospora
spp.
46. A kit for vaccinating a mammal against neosporosis, comprising
a first container having an immunologically effective amount of an
homogenate prepared from cells of Neospora, which homogenate is
capable of inducing a protective response against neosporosis in a
mammal, and a second container having a veterinarily acceptable
carrier or diluent.
47-51. (Canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a vaccine against the
pathogenic protozoan Neospora which vaccine is useful in the
prevention of clinical disease and abortion in mammals. The vaccine
of the invention comprises an homogenate prepared from cells of a
species of Neospora.
BACKGROUND OF THE INVENTION
[0002] Neosporosis in mammals is caused by infection with a
pathogenic strain of the protozoan parasite Neospora, and has been
recognized as a major cause of abortion, neonatal death, congenital
infection, and encephalitic disease. Dubey and Lindsay, 1996, Vet
Parasitol. 67:1-59; Dubey and Lindsay, 1993, Parasitol. Today
9:452-458. Neospora caninum infects dogs and congenitally infects
pups, often leading to paralysis. Tachyzoites of N. caninum have
been isolated from naturally infected pups. Lindsay and Dubey,
1989, J. Parasitol. 75:163-165. Infection with Neospora is also a
major cause of abortion in dairy cattle. Cases of neosporosis have
also been reported in goats, sheep and horses.
[0003] Although N. caninum is superficially similar to the pathogen
Toxoplasma gondii, N. caninum and T. gondii are distinguishable
from each other both antigenically and ultrastructurally. Dubey and
Lindsay, 1993, above. In addition, Neospora-like protozoan
parasites isolated from the brains of aborted bovine fetuses and
continuously cultured in vitro were shown to be antigenically and
ultrastructurally distinct from both T. gondii and Hammondia
hammondi, and most similar to N. caninum. Conrad et al., 1993.
Parasitology 106:239-249. Furthermore, analysis of nuclear small
subunit ribosomal RNA genes revealed no nucleotide differences
between Neospora strains isolated from cattle and dogs, but showed
consistent differences when compared to T. gondii, thus confirming
the distinction between pathogens. Marsh et al., 1995, J.
Parasitol. 81:530-535.
[0004] The etiologic role of a bovine isolate of Neospora in bovine
abortion and congenital disease has been confirmed. Barr et al.
1994, J. Vet. Diag. Invest. 6:207-215. A rodent model of central
nervous system neosporosis has been developed using inbred BALB/c
mice infected with N. caninum. Lindsay et al., 1995, J. Parasitol.
81:313-315. In addition, models to study transplacental
transmission of N. caninum in pregnant outbred and inbred mice have
been described by Cole et al., 1995, J. Parasitol. 81:730-732, and
by Long et al., 1996, J. Parasitol. 82:608-611, respectively.
Furthermore, an experimental N. caninum pygmy goat model closely
resembling naturally acquired Neospora-induced cattle abortion has
been developed. Lindsay et al., 1995, Am. J. Vet. Res.
56:1176-1180.
[0005] WO 9525541 discloses a biologically pure culture of bovine
Neospora, methods of detecting anti-Neospora antibodies and
Neospora-specific nucleic acids, and a composition containing a
bovine Neospora antigen and carrier for use as a vaccine.
[0006] An effective vaccine against neosporosis comprising an
homogenate prepared from cells of Neospora has not previously been
disclosed.
SUMMARY OF THE INVENTION
[0007] In a first aspect, the present invention provides an
homogenate prepared from cells of Neospora, which homogenate is
capable of inducing a protective response against neosporosis in a
mammal.
[0008] In a second aspect, the present invention provides a vaccine
to protect a mammal against neosporosis, comprising an
immunologically effective amount of an homogenate prepared from
cells of Neospora, which homogenate is capable of inducing a
protective response against neosporosis in a mammal, and a
veterinarily acceptable carrier. The vaccine of the present
invention may further comprise one or more additional
immunomodulatory components including, e.g., an adjuvant or
cytokine.
[0009] In a third aspect, the present invention provides a method
for preparing a vaccine that protects a mammal against neosporosis,
comprising homogenizing cells of Neospora to form an homogenate
capable of inducing a protective response against neosporosis in a
mammal, and combining an immunologically effective amount of the
homogenate with a veterinarily acceptable carrier in a form
suitable for administration to the mammal.
[0010] In a fourth aspect, the present invention provides a method
for protecting a mammal against neosporosis, comprising
administering to the mammal a vaccine comprising an immunologically
effective amount of an homogenate prepared from cells of Neospora,
which homogenate is capable of inducing a protective response
against neosporosis in a mammal, and a veterinarily acceptable
carrier. The vaccine of the present invention may be administered
to any mammalian species susceptible to infection and disease
caused by Neospora, including but not limited to dogs, cows, goats,
sheep and horses.
[0011] In a fifth aspect, the present invention provides a
combination vaccine for protecting a mammal against neosporosis
and, optionally, one or more other diseases or pathological
conditions that can afflict the mammal, which combination vaccine
comprises an immunologically effective amount of a first
composition comprising an homogenate prepared from cells of
Neospora, which homogenate is capable of inducing a protective
response against neosporosis in a mammal; an immunologically
effective amount of a second composition capable of inducing a
protective response against a disease or pathological condition
that can afflict the mammal; and a veterinarily acceptable
carrier.
[0012] The second composition of the combination vaccine is
selected based on its ability to induce a protective response
against either neosporosis or another disease or pathological
condition that can afflict members of the mammalian species, as
known in the art. The combination vaccine of the present invention
may further comprise one or more additional immunomodulatory
components including, e.g.. an adjuvant or cytokine, among
others.
[0013] In a sixth aspect, the present invention provides a kit for
vaccinating a mammal against neosporosis, comprising a first
container having an immunologically effective amount of an
homogenate prepared from cells of Neospora, which homogenate is
capable of inducing a protective response against neosporosis in a
mammal, and a second container having a veterinarily acceptable
carrier or diluent suitable for mixing with the contents of the
first container.
[0014] In a seventh aspect, the present invention provides
antibodies specific to one or more antigenic components present in
an homogenate prepared from cells of Neospora.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1. Pre-challenge Western blot analysis of serum from
BALB/c mice. A whole cell homogenate of NC-1 tachyzoites (the "NSA
preparation") was fractionated by SDS-PAGE and transferred to PVDF
membrane, which was then incubated with primary antiserum samples,
followed by alkaline-phosphatase conjugated goat anti-mouse IgG,
and developed using the chromogenic substrate BCIP/NBT. Lanes
1-3=serum from mice administered adjuvant alone (control); lanes
4-6=serum from mice administered the NSA preparation plus adjuvant
(vaccine); molecular weight standards indicated. Serum from
immunized mice contains antibodies that are reactive with NSA
preparation proteins having molecular weights of about 17-19,
28-30, 33, 37, 46, 48 and 56 kD.
