Note: Descriptions are shown in the official language in which they were submitted.
~1~;i5
eJVO 94118311 PCTIUS94/00951
-1-
PROCESS FOR GROWING PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS AND
ITS USE IN VACCINES.
BACKGROUND OF THE INVENTION
Field of the Invention: The present invention relates to a
porcine reproductive and respiratory syndrome virus (PRRSV)
isolate. More specifically, the present invention relates to
diagnostic and protective antigens and vaccine for porcine
reproductive and respiratory disease of pigs, and the methods
of making and using the same.
Brief Description of the Prior Art: Porcine reproductive
and respiratory syndrome is rapidly emerging as an economically
devastating disease problem for U.S. and European swine produc-
t o ers. The syndrome was first described in the U.S. in 1987,
with similar descriptions surfacing in Europe and Canada, two
to three years later. The disease syndrome has been referred
to by many different names including mystery swine disease,
abortus blau, blue eared pig disease, porcine epidemic abortion
15 and respiratory syndrome (PEARS), swine infertility and respi-
ratory syndrome (SIRS). The name porcins~ reproductive and
respiratory syndrome (PRRS) is employed hereinafter.
The etiological agent capable of reF~roducing the disease
syndrome has been identified as a small s:nveloped spherical RNA
2o virus, with an average virion diameter oi-' 62 nm and a 25-30 nm
core surrounded by an envelope. This virus has been
tentatively grouped as a member of the genus Arterivirus within
the Togaviridae family. The porcine reproductive and
respiratory syndrome virus (PRRSV) isolate, described herein,
25 fits this tentative classification and has been shown to
reproduce the disease syndrome.
CA 02155533 2004-05-04
30138-3
2
The disc=_ase syndrome associated with PRRSV is
characterized by acute and chronic reproductive failure in
adult female swim? and severe to mild respiratory disease in
young pigs. Reproductive failure is characterized by late
term abortions resulting in increased incidence of
mummified, stillborn and weak born pigs with markedly
reduced chances for survival. Chronic problems with delayed
return to estrus in infected sows has also been described.
Respiratory disease sequela range from marked fever and
interstitial pneumonitis to mild upper respiratory signs
(i.e. sneezing, coughing, and nasal or ocular discharge) in
young pigs. Recent (1990) serological and herd history
studies indicate that at least 50% of U.S. swine herds have
been exposed to FRRSV or have experienced reproductive
failure and respiratory disease indicative of PRRSV
infection.
Due to the recent emergence of this disease
syndrome, studies on the pathogenesis, epidemiology and
control of disea~~e have been limited. As would be realized,
an efficient propagation and processing of the antigens
comprising PRRSV, will facilitate the development of a PRRSV
vaccine as an aid in the prevention and control of porcine
reproductive and respiratory syndrome.
SUMMARY OF THE INVENTION
In accordance with the foregoing, the present
invention encompasses a virus isolate designated as ISU-P
which is useful in the preparation of antigens for the
diagnosis of porcine reproductive and respiratory syndrome
and the induction of a protective immune response to porcine
reproductive and respiratory syndrome virus (PRRSV). Also
encompassed by the invention are the processes for making
and using the antigens.
CA 02155533 2004-05-04
30138-3
2a
In one aspect, the invention provides a process
for growing a porcine reproductive and respiratory syndrome
virus (PRRSV) designated as ATCC VR 2402 by growing the
virus in a tissue culture to an amount sufficient to protect
animals against PRRS, to diagnose PRRS or to identify the
molecular structure of PRRSV for subunit or recombinant
products, comprising inoculating PRRSV onto a tissue culture
derived from MA 104 cells and harvesting the grown virus.
