Note: Descriptions are shown in the official language in which they were submitted.
2~8~6~
Preparation of antigens of and of vaccines for
the virus of Mystery Disease, antigens and vaccines
obtained for the prevention of this disease.
The present invention relateæ to the preparation
of antigens of and of vaccines for the virus of Mystery
Disea~e, as well as to the antigens and to the vaccines
obtained.
The disease called Mystery Disea~e (M.D.) or also
Porcine Reproductive Re~piratory Syndrome (P.R.R.S.)
began to acquire a identity of its own in pigs, in 1986
in the United StateR and in 1990 in Europe. This disease
manifests itself essentially in pig~ by ~ign~ of exhaus-
tion, anorexia and hyperthermia of the order of 40C,
which a~e conventionally observed in ~OW8 in pig farms
affected by the disease. The~e signs are accompanied or
followed by reproductive disorders (premature or late
farrowing and bi.rth of stillborn, mummified or sickly
piglets, and return of the 50W~ to heat). A respiratory
syndrome can be observed in piglet with interstitial
pneumonia lesions. Older pigs can al~o be affected by
respiratory di~orders. All this symptomatology can be
accompani~d by diseases caused by chance infections
conventionally observed in pigs.
The inventors have succeeded Ln isolating and in
identifying a new virus, responsible for this disease.
Thi~ virus is of Myxovirus type, according to the analy-
sis carried out in the electron microscope, and has the
characteristic of not being neutralised by porcine anti-
influenza sera HlNl and H3N2.
A strain of this virus identified under the name
Pl29-Z9~ was deposited in the Collection Nationale de
Cultures de Micro-organismes held at the Institut Pasteur
under No. I-1153.
Moreover, it then turns out that this virus has
a certain number of features of the virus of Newcastle
disease, while being different from the known strains of
this virus.
The subject of the pres nt invention is a process
for isolating the virus and its use for the preparation
of antigens.
Such a process comprises taking samples from
S organs of sick or infected animals, milling the samples,
and passing the supernatant through sensitive
heterologous or homologous cells such as, in particular,
cells of the Vero, MDC~ (in the presence of trypsin), ST,
or BHK lines or also through chick embryos.
Th~ samples are preferably taken from the lung of
the infected animal or else from a pool of organs com-
prising, for example, heart, spleen, liver, kidney and
lymphoid tissues.
The harvests are not neutralised by porcine anti-
influenza sera HlNl and H3N2. The haemagglutination testsare positive from the an~igen produced in chick embryos.
Another subject of the invention is a process for
the industrial production of this virus in which the
virUc is cultured in sensitive heterologous or homologous
cells such as, especially, cells of the Vero, MDCK tin
the presence of trypsin), ST or BHR lines or also in
chick embryos.
The harvested virus can be used for the prepar-
ation of antigens. To thi~ end, it is, preferably,
suitably purified accordiny to conventional procedures
for the Myxoviruses, for example ultra-centrifugation or
chromatography. It can also be concentrated by conven-
tional techniques.
According to the use envisaged, the antigen
preparations according to the invention can consist of
living viral particles, optionally attenuated, of inac-
tivated particles, of sub-unit antigens or even of
antigens obtained by genetic recombination from genes of
the isola~ed virus, which are inserted in order to be
expres~ed in the genome of recombining procaryote or
eucaryote hosts.
Another sub~ect of the invention is the vaccines
made from the abovementioned antigens, containing an
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effective vaccinating quantity of antigens in suitable
carriers.
These vaccines can be provided to be administered
to pigs but also to other animals which could turn out to
be sensitive to this virus.
An attenuated vaccine can be prepared by pas~ing
the virus through a cell culture.
The quantity of viru~ per vaccinal dose is
preferably of between 103 and lOa TCID50 pex dose.
The living attenuated vaccine can be pre~ented in
the liquid or lyophilised form in the presence of
stabili~ers of very varied formulations, which can
include sugars, proteins and buffer~. Inorganic or
organic adjuvants can be added to the vaccine.
The living vaccine can be administered to anLmals
to protect them from the beginning of the fattening
period or before artificial insemination or covering, or
during gestation, in one and preferably two injections
at intervals of three or four weeks.
An inactivated vaccine csn be prepar d from viral
suspensions obtained by passing through homologous
(porcine) or heterologous cell systems and then inac-
tivation by conventional chemical inactivating agents
such as beta-propiolactone, enzymes or organic solvents
or detergent~O Inactivation can also be obtained by
physical action such as ultraviolet radiation or gamma
or X-ray irradiations. The inactivating agent can be
neutrali~ed if neces~ary.
The inactivated vaccine contains preferably at
least the equivalent of 105 TCIDso per vaccinal dose, a
concentration determined before inactivation.
The inactivated vaccine can be administered to
animals to protect them from the beginning of the
fattening period, or before artificial insemination or
covering, or during gestation, in one and preferably two
injections at intervals of three or four weeks. I~ is
pre~erable that the vaccine contains an adjuvant of
inorganic or organic origin.
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A recombinant vaccine can be obtained, for
example, by insertion of the sequence coding for th~
desired antigen of the ~irus in ~he genome of the host.
The host can be a virus, especially for the preparation
of a living vaccine.
This host can also be a bacterial system, a yeast
system or a system of other eucaryote cells. In this
caser the host is preferably used for the production of
antigens which are then purified and conditioned in order
to make the vaccine.