[0016] FIG. 2. Pre-challenge (A) and post-challenge (B)
immunofluorescence antibody (IFA) titers. Serum from animals on day
21 post-immunization (day 0 pre-challenge) and day 21
post-challenge were added to wells containing NC-1 tachyzoites.
Wells were then incubated with (Fab).sub.2 fluorescein
isothiocyanate-conjugated anti-mouse IgG.+IgM (Kirkegaard &
Perry, Gaithersburg, Md.), washed, and examined by epifluorescent
microscopy. Antibody titer is based on the highest dilution of
immune serum producing a detectable fluorescence. Results show
higher mean IFA antibody titers in vaccinated animals pre-challenge
(2A) and significantly higher IFA antibody titers post-challenge
(2B) (P<0.001) when compared to controls. In 2B, with 10.sup.6
challenge, control geometric mean titer (GMT)=2,691; vaccine
GMT=25,600. With 10.sup.7 challenge, control GMT=5,382; vaccine
GMT=72,408.
[0017] FIG. 3. Pre-challenge splenic antigen-specific proliferation
assay. On day 21 post-immunization, splenocytes from mice
administered adjuvant alone (control) or the NSA preparation plus
adjuvant (vaccine) were prepared and T-cell proliferation assays
conducted by incubating splenocytes in the presence of the NSA
preparation and pulsing splenocyte cultures with
[.sup.3H]thymidine, as described below (Example 2). Results are
expressed as .DELTA. cpm (mean cpm with NSA minus mean cpm with
medium alone), and demonstrate that a cell homogenate of Neospora
can induce a T-cell population in vivo which is capable of
proliferating in vitro following stimulation with the NSA
preparation.
[0018] FIG. 4. Donor pre-challenge splenic antigen-specific
cytokine production. On day 21 post-immunization, splenocytes from
mice administered adjuvant alone (control) or the NSA preparation
plus adjuvant (vaccine) were prepared and levels of cytokine
production determined by incubating splenocytes in the presence of
the NSA preparation, collecting cell-free supernatants and assaying
for specific cytokines using commercial cytokine-specific
antibodies following manufacturer's instructions (PharMingen, San
Diego, Calif.). Results demonstrate that a cell homogenate of
Neospora can induce a T-cell population in vivo which is capable of
producing both type-1 (IFN-.gamma., IL-2) and type-2 (IL-6, IL-10)
cytokines in vitro following stimulation with the NSA
preparation.
[0019] FIG. 5. Post-challenge splenic antigen-specific
proliferation assay. At day 21 post-challenge, splenocytes from
BALB/c mice administered adjuvant alone (control) or the NSA
preparation plus adjuvant (vaccine) were prepared and T-cell
proliferation assays conducted by pulsing splenocyte cultures with
[3H]thymidine as described below. Following challenge with
1.times.10.sup.6 (A) or 1.times.10.sup.7 (B) NC-1 tachyzoites,
significantly higher antigen-specific responses (*=P<0.05;
**=P<0.01) were detected using splenocytes from vaccinated mice
compared to control mice (n=4/group).
[0020] FIG. 6. Post-challenge lung (A, C) and brain (B, D) lesion
scores. Sections of lung and brain tissue from day 21
post-challenge control (n=6) and vaccine (n=6) BALB/c mice
challenged with either 1.times.10.sup.6 (A. B) or 1.times.10.sup.7
(C. D) NC-1 tachyzoites were prepared and scored as described
below. Lesion scores for individual animals are presented. Dotted
line represents mean lesion score for each group. Results
demonstrate that animals immunized with a cell homogenate of
Neospora and challenged with 1.times.10.sup.7 NC-1 tachyzoites have
significantly lower mean lung (p<0.01) and brain (P<0.05)
lesions scores compared to challenge controls. A. control mean=0.5,
vaccine mean=0.33. B. control mean=1.0, vaccine mean =0.33. C.
control mean=1.83, vaccine mean=0.67 (P<0.01). D. control
mean=1.83, vaccine mean=1.0 (P<0.05).
[0021] FIG. 7. Post-challenge survival curves of athymic nude mice.
Nude mice receiving DPBS alone (no splenocytes="no cells"); nude
mice receiving splenocytes from BALB/c mice that were injected with
adjuvant alone ("adjuvant"); nude mice receiving splenocytes from
BALB/c mice that were injected with the NSA preparation plus
adjuvant ("vaccine") (n=6-7 mice/group). Results demonstrate that
the transfer of cells from vaccinated BALB/c mice to nude mice
results in adoptive protective immunity as shown by prolonged
survival and significant protection against an NC-1 virulent
challenge.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Applicants have discovered that an homogenate prepared from
cells of Neospora is capable of inducing a protective response
against neosporosis in mammals. The present invention thus provides
an homogenate prepared from cells of Neospora, which homogenate is
capable of inducing a protective response against neosporosis in a
mammal.
[0023] The present invention further provides a vaccine to protect
a mammal against neosporosis, comprising an immunologically
effective amount of an homogenate prepared from cells of Neospora,
which homogenate is capable of inducing a protective response
against neosporosis in a mammal, and a veterinarily acceptable
carrier.
[0024] As used herein, the term "neosporosis" refers to infection
of a mammal by cells of a species or strain of Neospora, or to any
clinical symptom, condition, event or pathology associated with or
resulting from infection of the mammal by cells of a species or
strain of Neospora.
[0025] The phrase "capable of inducing a protective response" is
used broadly herein to include the induction or enhancement of any
immune-based response in the animal in response to vaccination,
including either an antibody or cell-mediated immune response, or
both, that serves to protect the vaccinated animal against
neosporosis. The terms "protective response," "protection against,"
"protect," etc., as used herein, refer not only to the absolute
prevention of neosporosis or absolute prevention of infection by a
neosporosis-causing pathogen, but also to any detectable reduction
in the degree or rate of infection by such a pathogen, any
detectable reduction in the incidence of death or any detectable
increase in survival time following infection with a virulent
strain of the pathogen, any detectable reduction in the severity of
the disease or in any symptom or condition resulting from infection
with the pathogen, including, e.g., any detectable reduction in the
rate of formation or in the absolute number of lesions in one or
more tissues in the vaccinated animal, or any detectable reduction
in the occurrence of abortion or the transmission of infection from
a parent mammal to its offspring.
[0026] The phrase "immunologically effective amount" refers to that
amount or dose of vaccine, homogenate, antigen or NSA preparation
capable of inducing a protective response against neosporosis when
administered to a member of a mammalian species after either a
single administration, or after multiple administrations.
[0027] Preparation of Neospora Antigen
[0028] The invention is based on the discovery that an homogenate
prepared from cells of Neospora is capable of inducing a protective
response against neosporosis in mammals. The cells used to produce
the homogenate in the vaccine of the present invention may be
derived from any strain of any species of the genus Neospora, which
cells may or may not be pathogenic, where the homogenate is capable
of inducing a protective response against neosporosis in mammals.
