Note: Descriptions are shown in the official language in which they were submitted.
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PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VACCINE
BACKGROUND OF THE INVENTION
The present invention relates to a vaccine for the treatment of
porcine reproductive and respiratory syndrome (PRRS).
In 1987, the swine-producing industry in the United States
experienced an unknown infectious disease which had a serious
economic impact on the swine industry. The disease syndrome was
reported in Europe including Germany, Belgium, the Netherlands,
Spain and England.
The disease is characterized by reproductive failure,
respiratory disease and various clinical signs including loss of
appetite, fever, dyspnea, and mild neurologic signs. A major
component of the syndrome is reproductive failure which manifests
itself as premature births, late term abortions, pigs born weak,
stillbirths, mummified fetuses, decreased farrowing rates, and
delayed return of estrus. Clinical signs of respiratory disease
are most pronounced in pigs under 3-weeks-of-age but are reported
to occur in pigs at all stages of production. Affected piglets
grow slowly, have roughened hair coats, respiratory distress
("thumping"), and increased mortality.
The disease syndrome has been referred to by many different
terms including mystery swine disease (MSD), porcine epidemic
abortion and respiratory syndrome (PEARS), swine infertility and
respiratory syndrome (SIRS). The name now commonly used is
porcine reproductive and respiratory syndrome (PRRS); this term
will be employed throughout this patent application.
It is an object of the invention to provide a vaccine which
protects a pig against clinical disease caused by PRRS. Another
object is to provide a vaccine which, when administered to a
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breeding swine herd, will reduce the presence of PRRS in their
population.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a novel
vaccine which protects a pig against clinical disease caused by
porcine reproductive and respiratory syndrome (PRRS) virus.
A further object of the present invention is to provide a
vaccine which protects a pig against the strain NEB-1 of PRRS
virus.
It is a further object of the present invention to provide a
method of protecting a pig against clinical disease caused by a
porcine reproductive and respiratory disease virus.
It is a further object of this invention to provide a
vaccine virus with phenotypic characteristics which can be
distinguished from wild type PRRS virus.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a composition of matter comprising an
attenuated Porcine Respiratory and Reproductive Syndrome (PRRS)
Virus which has been modified by laboratory manipulation for use
in vaccination. Further the composition has phenotypic
properties which allow its use for diagnostic purposes to
distinguish between swine that have been naturally-infected with
PRRS virus versus animals that have only been exposed to the
vaccine strain. The PRRS virus isolate NEB-1-P94 has been
deposited with American Type Culture Collection (Accession No.
VR-2525).
A virulent isolate of PRRS virus was obtained from tissue
samples from a dead pig presented to the University of Nebraska
Diagnostic Laboratory. A tissue homogenate from the dead pig was
inoculated onto primary swine alveolar macrophages and the
presence of virus detected by cytopathic effects on inoculated
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- but not control cultures. The isolated virus (designated NEB-1)
was subsequently characterized as a PRRS virus based on physical
properties (ether and chloroform sensitivity, buoyant density,
and lack of hemagglutinating activity), reactivity with specific
antibodies, and genetic analysis. Inoculation of the virus into
nursing piglets resulted in a respiratory disease characterized
by high fever, altered respiration and lung pathology consistent
with viral interstitial pneumonia. Additionally, inoculation of
the virus into pregnant sows resulted in reproductive disease
characterized by mummification of fetuses, stillborn piglets, and
piglets born weak that subsequently died. The respiratory and
reproductive disease caused by this virus was typical of the
syndrome reported for PRRS virus.
The NEB-1 virus was attenuated by serial passage in tissue
culture. The virus was initially passed by inoculation of
primary swine alveolar macrophage (SAM) cultures (for the first
two passages) and then by serial passage on MA104 cells
(available from Microbiological Associates, Inc., Rockville, MD)
for a total of 94 passages. During this process, virus clones
were isolated by plaque purification and characterized for
phenotypic properties. The vaccine clone, designated NEB-1-P94,
was selected for impaired growth on swine alveolar macrophages,
lack of reactivity with the PRRS-specific monoclonal antibody
SDOW17 (ATCC HB10997), and lack of disease induction in piglets
and pregnant sows. The NEB-1-P94 was expanded on MA104 cells and
frozen as a master seed virus, also designated PRRS-MSV-94-1, for
use in vaccine development studies.