In another aspect, the invention provides a
process for growing a porcine reproductive and respiratory
syndrome virus (PRRSV) that is reactive with antiserum to an
isolate ISU-P designated as ATCC VR 2402 by growing the
virus in a tissue culture to an amount sufficient to protect
animals against PRRS, to diagnose PRRS or to identify the
molecular structure of PRRSV for subunit or recombinant
products, comprising inoculating PRRSV onto a tissue culture
which is of a cloned African Green Monkey Kidney cell line
clone 9009B designated as ATCC CRL 11302 and harvesting the
grown virus.
In a further aspect, the invention provides a
tissue culture containing the PRRSV produced according to
the process defined above.
In a further aspect, the invention provides a
process for preparing an effective vaccine for protecting
pigs against PRRS comprising providing PRRSV as defined
above, releasing the PRRSV from the tissue culture cells and
adjusting antigenic mass by dilution, concentration or
extraction to produce an immunologically effective amount of
the antigenic mass for a subunit or recombinant product.
In a further aspect, the invention provides a
vaccine prepared by the process of growing a porcine
reproductive and respiratory syndrome virus (PRRSV) in a
CA 02155533 2004-05-04
30138-3
2b
tissue which is susceptible to infection and replication of
PRRSV to an amount sufficient to protect animals against
PRRS comprising inoculating the PRRSV onto tissue cells and
harvesting the replicated virus, releasing the virus from
the tissue cells and adjusting the virus antigenic mass by
dilution, concentration or extraction to produce an
immunologically effective amount of the antigenic mass for a
modified live or _Lnactivated formulation of the vaccine
wherein the PRRSV is an isolate designated as ISU-P and
having an Accession number ATCC VR 2402.
In a further aspect, the invention provides a
porcine reproduct:~ve and respiratory syndrome virus isolate
designated as ISU-P and having an Accession number
ATCC VR 2402.
In a further aspect, the invention provides a
continuous cell line designated as 9009B and having an
Accession Number ATCC CRL 11302.
In a present embodiment of the invention, PRRSV
isolate can be obtained by a process comprising isolating
the PRRSV by co-cultivation of 10% weight/volume (w/v) lung
homogenates from
f
'~ ~ ~ PCT/US94/00951
~'VO 94118311
-3-
virus infected pigs with primary porcine <<lveolar macrophage or
continuous cell line cultures at 35'C.
The present invention further encompasses propagation of
the virus to high titer in certain cell lines such as an
African Green Monkey Kidney continuous cell line (MA 104) and a
unique cloned derivative of the same (900!~B).
Also encompassed by the invention arse processes for
preparing and using live virus or killed ~rirus antigens
(conventional or recombinant) and the vac~~ines resulting
to therefrom by combining an immunologically effective amount of
the virus with diluent and/or adjuvant, respectively.
It has been found that direct experimental immunization of
sows with live virus or contact with live virus immunized sows
prevented reproductive failure following an intranasal chal-
1 5 lenge with virulent PRRSV.
It has also been found that inactivated, adjuvanted PRRSV
vaccines protected pigs from PRRS infection post challenge as
measured by lack of virus replication in vaccinates as compared
with non-vaccinated control pigs.
DETAILED DESCRIPTION OF THE INVENTION
As set forth above, the present invs:ntion is directed to
the isolation of the PRRSV virus isolate ISU-P for the purpose
of obtaining viral antigens for use in vaiccines and diagnostic
assays. The ISU-P isolate was obtained from the lung tissue of
2s a weak born pig. Illustratively, a 10% w/v tissue homogenate
from lung, spleen, and lymph node was prs~pared in Minimal
Essential Media supplemented with antibiotics (MEM+A) to
minimize the potential for contamination. The crude homogenate
was clarified by centrifugation at 4°C for 15 minutes at
1500 x g. Clarified supernatant was diluted 1:5 in MEM+A and
l.OmL was adsorbed onto a 48 hour old confluent monolayer of MA
104 cells or primary alveolar macrophages in 25 cm2 flasks.