In a preferred embodiment, the species of Neospora is N. caninum. A
non-limiting example of a strain of N. caninum from which an
homogenate may usefully be prepared is strain NC-1, which is
available in infected MARC145 monkey kidney cells from the American
Type Culture Collection, located at 12301 Parklawn Drive,
Rockville, Md. 20852, USA (ATCC Accession No. CRL-12231), and which
encompasses strains derived from NC-1 by one or more in vitro
and/or in vivo passages. Strain NC-1 is also described in Dubey et
al., 1988, J. Am. Vet. Med. Assoc. 193:1259-63, which publication
is incorporated herein by reference. Strains of Neospora for use
according to the present invention may alternatively be isolated
from organs, tissues or body fluids of infected animals using
standard isolation techniques, such as those described in the
publications reviewed above.
[0029] In a non-limiting embodiment, the vaccine of the present
invention may be prepared using homogenates of cells of other
species of Neospora that are immunologically equivalent to N.
caninum or using homogenates of cells of other strains of N.
caninum that are immunologically equivalent to N. caninum strain
NC-1. A species of Neospora is "immunologically equivalent" to N.
caninum where an homogenate prepared from the cells of the
immunologically equivalent species is capable of inducing in a
mammal the production of antibodies that recognize one or more
antigenic components present in an homogenate of cells of N.
caninum, as determined, e.g., by Western blot analysis, and where
the homogenate of cells of the immunologically equivalent species
is capable of inducing a protective response against neosporosis in
mammals. Likewise, a strain of N. caninum is "immunologically
equivalent" to N. caninum strain NC-1 where an homogenate prepared
from the cells of the immunologically equivalent strain is capable
of inducing in a mammal the production of antibodies that recognize
one or more antigenic components present in an homogenate of cells
of N. caninum strain NC-1 (see FIG. 1), and where the homogenate
prepared from cells of the immunologically equivalent strain is
capable of inducing a protective response against neosporosis in
mammals.
[0030] Cells of Neospora for use according to the present invention
may be utilized directly and without further modification.
Alternatively, such cells may be modified, e.g., by genetic
manipulation, to add, increase, delete or reduce the expression of
one or more metabolic pathways or products, or antigenic
properties, such as, e.g., a particular surface antigen or
virulence factor, or otherwise modify said pathways, products or
properties. Such pathways, products or properties, the expression
of which may usefully be added, increased or otherwise modified in
the cells, are preferably those which serve to trigger or enhance
the induction of a protective response against neosporosis in a
mammal vaccinated with the corresponding homogenate. For example,
cells of Neospora may be genetically modified to add or detectably
increase the expression of one or more antigenic components which
are useful to trigger or enhance the induction of a protective
response. In a non-limiting embodiment, cells of Neospora are
genetically modified to add or detectably increase the expression
of one or more immunodominant antigens, such as those visualized by
SDS-PAGE separation and Western blot analysis of the NSA
preparation as described below, including those antigens identified
as having molecular weights selected from the group consisting of
about 17-19, 28-30, 33, 37, 46, 48 and 56 kD.
[0031] Alternatively or additionally, cells may be modified to
delete or detectably reduce the expression of one or more antigenic
components normally associated with unmodified cells of Neospora or
an homogenate prepared therefrom. In a non-limiting embodiment,
cells of Neospora are genetically modified to delete or detectably
reduce the expression of one or more antigenic components, such as
those that may be visualized by SDS-PAGE separation and Western
blot analysis of the NSA preparation as described below, and
including those antigenic components identified as having molecular
weights selected from the group consisting of about 17-19, 28-30,
33, 37, 46, 48 and 56 kD. In this manner, vaccines may be produced
that are "marked" or "tagged," thereby allowing for animals that
have been vaccinated to be distinguished from those that have
naturally been infected with the pathogen.
[0032] Methods by which protozoan cells, such as those of Neospora,
may be genetically modified are generally known in the art, and
include the introduction of random mutations, e.g., by exposure to
chemical mutagens or radiation, followed by selection for a desired
mutant phenotype. Alternatively, or additionally, Neospora cells
may be modified by targeted genetic modification as carried out by
known procedures such as, e.g., by homologous recombination as
described, e.g., by Cruz and Beverley, 1990, Nature 348:171-173;
Cruz et al., 1991, Proc. Natl. Acad. Sci. USA 88:7170-7174; Donald
and Roos, 1994, Mol. Biochem. Parasitol. 63:243-253; and Titus et
al., 1995, Proc. Natl. Acad. Sci. USA 92:10267-10271, which
publications are incorporated herein by reference. Such genetic
modification is within the skill in the art and may be carried out
using generally known recombinant techniques such as those
described, e.g., in Maniatis, et al., 1989, Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; Ausubel, et al., 1989, Current Protocols In Molecular
Biology, Greene Publishing Associates & Wiley lnterscience,
N.Y.; and Sambrook, et al., 1989, Molecular Cloning: A Laboratory
Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., which publications are incorporated herein by
reference.
[0033] Once obtained, cells of Neospora for use in the present
invention may be cultured in vitro by infecting any receptive cell
line, preferably a mammalian cell line, with tachyzoites of the
species or strain of Neospora according to known techniques
described in the art. Mammalian cell lines in which tachyzoites of
Neospora can be cultured include, e.g., human foreskin fibroblasts
(Lindsay et al., 1993, Am. J. Vet. Res. 54:103-106), bovine
cardiopulmonary aortic endothelial cells (Marsh et al., 1995,
above), bovine monocytes (Lindsay and Dubey, 1989, above), monkey
kidney cells, among others. For example, tachyzoites of N. caninum
may be cultured in monolayers of Hs68 human foreskin fibroblast
cells (ATCC Accession No. CRL-1635) (Lindsay et al., 1993, above).
Bradyzoites may be similarly cultured and manipulated.
[0034] Mammalian cell cultures can be grown, and cell cultures that
have been infected with Neospora can be maintained, in any of
several types of culture media described in the art. For example,
stationary monolayer cultures of bovine cardiopulmonary aortic
endothelial cells infected with tachyzoites of N. caninum may be
grown in Dulbecco's Minimum Essential Medium (DMEM: Gibco
Laboratories, N.Y.), supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (FBS) or adult equine serum (ES), 2 mM
L-glutamine, 50 U/ml penicillin, and 50 .mu.g/ml streptomycin
(Conrad et al., 1993, above). Monolayers of Hs68 human foreskin
fibroblast cells may be maintained in RPMI 1640 containing 2% (v/v)
FBS, 1.0 mM sodium pyruvate, 1.times.10.sup.4 U/ml penicillin,
1.times.10.sup.4 .mu.g/ml streptomycin, 5.times.10.sup.-2 mM
2-mercaptoethanol and 0.3 mg/ml L-glutamine (maintenance medium).