Vaccine is prepared using MA104 cells as the substrate
(however alternate cell lines that support the growth of PRRS
virus such as MARL 145 [available from Dr. Wang, Agriculture
Research Station, Clay Center NE] cells can also be used). MA104
cells are grown to confluency in suitable tissue culture vessels,
e.g. 850 cm2 roller bottles, using Eagle's minimum essential
media (EMEM) containing 5 to 10~ bovine serum, 30 mM HEPES (N-2-
hydroxyethylpiperazine-N~-2-ethanesulfonic acid, 2 mM L-
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glutamine, and antibiotics (such as 30 ~g/ml gentamicin).
Alternate tissue culture media that can support the growth of
MA104 cells such as Dulbecco's modified essential media [DMEM],
Medium 199, or others can also be used. Confluent monolayers of
MA104 cells are inoculated with NEB-1-P94 virus at a multiplicity
of infection (MOI) in the range of 1:5 to 1:1000, and, preferably
in the range of 1:10. Following incubation for three to five
days at 37°C, culture supernatant fluids are harvested by
decanting.
Virus fluids are titered by making serial dilutions in EMEM
supplemented as above and inoculation of 0.2 ml per well into at
least four replicate wells of confluent MA104 or MARC 145 cells
in a 96-well tissue culture plate. Cultures are incubated for
five days at 37°C, 3-5~ C02 in a humidified chamber and observed
for cytopathic effects. Titers (50~ endpoints) are calculated
according to the methods of Spearman and Karber (Methods in
Virology, Volume IV, K. Maramorosch and H. Koprowski (Eds).
Academic Press, New York, 1977). Cells may be fixed with 80~
acetone and tested for lack of reactivity with SDOW17 and
positive reactivity with V017 or EP147 (available from Dr. E.
Nelson, South Dakota State University, Brookings, SD) (expected
positive result) monoclonal antibodies to confirm phenotypic
identity of the virus.
For the preparation of a killed vaccine, virus fluids are
incubated with a chemical inactivation agent. Examples of
inactivation agents include formaldehyde, glutaraldehyde, binary
ethyleneimine, or beta-propiolactone. Virus fluids are then
stored at 4°C until formulated into vaccine. Vaccine is prepared
by mixing virus fluids (containing 106 to 109 TCIDSp of virus;
based on preinactivation titers) with a physiologically
acceptable diluent (such as EMEM, Hank's Balanced Salt Solution,
Phosphate Buffered Saline) and an immune-stimulating adjuvant
(such as mineral oil, vegetable oil, aluminum hydroxide, saponin,
non-ionic detergents, squalene, block co-polymers or other
compounds known in the art, used alone or in combination). A
vaccine dose is typically between 1 and 5 ml.
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For a live vaccine formulation, virus fluids are stored
frozen at -50°C or colder until use. Virus fluids within the
range of lOW p and 10W p TCIDSp/dose and preferably containing
106~p TCIDSp/dose are diluted with a physiologically suitable
diluent (such as EMEM, Hank's Balanced Salt Solution, Phosphate
Buffered Saline) and a physiologically suitable mixture of
compounds designed to stabilize the virus. Compounds known in
the art that can be used alone or in combination to stabilize
viruses include sucrose, lactose, N-Z amine, glutathione,
neopeptone, gelatin, dextran and tryptone. Vaccine is stored
frozen (-50°C or colder) or lyophilized with storage at 4°C
until
use. The vaccine typically has a dose size range of 1 to 5 ml,
and preferably 2 ml.
For prophylaxis against PRRS-induced disease, the vaccine is
administered to the pig orally, intranasally or parenterally.
Examples of parenteral routes of administration include
intradermal, intramuscular, intravenous, intraperitoneal and
subcutaneous routes of administration.
When administered as a solution, the present vaccine may be
prepared in the form of an aqueous solution, a syrup, an elixir,
or a tincture. Such formulations are known in the art, and are
prepared by dissolution of the antigen and other appropriate
additives in the appropriate solvent systems. Such solvents
include water, saline, ethanol, ethylene glycol, glycerol, A1
fluid, etc. Suitable additives known in the art include
certified dyes, flavors, sweeteners, and antimicrobial
preservatives, such as thimerosal (sodium ethylmercuri-
thiosalicylate). Such solutions may be stabilized, for example,
by addition of partially hydrolyzed gelatin, sorbitol, or cell
culture medium, and may be buffered by methods known in the art,
using reagents known in the art, such as sodium hydrogen
phosphate, sodium dihydrogen phosphate, potassium hydrogen
phosphate and/or potassium dihydrogen phosphate.