Following a 2 hour adsorption period at 35°C monolayers were
_ washed twice
2 ~.~~ ~ ~~~~' jy
WO 94/18311 PCT/US94/00951
-4-
with MEM+A, and 5.0 mL of MEM+A supplemented with 5.0%
gamma-irradiated fetal calf serum was added. Virus isolation
flasks were incubated at 35°C in a 5% C02 atmosphere and
observed daily for evidence of cytopathic effect (CPE). The ,
PRRSV ISU-P cytopathic effect began with the rounding of small
foci of 5 to 10 cells and progressed as many cells became
pyknotic and detached from the substrate over the course of 4
to 7 days post infection.
The ISU-P isolate was confirmed as a PRRSV isolate by
Io fluorescent antibody staining with PRRSV group specific
monoclonal antibody, designated in the public domain as SDOWI7.
In addition, negative stained virus infected cells were
examined by transmission electron microscopy for the presence
of virus particles. The virus particles observed were
spherical and enveloped with an average virion diameter of 65
nm and core diameter of 27 nm. Tissue culture virus
characterized in this manner was used to experimentally
reproduce late term abortion and reproductive failure in
pregnant sows (Table I). Table 1 shows that pregnant gilts
2o exposed to either ISU-P-infected lung homogenates (PRRS-hm) or
ISU-P grown in a cell culture (PRRS-tc) demonstrate typical
disease signs of porcine reproductive and respiratory syndrome
(PRRS). In the PRRS-hm groups there were no normal pigs
farrowed. In the PRRS-tc groups only 4 of 33 pigs were
farrowed normal. In comparison, 29 of 30 (96.7%) of the
non-exposed pigs were normal at farrowing. The fact that
ISU-P, even after purification in tissue culture, can reproduce
the disease of PRRS confirms that this isolate is correctly
classified as a member of the genus Arterivirus within the
3o To4aviridae family. This isolate has also been found to
produce a mild interstitial pneumonia in experimentally
infected pigs.
It is a feature of the invention that the PRRSV in accor-
dance with the invention can be grown in certain tissue culture
cells which effectively replicate the virus to a level
~WO 94118311
~' PCT/US9410095I
-5-
sufficient enough to provide the antigenic mass necessary
to be
incorporated in vaccines which can protect pigs and/or
pregnant
gifts or sows from PRRS challenge. Illustratively, the
PRRSV
can be grown in a tissue culture cell designated MA 104
by
Whittaker BioProducts. Virus propagation experiments,
. employing identical culture conditions, with MA 104 cells
obtained from three different sources indicate that a
high
degree of variability exists among MA 104 cells in terms
of the
ability to support PRRSV replication to high titer. One
MA 104
cell culture designated MA 104(M) by Miles Inc., Agriculture
1 o Division, Animal Health Products Group, and used in the
studies
described herein, consistently supported PRRSV replication
to
high titer. Furthermore, a cell line clone, generated
at Miles
Inc., Agriculture Division, Animal Health Products Group,
which derived from MA 104(M), designated 90098, supports
15
replication of PRRSV to titers higher thin the parental
cell
line. Therefore, the preferred tissue culture cell for
growth
of ISU-P is 90098. The following more fully illustrates
cell
culture propagation of the PRRSV ISU-P.
Tissue culture cells [MA 104 (M) and 90098] were grown
and
2o maintained in Dulbecco's modified minimum essential media,
high
glucose (DMEM/HG), supplemented with 5% fetal calf serum
and
buffered with 1.3g/L sodium bicarbonate. Cell passages
were
made with sufficient cell counts to attain a 80-100%
confluent
monolayer in 48 hours. PRRSV ISU-P virus stocks were
diluted
2s in DMEM/HG + 5% fetal calf serum buffered to pH 6.8 with
PIPES.
Virus was inoculated onto the cell sheet at a low multiplicity
of infection (MOI) and incubated at 36C. Infected cultures
were observed daily for CPE for 4-7 days. Infected cultures
were harvested when 90-100% CPE was evident. Maximum
virus
3o antigenic mass was obtained after one -70C freeze/thaw
cycle.