Monolayer cultures of Hs68 human foreskin fibroblast cells infected
with Neospora may be maintained in identical media, but in which
the FBS is increased to 10% (v/v) (growth medium).
[0035] Neospora-infected monolayer cultures of mammalian cells are
typically maintained under standard tissue culture conditions such
as, e.g., at 37.degree. C. and 5% CO.sub.2. Tachyzoites are
typically passaged to uninfected monolayer cultures when 70-90% of
the mammalian cells in the culture have become infected, which may
be determined microscopically using standard techniques.
Tachyzoites may be collected from the infected mammalian cell
cultures by lysing the host cells using any standard technique and
collecting the tachyzoites, e.g., by filtration or by
centrifugation.
[0036] Cells which may be used to produce the cell homogenate of
the invention are preferably tachyzoites, but may alternatively be
bradyzoites or oocysts, or some combination thereof. In addition,
cells for use in the present invention may either be viable cells
or cells which have previously been inactivated, e.g., by treatment
with a chemical inactivating agents such as formaldehyde or
glutaraldehyde, among others, or by treatment with radiation, or by
exposure to extreme pH or temperature, or some combination
thereof.
[0037] The production of the homogenate of the invention is not
limited to any particular method of homogenization or disruption.
Rather, cells of Neospora may be homogenized or disrupted using any
technique known in the art including but not limited to
freeze/thawing, osmotic bursting, grinding, sonication, use of a
polytron, blender or tissue homogenizer, or some combination
thereof.
[0038] As used herein, the term "homogenate" refers to a
preparation prepared by homogenizing or disrupting whole cells of
Neospora. The homogenate of the present invention may comprise all
of the components produced by the homogenization or disruption of
whole Neospora cells, thus representing a "whole cell" preparation.
Alternatively, the homogenate of the present invention may consist
of a fraction of the total contents of homogenized or disrupted
Neospora cells, which fraction is prepared from the whole cell
preparation using one or more fractionation, isolation or
purification steps known in the art, including, e.g.,
centrifugation, filtration, dialysis, preparative gel
electrophoresis, affinity chromatography, ion exchange
chromatography, size exclusion chromatography, ammonium sulfate
precipitation, or some combination thereof, where the resulting
fraction of the whole cell preparation retains the ability to
induce a protective response against neosporosis in mammals. Such a
fraction may be an enriched membrane fraction or, alternatively, a
fraction enriched in soluble cytoplasmic components. Such fractions
are easily prepared and tested using nothing more than routine
preparative and screening procedures.
[0039] Preparation And Use Of Vaccines
[0040] The present invention provides a vaccine against
neosporosis, comprising an immunologically effective amount of an
homogenate prepared from cells of Neospora, which homogenate is
capable of inducing a protective response against neosporosis in a
mammal, and a veterinarily acceptable carrier.
[0041] The present invention further provides a method for
preparing a vaccine that protects a mammal against neosporosis,
comprising homogenizing cells from Neospora to produce a homogenate
capable of inducing a protective response against neosporosis in a
mammal, and combining an immunologically effective amount of the
homogenate with a veterinarily acceptable carrier in a form
suitable for administration to the mammal.
[0042] The vaccine may simply comprise a cell homogenate prepared
in culture fluid taken directly from a Neospora cell culture, which
is then administered directly to the mammal, or may instead
comprise a cell homogenate combined with a veterinarily acceptable
carrier selected from those known in the art appropriate to the
route of administration. For example, the vaccine of the present
invention may be formulated following accepted convention by
combining the homogenate or a fraction thereof with standard
buffers, carriers, stabilizers, diluents, preservatives, and/or
solubilizers. The vaccine may also be formulated to facilitate
sustained release. Diluents may include water, saline, dextrose,
ethanol, glycerol, and the like. Additives for isotonicity may
include sodium chloride, dextrose, mannitol, sorbitol, and lactose,
among others. Stabilizers may include albumin, among others.
Suitable other vaccine vehicles and additives are known, or will be
apparent, to those skilled in the art. See, e.g., Remington's
Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is
incorporated herein by reference.
[0043] The vaccine of the present invention may further comprise
one or more additional immunomodulatory components such as, e.g.,
an adjuvant or cytokine. Non-limiting examples of adjuvants include
the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum,
mineral gels such as aluminum hydroxide gel, oil-in-water
emulsions, water-in-oil emulsions such as, e.g., Freund's complete
and incomplete adjuvants, Block co polymer (CytRx, Atlanta Ga.),
QS-21 (Cambridge Biotech Inc., Cambridge Mass.) and SAF-M (Chiron,
Emeryville Calif.), AMPHIGEN.RTM. adjuvant, saponin, Quil A or
other saponin fraction, monophosphoryl lipid A, and Avridine
lipid-amine adjuvant. Specific non-limiting examples of
oil-in-water emulsions useful in the vaccine of the invention
include SEAM62 and SEAM 1/2, the components of which are set forth
below. Other immunomodulatory agents which may be included in the
vaccine include, e.g., one or more interleukins, interferons, or
other known cytokines. The vaccine may be stored in solution or,
alternatively, in lyophilized form to be reconstituted with a
sterile diluent solution prior to administration.
[0044] The vaccine of the present invention may optionally be
formulated for the sustained release of the antigen. Examples of
such sustained release formulations include homogenate in
combination with composites of biocompatible polymers, such as,
e.g., poly(lactic acid), poly(lactic-co-glycolic acid),
methylcellulose, hyaluronic acid, collagen and the like. The
structure, selection and use of degradable polymers in drug
delivery vehicles have been reviewed in several publications,
including A. Domb et al., 1992, Polymers for Advanced Technologies
3: 279-292, which is incorporated herein by reference. Additional
guidance in selecting and using polymers in pharmaceutical
formulations can be found in the text by M. Chasin and R. Langer
(eds), 1990, "Biodegradable Polymers as Drug Delivery Systems" in:
Drugs and the Pharmaceutical Sciences, Vol. 45, M. Dekker, NY,
which is also incorporated herein by reference. Alternatively, or
additionally, the homogenate may be microencapsulated to improve
administration and efficacy. Methods for microencapsulating
antigens are well-known in the art, and include techniques
described, e.g., in U.S. Pat. No. 3,137,631; U.S. Pat No.
3,959,457; U.S. Pat. No. 4,205,060; U.S. Pat. No. 4,606,940; U.S.
Pat. No. 4,744,933; U.S. Pat. No. 5,132,117; and International Pub.
WO 95/28227, all of which are incorporated herein by reference.
[0045] Liposomes may also be used to provide for the sustained
release of the homogenate of the invention. Details concerning how
to make and use liposomal formulations can be found in, among other
places, U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,452,747; U.S. Pat.
No. 4,921,706; U.S. Pat No. 4,927,637; U.S. Pat No. 4,944,948; U.S.