The vaccine can be presented in the monovalent
form or combined with other viral or bacterial agents
responsible for diseases in pigs.
Another subject of the invention, as an equi-
valent vaccine against Mystery Disease in pig8, iS avaccine characterised in that it comprises a quantity
which is therapeutically effective in pigR, of a virulent
strain of Newcastle virus, suitably inactivated, ox of
vaccinating antigens of such a strain, with, preferably,
a conventional ad~uvant.
Other advantages and characteristics of the
invention will become apparent on reading the following
description, given by way of non-limiting example.
Example 1 : Isol tion of the viral Rtrain
Sampleq of organs were taken from a sow origin-
ating from ~ pig farm in Germany and identified under No.
29~.
The samples came from the heart, spleen, liver,
kidney, lymphoid tissue and lung, and were used without
freezing.
a) Method:
Milled preparations of organs: each sample is
individually milled in ~EM medium + antibiotics.
Dilution: organ weight per volume of approxi-
mately 1 to 10.
Clarifying centrifugation for recovery of thesupernatant liquid (SL).
Filtration at 0.22 ~.
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b) Tests on cell cultures.
First passages
From:
1) SL of milled lung preparation
2) SL of pool of organs: heart, spleen, liver,
kidney and lymphoid tissue
cell8 u~ed: Vero, IRO.3, ST, MDCK (in the
presence of trypsin), BHK, and primary pig kidney cell
lines
. inoculation in cultures in established layer
(25 cm2 Falcons)
. ob~ervation of the cells and freezing at -70C.
Second passages
From the first unfrozen passages.
On the ~ame type of cells as the first passages;
the cells are used in simultaneous inoculation.
Observation and freezing.
c) Tests on chick embryos
. Inoculation in SPF ch}ck embryo~ of 9 days
. Inoculum: SL of milled lung prepsration from
the sow 294. 0.1 ml of undiluted inoculum or inoculum
diluted to 1/10 in PBS + antibiotics is inoculated intra-
allantoidally.
After incubating for 3 day~, the eggs are
emptied and the allantoic liquid is harve~ted (individual
harvesting from each egg).
. A haemagglutination test with respect to
chicken red cells (concentration 20-106 red cell~ per ml)
i8 carried out on all the harvestéd samples.
. The successive passages are carried out under
the condition~ described above.
d) Results
1) Results of cell cultures
In two cell types, change~ in the cell layer
could be observed:
- in Vero cells: the cells of the inoculated
Falcons show, during the second passage, an appearance of
the cell lawn different from the cell~ of the standard
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.. .
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Falcon. This abnormal appearance is observed at +7 days
after inoculation.
- in MDCK cells in the presence of t~ypsin
(concentration 20 ~g/ml): the cells inoculated by the
pool of organs show an appearance different from the
standard cells at +6 days of culture.
2) Results in egg~
First passage: no haemagglutination could be
shown.
Second passags: 1 egg showq haemagglutination.
The haemagglutination titre HA is 640.
Third passage:
. HA titre which can reach 1024
presence of HA not neutxalised by porcine anti-
influenza sera HlN1 and H3N2.
Fourth passage: haemagglutination confirmed withtitres which can reach 2048.
Additionally, a significant mortality in the eggs
could be observed in the second, third and fourth pas-
sages, it being poRsible for the mortality to exceed 50%of the inoculated eggs.
A positive haemagglutination test was carried out
with respect to guinea pig xed cells. Using the haemag-
glutina~ing antigen of the 3rd passage, cells of ST and
Vero lines and E~rimary pig kidney cells SPF were inocu-
lated, a cytopal:hogenic effect was observed in these 3
cell~, a cytopal:hogenic effect more marked than in the
cells inoculated directly with lung SL. The haemagglutin-
ating effect had disappeared after passages through
cells.
From the viral suspension of ~he 3rd passage
through eggs, 2 pigs with a weight of 30 kg were
inoculated intravenously with a volume of 1 cc. These 2
pigs showed signs of exhaustion, anorexia and hyper-
thermia of the order of 40C for at most 7 days. Thesesymptoms are those commonly observed in sows in pig farms
attacked by P.R.R.S. or M. D .
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The isolated agent thus has the following charac-
teristics:
- it causes haemagglutination of g~inea pig red
cells from antigen multiplied in chick embryos,
- it is not inhibited by porcine anti-influenza
sera,
- it produces a cytopathogenic effect in various
cells,
- its properties, made visible in the electron
iO microsrope, ally it to the Myxovirus group.
These properties are also those of the strain
P129~294 deposited in the CNCM.
Example_2 : Preparation of a livinq vaccine
The living vaccine i~ prep~red from the strain
P129-294. The strain i~ multiplied by passages through
Vero cells. The harve~ing is carried out afterward~. The
harvested and processed supernatan~ is lyophilised with
a stabiliser SPGA.
The vaccine is packaged in doses of 10, 50 and
100.
Example 3 : Prepara~ion of an inactivated vaccine
The virus of the strain P129-294 is multiplied in
ST cells. Following the fifth passage, the supernatant
containing the virus is harvested, concentrated and
purified. It is inactivated by beta-propiolactone and
then aluminium hydroxide is added thereto.
The vaccine is packaged in doses of 10 and 50.