Mean virus titers resulting from propagation in MA 104(M)
cells
ranged from 103'0 to 104'5 flourescent antibody infectious
dose
/mL). Virus titers achieved by propagation in
/mL (FAID
50
the 90098 cell clone ranges from 105'5 i;o 106'5 FAID
50 /mL.
3s
WO 94/18311 ~ PCT/US94/00951
-6-
Titers of this magnitude make it possible to incorporate
sufficient antigenic mass into commercial scale PRRSU vaccine
formulations of greater than 50L in volume.
As would be realized by those skilled in the art, once the ,
s PRRSU can be propagated to high levels of antigenic mass,
derivatives of this virus including subunits thereof which are
effective in accordance with the invention, can be obtained by
means known to the art such as extraction from the virus.
Additionally, the protective antigens can be identified at the
1 o molecular level and reproduced and expressed using recombinant
technology.
Viral fluids for vaccine formulation were inactivated by
two methods. The inactivants chosen were formalin and binary
ethylenimine (BEI). Inactivation was performed by standard
1 s methods described more fully herein. Formalin inactivation was
accomplished by mixing viral fluids with stock 37% formaldehyde
to a final formalin concentration of 0.05%. The formalin-virus
mixture was held at room temperature (approximately 25°C) with
constant mixing for 24 hours. Samples were taken at times 0,
20 8, 12 and 24 hours and assayed for live virus in 9009B cells.
Cytopathic effect and fluorescent staining with group specific
monoclonal antibody detected live PRRSV only at time = 0 hours.
Inactivation with BEI was accomplished by combining stock
0.1 M BEI (20.5 g/L 2-bromo-ethylamine HBR in 0.175 N NaOH)
2s with viral fluids to a final concentration of 1.0 mM BEI.
Inactivation was performed by holding the BEI-virus mixture at
room temperature with constant mixing for 48 hours. Virus
inactivation was halted by the addition of 1.0 M sodium
thiosulfate to a final concentration of 0.1 mM with mixing for
~0 2 hours. Samples were taken at times 0, 2, 4, 8, 12, 24 and 48
hours and assayed for live virus as described above. Live
PRRSV was detected only at times = 0, 2 and 4 hours. .
In the preparation of a modified live or attenuated
vaccine of the invention, the PRRSV is altered so that it can
still infect the host in a limited manner but cannot cause
~WO 94/18311 ~ ~S'' PCT/US94/00951
signs of disease in the host. Normally, such alteration of the
virus takes place by passaging it through a non-host cell such
as African Green Monkey Kidney cells. Initially, the virus may
not grow well or even not all. In the latter case, it may be
necessary to force the virus to adapt to growth in cell
culture. This can be done by adding the virus to the cell
culture at a high or low multiplicity of infection (MOI).
Alternately, the virus can be force-passaged or blind passaged
along with the cells until enough cytopathic effect is
1 0 observed, thus indicating that virus is being adapted. As
adaptation occurs, the virus titer will increase as is observed
when the PRRSV isolate ISU-P is passaged from the MA 104(M)
cell line to the 9009B cell line. Table 1 shows that the
tissue culture passaged PRRSV ISU-P (PRRS-tc) may be less
1 s virulent than that isolated from lung horr~ogenates, a typical
result of adaptation. Data disclosed in Table 2 indicate that
the method of making vaccines in accordance with this invention
are especially effective in conferring protection against PRRS.
The inactivated PRRSV ISU-P was adjuvanted as follows.
2 o Inactivated fluids were adjuvanted with <<djuvants selected from
the group consisting of Freund's complete adjuvant (FCA), ,
Freund's incomplete adjuvant (FIA), a carbopol based adjuvant,
and Diamond Scientific Adjuvant B (Adj B;I. These adjuvants
represent the major types used in vaccinE~ manufacture.
2 s Oil-based adjuvants are represented by F(:A, FIA and ADJ B.