Pat. No. 5,008,050; and U.S. Pat. No. 5,009,956, all of which are
incorporated herein by reference.
[0046] The present invention further provides a method for
protecting a mammal against neosporosis, comprising administering
to the mammal a vaccine comprising an immunologically effective
amount of an homogenate prepared from cells of Neospora, which
homogenate is capable of inducing a protective response against
neosporosis in a mammal, and a veterinarily acceptable carrier.
[0047] The vaccine is preferably administered parenterally, e.g.,
either by subcutaneous or intramuscular injection. However, the
vaccine may instead be administered by intraperitoneal or
intravenous injection, or by other routes including, e.g., orally,
intranasally, rectally, vaginally, intra-ocularly, or by a
combination of routes. The skilled artisan will know how to
formulate the vaccine composition according to the route
chosen.
[0048] An effective dosage may be determined by conventional means,
starting with a low dose of homogenate and then increasing the
dosage while monitoring the effects. Numerous factors may be taken
into consideration when determining an optimal dose per animal.
Primary among these is the species, the size of the animal, the age
of the animal, the general condition of the animal, the presence of
other drugs in the animal, the virulence of a particular species or
strain of Neospora against which the animal is being vaccinated,
and the like. The actual dose is preferably chosen after
consideration of the results from other animal studies.
[0049] Vaccine regimens may also be selected based on the
above-described factors. The vaccine of the invention may be
administered at any time during the life of a particular animal
depending upon several factors including, e.g., the timing of an
outbreak of neosporosis among other animals, etc. The vaccine may
be administered to animals of weaning age or younger, or to more
mature animals, e.g., as a pre-breeding vaccine to protect against
Neospora-related congenital disease or abortion.
[0050] Effective protection may require only a primary vaccination,
or one or more booster vaccinations may be needed for full
protection. One method of detecting whether adequate immune
protection has been achieved is to determine seroconversion and
antibody titers in the animal after vaccination. The timing of
vaccination and the number of boosters, if any, will preferably be
determined by a veterinarian based on analysis of all relevant
factors, some of which are described above.
[0051] The amount of homogenate in the vaccine preferably ranges
from about 10 to about 1,000 .mu.g protein/ml, and more preferably
from about 100 to about 500 .mu.g protein/ml. A suitable dosage
size ranges from about 0.5 ml to about 10.0 ml, and more preferably
from about 1.0 ml to about 5.0 ml. Generally, on a per dose basis,
the amount of cell homogenate administered to an animal is
preferably from about 10 to about 1,000 .mu.g protein, and more
preferably from about 100 to about 500 .mu.g protein.
[0052] The vaccine of the present invention is useful to protect
mammals against infection or disease caused by Neospora. As used
herein, the term "mammal" refers to any mammalian species that can
be protected against neosporosis using the vaccine of the
invention, including dogs, cows, goats, sheep and horses, among
others. The vaccine is useful to protect both pregnant and
non-pregnant mammals.
[0053] The present invention further provides a combination vaccine
for protecting a mammal against neosporosis and, optionally, one or
more other diseases or pathological conditions that can afflict the
mammal, which combination vaccine comprises an immunologically
effective amount of a first composition comprising an homogenate
prepared from cells of Neospora, which homogenate is capable of
inducing a protective response against neosporosis in a mammal; an
immunologically effective amount of a second composition capable of
inducing a protective response against a disease or pathological
condition that can afflict the mammal; and a veterinarily
acceptable carrier, such as described above.
[0054] The second composition of the combination vaccine is
selected based on its ability to induce a protective response
against either neosporosis or another disease or pathological
condition which afflicts members of the mammalian species, as known
in the art. Any immunogenic composition known to be useful in a
vaccine composition in the particular mammalian species may be used
in the second composition of the combination vaccine. Such
immunogenic compositions include but are not limited to those that
provide protection against pathogens selected from the group
consisting of bovine herpes virus (infectious bovine
rhinotracheitis), bovine respiratory syncitial virus, bovine viral
diarrhea virus, parainfluenza virus types I, II, or III, Leptospira
spp., Campylobacter spp., Staphylococcus aureus, Streptococcus
agalactiae, Mycoplasma spp., Klebsiella spp., Salmonella spp.,
rotavirus, coronavirus, rabies, Pasteurella haemolytica,
Pasteurella multocida, Clostridia spp., Tetanus toxoid, E. coli,
Cryptosporidium spp., Eimena spp., and other eukaryotic parasites,
among others.
[0055] The combination vaccine of the present invention may further
comprise one or more additional immunomodulatory components
including, e.g., an adjuvant or cytokine, and may optionally be
formulated for sustained release or be stored in lyophilized form
or both, as described above.
[0056] The present invention further provides a kit for vaccinating
a mammal against neosporosis, comprising a first container having a
vaccine comprising an immunologically effective amount of an
homogenate prepared from cells of Neospora, which homogenate is
capable of inducing a protective response against neosporosis in a
mammal, and a second container having a veterinarily acceptable
carrier or diluent suitable for mixing with the contents of the
first container. The vaccine may be stored in lyophilized form to
be reconstituted by addition of the carrier or diluent.
[0057] Anti-Neospora Antibodies
[0058] The production of polyclonal and monoclonal antibodies that
bind to one or more Neospora-specific antigenic components falls
within the scope of the invention. Such antibodies may be specific
to any of the antigenic components associated with Neospora cells
or an homogenate prepared therefrom. In a non-limiting embodiment,
antibodies may be raised against one or more of the antigenic
components visualized in the Western blot of FIG. 1, including
those antigenic components identified as having molecular weights
selected from the group consisting of about 17-19, 28-30, 33, 37,
46, 48 and 56 kD. Such antibodies may be useful as reagents for the
differential diagnosis of neosporosis, such as for detecting
Neospora-specific antigens in histological sections, or in cell,
tissue or fluid samples from an animal, such as, e.g., in ELISA or
Western blot assays, or to quantify the amount of antigen in a
vaccine preparation.
[0059] Antibodies may be raised against any of the antigenic
components present in a homogenate of Neospora cells, such as those
in the NSA preparation described below. Various host animals,
including but not limited to cows, horses, rabbits, goats, sheep,
and mice, may be used according to known immunological techniques
to produce antibodies against one or more Neospora-specific
antigenic components. Various adjuvants, such as those described
above, may be used to increase the immunological response to
enhance antibody production.
[0060] Polyclonal antibodies may be obtained from immunized animals
and tested for specificity against Neospora-specific antigenic
components using standard techniques. Alternatively, monoclonal
antibodies to a Neospora-specific antigenic component may be
prepared using any technique which provides for the production of
antibody molecules by continuous cell lines in culture. These
include but are not limited to the hybridoma technique originally
described by Kohler and Milstein (Nature, 1975, 256: 495-497); the
human B-cell hybridoma technique (Kosbor et al., 1983, Immunology
Today 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80:
2026-2030); and the EBV-hybridoma technique (Cole et al., 1985,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96). Alternatively, techniques described for the production of
single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be
adapted to produce Neospora antigen-specific single chain
antibodies. These publications are incorporated herein by
reference.