Liquid formulations may also include suspensions and
emulsions. The preparation of suspensions, for example using a
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colloid mill, and emulsions, for example using a homogenizer, is
known in the art.
Parenteral dosage forms, designed for injection into body
fluid systems, require proper isotonicity and pH buffering to the
corresponding levels of porcine body fluids. Parenteral
formulations must also be sterilized prior to use.
Isotonicity can be adjusted with sodium chloride and other
salts as needed. Other solvents, such as ethanol or propylene
glycol, can be used to increase solubility of ingredients of the
composition and stability of the solution. Further additives
which can be used in the present formulation include dextrose,
conventional antioxidants and conventional chelating agents, such
as ethylenediamine tetraacetic acid (EDTA).
A booster vaccination may be administered two to four weeks
after the initial immunization. For the prevention of
reproductive disease, the vaccination regimen is typically
performed up to 6 weeks prior to and 1 week after breeding. For
the prevention of respiratory disease in piglets, vaccination may
be given as early as 3 weeks of age. The response to vaccination
can be monitored by measuring antibody titer directed against
PRRS virus using enzyme-linked immunosorbent assay (ELISA), serum
neutralization assay, indirect immunofluorescence, or Western
blot.
The vaccine strain has phenotypic properties that can be
used for diagnosis in swine of wild type PRRS infection versus
exposure to only the vaccine strain. Animals exposed to field
strains of PRRS virus may be distinguished from animals exposed
only to the NEB-1-P94 vaccine strain by measurement of the
antibody response to the epitope recognized by monoclonal
antibody (MAb) SDOW17. The presence of antibodies reactive with
the SDOW17 epitope is indicative of wild type virus exposure,
Measurement of antibodies to the SDOW17 epitope can be
accomplished using a competitive ELISA. Plates (96-well) are
coated with the NEB-1 PRRS virus (or other PRRS viruses
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expressing the SDOW17 epitope). Plates are then incubated with
pig serum from test animals and enzyme-labelled (for example
conjugated to horseradish peroxidase) SDOW17 monoclonal antibody.
The ability of pig sera to recognize the SDOW17 epitope is
measured by the inhibition of enzyme-linked SDOW17 MAb binding to
the plate as detected by lack of enzyme substrate color
conversion. Alternatively, a direct ELISA may be used. The
amino acid sequence comprising the SDOW17 epitope can be prepared
as a synthetic peptide or by recombinant DNA expression methods
in a suitable vector system such as E. coli. Plates coated with
the SDOW17 antigen are incubated with pig serum. Binding of
swine antibodies to the SDOW17 antigen is detected by incubation
with enzyme-conjugated anti-swine immunoglobulin antisera
followed by incubation with enzyme substrate and detection of a
color change.
The following examples describe in detail the invention. It
will be apparent to those skilled in the art that modification of
materials and methods may be practiced without departing from the
purpose and intent of this disclosure.
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EXAMPLE 1
Phenotypic Characterization of NEB-1-P94 for Growth on
Alveolar Macrophages
The NEB-1-P94 vaccine strain virus at five passages from the
master seed was characterized for growth on MA104, MARC 145, and
swine alveolar macrophages. Swine alveolar macrophages (SAM)
were obtained by bronchi-alveolar lavage with saline followed by
centrifugation to pellet the cells. Macrophages were resuspended
in EMEM with 10~ fetal bovine serum and 50 ~.g/ml gentamicin and
plated at approximately 7 x 104 cells per well of 96-well tissue
culture plates. MA104 cells and MARL 145 cells were plated in
96-well tissue culture plates in media (EMEM containing 10~ fetal
bovine serum, 30 mM FiEPES, 2 mM L-glutamine, and 50 ~.g/ml
gentamicin). NEB-1-P94 or the parental NEB-1 virus were serially
diluted in media and 0.2 ml of each dilution inoculated into
replicate wells of 96-well plates containing SAM, MA104, or MARL
145 cells. Cultures were incubated for 5 days at 37°C, 3~ to 5~
C02, in a humidified chamber and monitored for cytopathic effects
typical of PRRS virus. Titers (50~ endpoints) were calculated
according to the method of Spearman and Karber. The NEB-1-P94
showed reduced titers or unmeasurable titers on three separate
SAM cultures compared to the titers obtained on MA104 and MARL
145 cells (Table 1). This is a phenotypic change compared to the
parental strain which showed similar titers on all of the
cultures tested. Therefore, impaired growth on swine alveolar
macrophages was a selected phenotypic marker for the vaccine
strain NEB-1-P94.