Aqueous-based adjuvants are represented by carbopol. The
inactivated PRRSV ISU-P may optionally contain a suitable
preservative.
Vaccine formulations with FCA and FIA were prepared by
so emulsifying the inactivated virus, typically in equal volumes
of inactivated virus fluid and adjuvant using an 18 guage
double luer lock apparatus and two syringes. The fluids were
expressed repeatedly through the assembly between syringes
_ until a thickened semi-solid emulsion was formed. The
WO 94/18311 ~ PCT/US94/00951
;. -8-
1...
emulsions were loaded directly into syringes for
administration.
Vaccine formulations with Adj B were prepared by mixing
stock Adj B with an equal volume of inactivated PRRSV ISU-P ,
viral fluid. Adjuvant and virus was mixed by stirring at room
temperature for 1 hour. Adjuvanted vaccine was aseptically ,
transferred to sterile multidose vials and stored at 4°C prior
to administration.
Carbopol-based vaccine formulations were prepared by
to mixing stock carbopol adjuvant with inactivated viral fluids to
a final adjuvant concentration of 10% v/v. Adjuvant and virus
was mixed by stirring at room temperature for 4 hours.
Adjuvanted vaccine was aseptically transferred to sterile
multidose vials and stored at 4°C prior to administration.
PRRSV ISU-P virus at a preinactivation titer as low as
105'0 FAID50/mL was used in the various vaccine formulations
described.
In order to show that PRRSV isolate ISU-P could protect
against porcine reproduction and respiratory syndrome, two
2o vaccination/challenge studies were conducted. In the first
study, 4 gilts were immunized with live PRRSV ISU-P by oronasal
exposure to virus via either a direct route or contact with
diseased pigs. Two gilts of the same age were left unimmunized
(unexposed) and were housed separately to serve as controls.
The direct-exposure vaccinates (#57 and #58) received 3.0 mL of
virus inoculum intranasally. They seroconverted by 14 days
post exposure according to an Indirect Fluorescent Antibody
Test. The contact-exposed gilts (#476 and #480) were immunized
by the oronasal route after being placed in close contact with
3o diseased pigs. These pigs seroconverted by 90 days post
exposure. Seroconversion is indicated by an IFA titer greater
than 1:20.
After the immunized gifts seroconverted, their estrus was
synchronized and they were bred. Pregnancy was confirmed by
ultrasound. On gestation day 90, vaccinate and control
~WO 94J18311 PCTJUS94JUU951
_g_
pregnant gifts received 3.0 mL of PRRS challenge virus
at a
titer of 1 X 104 FAID50, intranasally.
Control gifts #52 and #477 demonstrai:ed early signs
of
disease in that they had reduced appetite<.> and elevated
body
temperatures (103-104F) for two to three days post challenge.
,
No clinical signs of disease were noted in the ISU-P
exposed
(vaccinated) gifts. These results demonsi~rate protection
from
the clinical respiratory disease syndrome.
All gifts were held until pigs were ~~reaned (4 weeks
post
farrowing). Table 2 shows the farrowing results from
this
1 o
experiment.
Farrowing results were recorded as t~~ gestation length
(gestation is normally shortened by PRRS infection),
still-
births or dead fetuses, weak pigs and mum~~nies. These
farrowing
difficulties are all indicative of PRRS infection in
pregnant
15
gifts or sows. Additionally, the number of normal pigs
were
noted.
Neither of the control gifts farrowed normal pigs (0
of 22
pigs were normal). Seven (7) of 22 pigs were born dead
and 15
of 22 were weak born pigs. The length of gestation of
the
20
control gifts was shortened by 2.5 days which is typical
of
PRRS in pregnant swine. In the immunized group (n = 50)
pigs or 80% were born normal. Six (6} of 50 pigs were
born
dead and 4 of 50 fetuses appeared mummified at farrowing.