[0061] Antibody fragments that contain specific binding sites to a
Neospora-specific antigenic component are also encompassed within
the present invention, and may be generated by known techniques.
Such fragments include but are not limited to F(ab').sub.2
fragments, which can be generated by pepsin digestion of an intact
antibody molecule, and Fab fragments, which can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragments.
Alternatively, Fab expression libraries may be constructed (Huse et
al., 1989, Science 246: 1275-1281) to allow rapid identification of
Fab fragments having the desired specificity to a Neospora-specific
antigen.
[0062] The antibodies and antibody fragments of the present
invention may further comprise a detectable label, such as a
fluorescent tag, radioactive label or enzyme, as known in the art,
to aid in the detection of specifically bound antibody in any of
the aforementioned diagnostic assays.
[0063] Techniques for the production and use of monoclonal
antibodies and antibody fragments are well-known in the art, and
are additionally described, among other places, in Harlow and Lane,
1988, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, and in J. W. Goding, 1986, Monoclonal Antibodies:
Principles and Practice, Academic Press, London, which are
incorporated herein by reference.
[0064] The following examples are offered to further illustrate,
but not limit the compositions and methods of the invention.
EXAMPLE 1
Preparation of Neospora Vaccine
[0065] Maintenance of Neospora Cultures
[0066] Tachyzoites of the NC-1 strain of N. caninum were maintained
in MARC-145 monkey kidney cell monolayers (USDA, ARS, Clay Center,
Nebr.) in tissue culture flasks at 37.degree. C. and 5% CO.sub.2 in
Opti-MEM.TM. medium (Gibco BRL) containing 1% (v/v) FBS, 100 U/ml
penicillin, 100 .mu.g/ml streptomycin, and 2 mM glutamine.
Tachyzoites were harvested from infected cell cultures when about
60-90% of the MARC-145 host cells had lysed, as determined by
microscopic examination of monolayers for cytopathic effects.
Remaining infected cells containing intracellular tachyzoites were
scraped off using a cell scraper and pooled with the culture medium
containing the free, extracellular tachyzoites. The preparation
(infected cells plus free tachyzoites) was centrifuged
(1,876.times.g, 10 min, 4.degree. C.), and the pellet was
resuspended in 5 ml Hank's balanced salt solution (HBSS) (Gibco
BRL). The suspension was passed five times through a 22 gauge
needle, centrifuged as before, resuspended in 5 ml HBSS, and passed
five times through a 28 gauge needle. Material was centrifuged as
before, and the pellet resuspended in 5 ml HBSS followed by passage
through a sterile 5 .mu.M filter to remove host cell debris.
Material was centrifuged as before, the parasite pellet containing
free tachyzoites was resuspended in HBSS, and the total number of
viable tachyzoites was determined using a hemacytometer and trypan
blue.
[0067] Preparation of Homogenate (Neospora Antigen)
[0068] Viable tachyzoites prepared as described above were adjusted
to a cell density of 2.times.10.sup.8/ml in Dulbecco's phosphate
buffered saline (DPBS). For each 1 ml of tachyzoite suspension, 5
.mu.l each of protease inhibitor stocks A and B were added.
Protease inhibitor stock A contains 1 ml EDTA solution (prepared by
adding 1.46 gm EDTA to 5 ml H.sub.2O) and 4 ml H.sub.2O. Protease
inhibitor stock B contains 1 ml NEM (N-ethyl maleimide), solution
(prepared by adding 312 mg NEM (Sigma Chemical Co.) to 2.5 ml
ethanol), 1 ml pepstatin solution (prepared by adding 3.43 mg
pepstatin (Sigma) to 5 ml ethanol), 3 ml PMSF (phenylmethylsulfonyl
fluoride) solution (prepared by adding 291 mg PMSF (Sigma) to 5 ml
ethanol), and 1 ml TPCK (N-tosyl-L-phenylalanine chloromethyl
ketone) solution (prepared by adding 176 mg TPCK (Sigma) to 5 ml
ethanol).
[0069] The tachyzoite preparation was frozen (-20.degree. C.) and
thawed (room temperature) three times, and then sonicated (Branson
Sonifer 250, Branson Inc.) at a constant output (4 minutes/cycle)
for three cycles on ice. The resulting homogenate was designated as
a Neospora antigen (NSA) preparation. The protein concentration of
the NSA preparation was determined using a commercial assay (Pierce
BCA). NSA preparation aliquots were prepared and stored at
-20.degree. C. or -70.degree. C. until further use in a vaccine and
for in vitro assays (e.g., Western blot, cell proliferation). The
NSA preparation did not contain any viable tachyzoites, as
determined by lack of in vitro growth in MARC-145 cells and the
inability to kill immunodeficient, nude mice.
[0070] Vaccine Formulation
[0071] The vaccine tested herein comprises the NSA preparation
prepared as above and a veterinarily acceptable adjuvant. One of
two different adjuvants, either SEAM62 or SEAM1/2, was used as
adjuvant. SEAM62 is an oil-in-water emulsion containing 5% (v/v)
squalene (Sigma), 1% (v/v) SPAN.RTM. 85 detergent (ICI
Surfactants), 0.7% (v/v) TWEEN.RTM. 80 detergent (ICI Surfactants),
2.5% (v/v) ethanol, 200 .mu.g/ml Quil A, 100 .mu.g/ml cholesterol,
0.5% (v/v) lecithin, and 0.01% Thimerosal (Sigma). SEAM 1/2 is an
oil-in-water emulsion containing 5% (v/v) squalene, 1% (v/v)
SPAN.RTM. 85 detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v)
ethanol, 100 .mu.g/ml Quil A, 50 .mu.g/ml cholesterol, and 0.01%
Thimerosal.
[0072] Vaccines were prepared by adding equal volumes of the NSA
preparation and adjuvant (SEAM62 or SEAM1/2), followed by gentle
mixing, and were stored at 4.degree. C. for primary and boost
immunizations. Final vaccine protein concentration in both
experiments was 250 .mu.g NSA protein/ml. Control vaccines
contained adjuvant alone (SEAM62 in Example 2, below; SEAM1/2 in
Example 3, below).
EXAMPLE 2
Immunization and Challenge of Immunocompetent Mice
[0073] The purpose of this two-part study was to demonstrate the
ability of a homogenate of Neopora cells, in this case tachyzoites
of N. caninum strain NC-1, to induce a protective immune response
in immunocompetent mice.