Table 1. Comparison of Growth of the NEB-1-P94 Virus on Various
Cell Types
Virus Titer* on Titer* on Titer* Titer* on Titer* on
on
MA104 MARC 145 SAM 7 SAM 8 SAM 22
NEB-1-P94 5.3 6.1 <1.2 2.5 <1.2
NEB-1 5.2 6.5 5.2 6.5 6.5
loglp detection in thisassay
*Titer TCIDSp/ml =
= (limit 1.2)
of
_g_
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EXAMPLE 2
Phenotypic Characterization of NEB-1-P94 for Lack of Virulence in
Swine
Four gnotobiotic piglets (seven to 10 days of age) from PRRS
seronegative sows were inoculated intranasally (3 ml/nare) with
NEB-1-P94 master seed virus (1053 TCIDSp/ml). Piglets were
observed for clinical signs of respiratory disease and the
vaccine strain virus was re-isolated from serum at five days
post-inoculation. Serum from the first group of pigs was used to
intranasally inoculate a second group of gnotobiotic pigs which
were monitored in the same way. This process was repeated for a
total of five serial backpassages in piglets in order to
determine whether the vaccine strain could revert to a virulent
state. Vaccine virus was recovered from each successive animal
passage, however, respiratory disease was not observed in the
gnotobiotic pigs. In addition, virus isolated from the fifth
backpassage pigs was intranasally inoculated into one-week-old
and three-week-old conventional piglets (approximately 1053
TCIDSp/ml was administered per piglet). Animals were monitored
for 42 days after virus inoculation and no clinical disease signs
(i.e. prolonged high fever, respiratory signs, lung lesions)
consistent with virulent PRRS infection were found. Therefore,
the NEB-1-P94 virus was concluded to be avirulent for induction
of respiratory disease in piglets.
Next the NEB-1-P94 virus was examined for its ability to cause
reproductive disease. PRRS seronegative sows at 85 days of
gestation were inoculated intranasally (3 ml/nare) with the
master seed vaccine strain (1045 TCIDSp/ml). All sows farrowed
at their expected time and 96~ of the piglets were born live and
healthy. By comparison, uninoculated control sows gave birth to
litters where 87~ of the piglets were live and healthy.
Therefore, the vaccine strain NEB-1-P94 failed to induced
reproduction disease typical of a virulent PRRS virus (See
Example 4). These data confirmed the avirulent phenotype of the
vaccine strain, NEB-1-P94.
_g_
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EXAMPLE 3
Phenotypic Characterization of NEB-1-P94 for Reactivity with PRRS
Virus-Specific Monoclonal Antibodies
MA104 cells infected with parental strain NEB-1 or the vaccine
strain NEB-1-P94 virus were examined for reactivity with
monoclonal antibodies specific for the PRRS virus by indirect
immunofluorescence. Briefly, 96-well plates of confluent MA104
cells were fixed with 80~ acetone for 10 minutes at 2 days after
infection with each virus. Monolayers were then incubated with
SDOW17, V017, or EP147 monoclonal antibodies. Following washing,
monoclonal antibody reactivity with each virus was detected by
incubation with fluorescein isothiocyanate conjugated anti-mouse
IgG followed by washing and examination for fluorescence by
microscopy. Positive fluorescence was noted with all three
monoclonal antibodies for the NEB-1 parental strain (Table 2).
However, the vaccine strain, NEB-1-P94, had lost reactivity with
the SDOW17 monoclonal antibody but tested positive with the other
two monoclonal antibodies. These data indicate that the NEB-1-
P94 strain had lost expression of epitope recognized by the
SDOW17 antibody. The loss of reactivity with this monoclonal
antibody most likely represents genetic mutation in the RNA
sequence of NEB-1-P94 which resulted in an altered amino acid
sequence in the nucleocapsid protein region recognized by SDOW17.
Table 2. Reactivity of Parental and Vaccine Strain PRRS with
Specific Monoclonal Antibodies.