There were no weak pigs in the vaccinate group. It is
clear
2s
that gifts previously exposed to live PRF:SV ISU-P which
are
bred and then challenged are protected from challenge
as
demonstrated by lack of direct clinical signs of disease
in the
gifts and by protection of the fetuses which they carry.
As would be realized, since the IFA test reagents which
so
were used to determine the serostatus of the gifts in
this
study were derived from PRRSV ISU-P and :>ince there
is a direct
correlation between the serostatus of thE~ gifts and
protection
from PRRS, PRRSV ISU-P isolate can be useful in diagnostic
tests such as Indirect Fluorescent Antibody tests as
WO 94/I831I ~ : ~ ~ s r.
PCT/US94/00951
-10-
demonstrated herein, ELISAs, serum neutralization assays,
Direct Fluoroescent Antibody assays and other art-known
diagnostic assays. Hence, a diagnostic assay derived from
antigen obtained from PRRSV ISU-P can be prepared and used in ,
accordance with the invention.
In the second vaccination/challenge study, young pigs (4 ,
to 6 weeks of age) were vaccinated with several inactivated,
adjuvanted PRRSV ISII-P vaccines. A vaccine antigen pool was
prepared by inactivation of the PRRSV ISU-P with BEI as de-
Io scribed earlier and adjuvanted with either Carbopol, FCA/FIA or
Adj B. A second vaccine antigen pool was prepared by inactiva-
tion of the PRRSV ISU-P with formalin as described earlier and
adjuvanted with the same 3 adjuvants. Three mock vaccines were
produced in order to make sure that the adjuvants and cells
1; could not stimulate a false immunological response. Mock
vaccines were prepared by adjuvanting uninfected tissue culture
cells. Two pigs were vaccinated with each vaccine. Vaccines
were administered subcutaneously on days 0, 21, 42 and 64.
Seven days after the last vaccination the pigs were challenged
2o intranasally with 103'5 FAID50 of virulent live PRRSV. All
pigs were observed daily post challenge for clinical signs of
disease. Additionally, blood samples were drawn and processed
for recovery of virus in tissue culture. Live virus found in
the blood stream post challenge signifies that a viremia has
occurred.
Serum neutralization titers were evaluated at the begin-
ning of the study, on the day of challenge and at 19 days post
challenge. It should be noted that the serum neutralization
test is less sensitive than the IFA test used in the previous
3o study. Therefore, one cannot compare these titer results with
the previous titer results.
Table 3 lists the results of the challenge. No clinical
signs of disease were observed in vaccinated or control pigs.
Additionally, neither vaccinates nor controls developed serum
neutralization titers during the vaccination period. This
.JVO 94118311 ~~'~ '~-~ PCT/US94/00951
-11-
indicates that there was no exposure to live virus during this
time period (controls remained negative) and also indicates
that the vaccines did not stimulate a humoral response high
enough to be measured by a serum neutralization test. The fact
s that serum neutralization titer response post challenge was
significant indicates that all pigs were effectively chal-
lenged with the live PRRS virus. A verification of this
challenge exposure is the percentage of ca~ntrol pigs demon-
strating viremia. All but one of the control pigs (87.5%)
to became infected. The immune response in vaccinated pigs was so
strong that they were able to block viremia. Only 1 of 12 of
the vaccinated pigs (8.3%) demonstrated viremia. Therefore,
the vaccines protected 91.7% of the pigs.
Without being bound to any particular theory of the
15 invention, it is believed that force-pass<<ging of the original
PRRSV isolate through a specially-adapted clone of MA104(M)
African Green Monkey Cells (9009B) may have successfully
altered one or more epitopes of the virus resulting in enhanced
immunogenicity of this virus so as to allow production of
2o vaccine in a field where other scientists have failed.
2s
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- I6 -
Although the invention has been described in detail in the
foregoing for the purpose of illustration, it is to be under-
stood that such detail is solely for that purpose and that
variations can be made therein by those skilled in the art
without departing from the spirit and scope of the invention
except as it may be limited by the claims.