[0074] In the first part of the study, 8 week old female BALB/c
mice (n=10/group) were immunized at day 0 and again at day 21 with
either the SEAM62 adjuvant alone (control) or the NSA preparation
plus the SEAM62 adjuvant (vaccine). Fifteen days after the last
immunization, individual serum samples were randomly collected from
3 mice per group and stored at -20.degree. C. for analysis of
parasite-specific antibodies by Western blot (FIG. 1). The
post-immunization Western blot analysis was conducted as follows.
The NSA preparation was fractionated alongside molecular weight
markers (Novex, San Diego, Calif.) under standard, nonreducing
conditions by preparative gel electrophoresis (SDS-PAGE) using 12%
sodium dodecyl sulfate-polyacrylamide precast gels (Novex).
Following electrophoresis, separated proteins were transferred to
PVDF membrane (Millipore, Bedford, Mass.), which was then rinsed in
wash buffer (phosphate buffered saline (pH 7.5)/0.5% Tween 20
detergent), air-dried, and individual membrane strips cut (approx.
8 .mu.g NSA protein/strip). Strips were incubated overnight at room
temp. in blocking buffer (wash buffer containing 5% skim milk).
Following two brief washes, strips were incubated for 1 hr at room
temp. with primary antiserum samples (1:200 dilution in wash
buffer) obtained at 15 days after the last immunization from either
3 individual adjuvant control mice (FIG. 1, lanes 1-3) or 3
vaccinated mice (FIG. 1, lanes 4-6). Following two rinses in wash
buffer, strips were incubated with alkaline-phosphatase conjugated
goat anti-mouse IgG (Kirkegaard & Perry) (1:10,000 dilution in
wash buffer) for 1 hr at room temp, rinsed twice in wash buffer,
and immunoreactive proteins detected using the chromogenic
substrate BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitroblue
tetrazolium) (Kirkegaard & Perry).
[0075] As shown in the Western blot in FIG. 1, serum antibodies
collected from the three mice immunized with the NSA preparation
plus adjuvant (lanes 4-6) were reactive with NSA preparation
proteins having molecular weights of about 17-19, 28-30, 33, 37,
46, 48 and 56 kD. The immunoreactivity profiles of the serum
samples from the three mice immunized with the NSA preparation plus
adjuvant were essentially identical to each other and due to
administration of the NSA preparation since no NSA-specific
antibodies were detected in control mice administered adjuvant
alone (lanes 1-3).
[0076] In the second part of the study, 4 week old female BALB/c
mice (n=16/group) were immunized on days 0 and 14 with either the
NSA preparation plus the SEAM1/2 adjuvant (vaccine), or with the
SEAM1/2 adjuvant alone (control). Seven days after the last
immunization, 4 donor mice were randomly selected from each group.
Serum was collected from each mouse, pooled, and stored at
-20.degree. C. until testing by immunofluorescence Splenocytes from
donor groups were then prepared using standard procedures. Briefly,
pooled spleens from each group were disrupted using tissue mesh
sieves to obtain a single-cell suspension, and erythrocytes were
lysed in Tris-buffered 0.83% NH.sub.4Cl. Following viable cell
counts, aliquots from each of the two pooled splenocyte
preparations (vaccine, control) were used for pre-challenge
antigen-specific lymphocyte proliferation and cytokine assays as
described below. Remaining pooled splenocytes were used for
adoptive transfer into T-cell deficient, athymic mice (see Example
3, below). Remaining mice were subdivided into 4 groups (n=6/group)
for subsequent challenge.
[0077] On day 22 post-immunization, groups (n=6/group) of BALB/c
mice administered the NSA preparation plus SEAM1/2 adjuvant, or the
SEAM1/2 adjuvant alone were challenged subcutaneously with either
1.times.10.sup.6 or 1.times.10.sup.7 NC-1 tachyzoites. Three weeks
post-challenge, individual serum samples from all mice were
collected, mice were euthanized, and tissues (spleen, lung and
brain) individually collected for subsequent processing and assays
as described below.
[0078] Pre- and post-challenge immunofluorescence antibody (IFA)
titer assays were conducted as follows. Viable NC-1 tachyzoites
(5.times.10.sup.4) were added to each well of a 96-well flat bottom
plate. Wells were air-dried, and plates were stored at -20.degree.
C. until used. Serum test samples collected on day 21
post-immunization (day 0 challenge) and day 21 post-challenge were
tested for IFA titers. Starting at an initial 1:50 serum dilution,
serial twofold dilutions were added to wells and incubated for 30
min at room temperature. Following two washes in carbonate rinse
buffer, wells were incubated with (Fab).sub.2 fluorescein
isothiocyanate-conjugated anti-mouse IgG+IgM (Southern
Biotechnology, Birmingham., Ala.) The plates were washed and 50
.mu.l of 50% glycerol diluted in rinse buffer was added to each
well. Plates were stored at 4.degree. C. until viewed under a
fluorescence microscope equipped with a filter for emission at 510
nm. Antibody titers are based on the highest dilution of immune
serum producing detectable fluorescence.
[0079] Based on the results of the IFA titer assays, the vaccine of
the invention is capable of inducing a humoral immune response
resulting in the production of antibodies reactive against whole
tachyzoites (FIG. 2). The mean IFA titer from 4 randomly pooled
serum samples from mice immunized with vaccine was >25,000
compared to a mean titer of <50 using pooled sera from control
mice. Post-challenge geometric mean titers were significantly
higher in vaccinated animals than in controls (P<0.001) and
higher than pre-challenge titers, indicating that the vaccine
induced a memory immune response in vaccinated animals that was
boosted upon subsequent parasite challenge.
[0080] The ability of the NSA preparation to induce cellular
(T-cell) immune responses was determined as follows. Seven days
after the last immunization, mice injected only with adjuvant
(control) and mice injected with the NSA preparation plus adjuvant
(vaccine) were euthanized and their spleens removed. Pooled spleens
(4 per group) were disrupted using tissue mesh sieves to obtain a
single-cell suspension, followed by lysis of erythrocytes in
Tris-buffered 0.83% NH.sub.4Cl. Following viable cell counts, cells
were suspended in complete RPMI 1640 medium containing 10%
heat-inactivated FBS, penicillin (100 U/ml), streptomycin (100
.mu.g/ml), 5.times.10.sup.5 M .beta.-mercaptoethanol, 2 mM
L-glutamine, 5 .mu.g/ml insulin, 10 .mu.g/ml transferrin, and 10
.mu.g/ml selenium. For proliferation assays; cells were plated in
96-well flat-bottomed plates at 5.times.10.sup.5 cells per well.
Cells were incubated with complete medium with or without 5-fold
serial dilutions of the NSA preparation starting at 10 .mu.g. NSA
protein/ml in quadruplicate wells in a final volume of 200 ml.
Plates were incubated at 37.degree. C. in 7% CO.sub.2 for 72 hr.