Reactivity with Reactivity with Reactivity with
SDOW17 V017 EP147
NEB-1-P94 - + +
NEB-1 + + +
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EXAMPLE 4
Prevention of Reproductive Disease by Vaccination of Sows with
NEB-1-P94
Vaccine was prepared by inoculation of confluent roller bottles
of MA104 cells with NEB-1-P94 (4 passages from the master seed)
at a multiplicity of infection of approximately 1:10 in EMEM
containing 10~ fetal bovine serum, 2 mM L-glutamine, and 30 ~,g/ml
gentamicin. Cultures were incubated for three days at 37°C and
then supernatant fluids were harvested by decanting. Virus
fluids were diluted 50~ (v/v) with stabilizer (75 g/L tryptone,
30 g/L dextran, 2 g/L gelatin, 100 g/L lactose, 2 g/L sodium
glutamate, 1.05 g/L KH2P04, 2.5 g/L K2HP04, 10g/L albumin
fraction V), frozen, and lyophilized. Vaccine was rehydrated
with sterile deionized water and 2 ml (1051 TCIDSp/ml)
administered intramuscularly to gifts four to six weeks prior to
breeding.
At 85 days of gestation, vaccinated and unvaccinated control
gifts were challenged by intranasal administration of NEB-1 virus
(approximately 1063 TCIDSp). Animals were monitored through
seven weeks after farrowing for signs of fetal or neonatal death
attributed to PRRS virus. PRRS viremia developed in 11/12 (92~)
of control sows and 100 of their live-born piglets. PRRS
infection during pregnancy resulted in 16~ death loss at
parturition (large mummies and stillborn pigs) in the control
group (Table 3) compared to only 6~ death loss in vaccinated
sows. In addition, vaccination resulted in a 50~ reduction in
the incidence of weak and shaky piglets and 94~ reduction in
piglets with low birth weights (weighing less than 2 pounds at
birth) when compared to controls. Vaccination prevented
congenital PRRS as evidenced by the absence of PRRS virus in
blood or tissues of any piglets from immunized sows and a 55~
prevention of death loss through 7 weeks of age (Table 3) when
- compared to controls. The statistically significant prevention
of death loss and virus infection in vaccinated sows and their
offspring clearly demonstrated the efficacy of this vaccine in
preventing the reproductive form of PRRS virus-induced disease.
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Table 3. Summary of Reproductive Disease Noted After PRRS Virus
Challenge of Vaccinated versus Control Sows
Group Number of Sows $ Large $ Born $ $ Piglets 7 Week
Weak
(Ave # Pigs/Litter)Mummies Live and Weighing Mortality
and but <2
Stillborn Died Sha lbs.
Vaccinated21 (10.8) 6$ 2$ 3$ 1$ 17$
Controls12 (10.1) 16$ 3$ 6$ 17$ 38$
EXAMPLE 5
Prevention of Respiratory Disease by Vaccination of Sows with
NEB-1-P94
Vaccine was prepared by inoculation of confluent roller bottles
of MA104 cells with NEB-1-P94 (at 4 passages from the master
seed) at a multiplicity of infection of approximately 1:10 in
EMEM containing 10~ fetal bovine serum, 2 mM L-glutamine, and 30
~.g/ml gentamicin. Cultures were incubated for five days at 37°C
and then supernatant fluids were harvested by decanting. Virus
fluids were diluted 50~ (v/v) with stabilizer (75 g/L tryptone,
30 g/L dextran, 2 g/L gelatin, 100 g/L lactose, 2 g/L sodium
glutamate, 1.05 g/L KH2P04, 2.5 g/L KzHP04, 10 g/L albumin
fraction V), frozen, and lyophilized. Vaccine was rehydrated
with sterile deionized water and 1 ml (1049 TCIDSp/ml)
administered intramuscularly to PRRS seronegative piglets at
three weeks of age.
Four weeks following vaccination, piglets were challenged with
virulent NEB-1 PRRS virus by intranasal route as described for
gilts (Example 4). Piglets were monitored for respiratory
disease signs for 14 days after challenge. All unvaccinated
control piglets developed clinical signs of respiratory disease
compared with 3/40 (8~) of vaccinated animals. Vaccination
resulted in a statistically significant reduction in fever,
respiratory signs, and clinical illness in vaccinated animals
compared to controls (Table 4). This study clearly demonstrated
the efficacy of the vaccine in the prevention of respiratory
disease caused by PRRS virus in young pigs.
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