Proliferation of T lymphocytes was assessed by pulsing splenocyte
cultures with 0.33 .mu.Ci of [.sup.3H]thymidine for an additional
18 to 24 hr. Cells were harvested onto filters using a MACHIII cell
harvester (TomTech, Orange, Conn.), and incorporation of
radioactivity was determined with a scintillation counter (Wallac,
Turku, Finland). Results are expressed in FIG. 3 as .DELTA. cpm
(mean cpm with NSA minus mean cpm with medium alone).
[0081] Splenocytes from vaccinated mice, but not from control mice,
proliferated in vitro following stimulation with the NSA
preparation. These in vitro cell proliferation assay results
demonstrate that the vaccine of the present invention can induce
cellular (T-cell) immune responses.
[0082] For cytokine assays, cells were plated in 96-well
flat-bottomed plates at 5.times.10.sup.5 cells per well. Cells were
incubated with complete medium with or without the NSA preparation
(10 .mu.g NSA protein/ml final concentration) in quadruplicate
wells in a final volume of 200 .mu.l. Plates were incubated at
37.degree. C. in 7% CO.sub.2, and cell-free supernatants collected
at 24 hr intervals for 4 days and stored at -20.degree. C. until
testing. The presence of specific cytokines in collected samples
was determined by two-site immunosorbent assay (ELISA) using a
panel of commercial cytokine-specific unconjugated and conjugated
antibodies, recombinant cytokine standards and protocols suggested
by the manufacturer (PharMingen, San Diego, Calif.). Results are
expressed as pg/ml in which the background cytokine activity in
wells of cells incubated without NSA was subtracted.
[0083] Donor pre-challenge splenic antigen-specific cytokine
production is shown in FIG. 4, demonstrating that the vaccine of
the present invention can induce both type 1 (IFN-.gamma., IL-2)
and type 2 (IL-6, IL-10) cellular immune responses following
immunization. The induction of IFN-.gamma. is especially
noteworthy, as this cytokine has recently been demonstrated to play
a protective role against murine neosporosis, (Khan et al., 1997,
Experimental Parasitology, 85:24-34). Moreover, IFN-.gamma. appears
to be required for host protection against the related Apicomplexa
parasite, T. gondii (Suzuki et al., 1988, Science 240:516-518). The
induction of IFN-.gamma. by the vaccine of the present invention
may also be involved in the vaccine's ability to protect
immunocompetent mice, as well as in it's ability to induce memory T
cells capable of secreting IFN-.gamma. following adoptive transfer
of such cells into immunodeficient athymic mice, as described below
in Example 3. The ability of the killed vaccine to induce IL-6 and
IL-10 may also be important in the vaccine's ability to protect
against neosporosis since both cytokines have been shown to play an
important role in host protection against T. gondii (Suzuki et al.,
1997, Infect. Immun. 65: 2339-2345.: Neyer et al., 1997, Infect.
Immun., 65:1675-1682).
[0084] The ability of the vaccine of the present invention to
induce cellular (T-cell) immune responses was further established
by examining day 21 post-challenge splenic antigen-specific
proliferation, as described above for day 21 post-immunization
mice. As shown in FIG. 5. T-lymphocyte responses to the NSA
preparation following challenge with either 1.times.10.sup.6 or
1.times.10.sup.7 NC-1 tachyzoites were significantly higher
(P<0.01, P<0.05) in vaccinated animals than in controls.
[0085] The ability of the vaccine of the invention to protect
animals against Neospora-induced encephalitis was determined by
measuring the number of lesions in lung and brain tissue sections
following infection of vaccinated and control mice at 2 different
challenge doses. On day 21 post-challenge, lung and brain tissues
were individually collected (6/group) and fixed in 10% neutral
buffered formalin, sectioned, and stained with hematoxylin and
eosin using routine histological techniques. Stained lung and brain
sections were coded and scored in a blinded fashion without prior
knowledge of treatment. Lung and brain lesions were scored using
the following system: (0) within normal limits, (1) slight, (2)
mild, (3) moderate, (4) severe and (5) marked.
[0086] Post-challenge brain and lung lesion scores from BALB/c mice
are presented in FIG. 6 (a-d), demonstrating that animals immunized
with the vaccine of the present invention have significantly lower
mean lung (p<0.01) and brain (P<0.05) lesions compared to
controls following a parasite challenge of 1.times.10.sup.7 NC-1
tachyzoites. In addition, mean brain and lung lesion scores are
numerically higher in control mice compared to vaccinated mice
following a parasite challenge of 1.times.10.sup.6 NC-1
tachyzoites. The lack of statistically significant difference in
lesion scores between vaccinated and control mice at the
1.times.10.sup.6 challenge dose can be attributed to one outlier
mouse in the vaccine group, which had both a lung and brain score
of 2.
EXAMPLE 3
Challenge of Immunodeficient Mice Following Adoptive Transfer of
Splenocytes
[0087] The purpose of this study was to determine if splenic
lymphocytes from vaccinated, immunocompetent mice could be used to
adoptively transfer protection to T-cell immunodeficient athymic
mice (nu/nu or `nude` mice, Charles River Labs), as demonstrated by
a prolongation of survival following a virulent, NC-1
challenge.
[0088] Each nude mouse (n=7/group) received an intravenous
injection of 1.times.10.sup.7 splenocytes (in 0.1 ml DPBS) obtained
from either vaccinated or adjuvant control BALB/c mice seven days
after the last immunization. Control nu/nu mice received only DPBS
(0.1 ml). On day 22 (24 hr post-transfer), all nude mice were
challenged subcutaneously with 5.times.10.sup.6 NC-1 tachyzoites.
Challenged nude mice were monitored for clinical signs of
neosporosis and death beginning on day 14 post-challenge.
[0089] Post-challenge survival curves of athymic nude mice are
presented in FIG. 7. Nude mice which received DPBS alone (no
splenocytes="no cells") were highly susceptible to challenge with
NC-1 tachyzoites. By day 21 post-challenge, none of the mice in
this group survived. Nude mice receiving splenocytes from BALB/c
mice that were injected either with the NSA preparation plus
adjuvant or adjuvant alone lived longer. Eighty percent of nude
mice receiving splenocytes from BALB/c mice that were injected with
adjuvant alone eventually succumbed to their infection, and only 1
mouse in this group was alive at the termination of the experiment
(day 48 post-challenge). By contrast, 100% of nude mice receiving
splenocytes from BALB/c mice that were injected with the NSA
preparation plus adjuvant survived parasite challenge through the
entire experimental period. These results demonstrate that
splenocytes from vaccinated donors are capable of conferring
adoptive protective immunity against neosporosis, and provide
further evidence for the efficacy of the vaccine of the present
invention.
[0090] All patents, patent applications, and publications cited
above are incorporated herein by reference in their entirety.
[0091] The present invention is not limited in scope by the
specific embodiments described, which are intended as single
illustrations of individual aspects of the invention. Functionally
equivalent compositions and methods are within the scope of the
invention. 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 foregoing description. Such
modifications are intended to fall within the scope of the appended
claims.
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