Note: Descriptions are shown in the official language in which they were submitted.
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1
A VACCINE PROTECTING AGAINST PIG HAEMOPHILOSIS
The invention relates to a vaccine
protecting against pig haemophilosis and a process for
obtaining the active principle included in said
vaccine.
Pig pleuropneumonia or pig haemophilosis
is a disease which is widely distributed geographically
and which has serious economic consequences. This
disease is notably characterized by a fibrinous
pleurisy and an haemor.rhagic pneumonia.
The germ which is responsible for this
disease isHaemophilus L-(Actinobacillus) pleuropneumoniae
which will hereafter be called H. pleuropneumoniae.
Twelve capsule. serotypes have been described in
the world.
Factors for the virulence of H. pleuropneu-
moniae are essentially three : the endotoxin
(LPS lipopolysaccharide), the capsule (capsule poly-
saccharide) and one or several toxins) responsible for
the cytotoxic activity as well as the haemolytic
activity of H. p,leuro n~ ice. Said toxins are
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still ill-defined and it is not known with certainty
whether these two activities are due to one or several
toxins.
Given the difficulties in implementing
sanitation programs, vaccination has often been contem-
plated. After the failure of vaccines prepared from
inactivated bacterial bodies (weak and specific
protection of serotypes included in the vaccine,
low innocuity),'sub-unit' type vaccines were studied.
However, efforts for the preparation of
sub-unit vaccines from the lipopolysaccharide, the
capsule polysaccharide and from outer membrane
proteins, were inconclusive : the protecting effect
is insufficient and specific.
Haemolysin (haemolytic toxin) was studied
after the research by Bendixen et al. (Toxicity of
Haemophilus pleuropneumoniae for porcine lung macro-
phages, peripheral blood monocyte~ and testicular
cells - Infect. Immun.. 1981, 33, 673-676) and Rosendal
2p et al. (H. pleuropneumoniae lung lesions induced
by sonicated bacteria and sterile culture supernatant --
Proceedings IPVS Copenhagen, 1980, p. 221) who showed
the presence of antihaemolysin antibodies in reco-
vering pig sera and the neutralising effect of an
hyper-immune rabbit serum as to the haemolytic
and cytotoxic activities.
Van Leengoed et al. (Vaccination of pigs
with.toxin. -containing supernatant of H. pleuropneumoniae -
1988, Thesis 'Pathogenic Studies on H. gleuropneumoniae',
State University of Utrecht) have shown that a
vaccine based on a serotype 9 toxin prepared in an
oily adjuvant provided a protection during an
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homologous virulent challenge in pigs. Vaccinated animals
present antibodies neutralizing haemolytic and cytotoxic
activities.
Conversely, R. Hesse et al. (H. pleuropneumoniae
vaccination - Challenge studies, 1994, IPVS Ghent, Belgium, p.
111) have shown that a vaccine based on serotype 1 cytotoxin as
inactivated by heating and without adjuvant did not provide a
protection during an homologous virulent challenge, and that a
vaccine made up of serotype 1 toxin with an emulsion as an
adjuvant, did not protect against a challenge by serotype 5.
They demonstrated a protection for a vaccine comprising both
the toxin and inactivated bacterial bodies.
Finally, J. Frey et al. (Biochemical and genetic
characterization of Actinobacillus pleuropneumoniae haemolysin,
1988, IPVS Rio de Janeiro, p. 79) have shown the importance of
the presence of Ca++ in the culture medium for the expression
of haemolysin in some serotypes, notably serotype 1. The
culture medium used by these authors is Columbia Broth to which
are added IsoVitaleX* and NAD.
Object of this invention is to provide a vaccine
against pig haemophilosis giving an efficient protection
against serotypes 1 and at least partial protection against
other serotypes of H. pleuropneumoniae and which is highly
innocuous.
Another object of the invention is to provide a
vaccine which is inexpensive and easy to make.
According to one aspect, the invention provides a
protective vaccine against porcine haemophilosis, comprising a
purified and inactivated toxin of serotype 1 of Haemophilus
(Actinobacillus) pleuropneumoniae, which appears in the form of
*Trade-mark
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20497-648
3a
a 105 kDa band in PAGE-SDS electrophoresis, and which, in its
non-activated form, confers cytotoxic and haemolytic activity
to H. pleuropneumoniae, and a vehicle or excipient of the type
used in the make up of vaccines.
According to another aspect of the present invention,
there is provided a process for preparing an inactivated toxin
of serotype 1 as defined herein comprising: a) selecting a
strain of H. pleuropneumoniae of serotype 1 which is a strong
producer of toxin; b) cultivating the strain under conditions
for toxin expression in a culture medium containing Ca++ and
NAD; c) recovering supernatant from the culture; d) purifying
the toxin, which appears in the form of a 105 kDa band in PAGE-
SDS electrophoresis and confers cytotoxic and haemolytic
activity to H. pleuropneumoniae, from the supernatant; and e)
inactivating the toxin with formalin.
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Object of the invention is a vaccine protecting
against pig haemophilosis, characterized in that
it compr'~osfes the inactivated toxin in the form of
anatoxin,/~'serotype 1 of H. pleuropneumoniae,
which is the foundation of H. pleuropneumoniae's
cytotoxic and haemolytic activity, in a vehicle
or excipient of the types used in the making of
vaccines. ,
The anatoxin is preferably obtained by inactivation
of the toxin by formalin.
To the anatoxin is preferably added an
adju.vant, notably aluminum hydroxide.
The anatoxin is preferably at a final
concentration of 50 ug/ml.
The vaccine is preferably administered
at a rate of 100 ug anatoxin per dose.
The anatoxin included in the vaccine
is preferably obtained by purifying the supernatant
ix~ a culture of serotype 1 _H. p'Leuropneumoniae strain
producing the toxin in a high amount in a medium
to which Ca++ and NAD are added.
In an improved embodiment, the inventive
vaccine may comprise, apart from the thus defined
serotype 1 anatoxin, the purified anatoxin of another
serotype, notably 2, 3, 4, 6, 8 and 12, from a toxin
which is duly inactivated, preferably with formalin.
The anatoxin of this other serotype may
be obtained by concentrating and purifying to a high
degree the supernatant in a culture of the serotype,
but it may also be obtained, especially for serotype
2, by expressing a genetic recombinant vector, and
under anatoxin of other serotypes, for instance 2,
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is also included the recombinant anatoxin as well
as its fragments, variants and peptides with relevant
epitopes, or else via a peptide synthesis.
The other serotype anatoxin concentration
5 in the vaccine is preferably about 50 ug/ml and
the dose which is administered also preferably includes
aw amount which is similar to that of the serotype
1 anatoxin.
In the inventive sense, an anatoxin of
a given serotype includes any polypeptide, or all
polypeptides, in the culture supernatant, to which
cytotoxic et/or haemolytic activities are attributed.
However, for serotype 1, the anatoxin comprises
or is made up by haemolysin and, for serotype 2,
if any, or the other serotypes, these preferably
contain or are made up by a polypeptide of about
120 KDa to which a cytotoxic activity is attributed.
The inventive vaccine will preferably be
administered intra-muscularly, sub-cutaneously
or intradermally in one or two injections.
Object of this invention is also a process
for obtaining the toxin responsible for the cytotoxic
and haemolytic activity in serotype 1 H. pleuro-
gneumoniae, so as to prepare protective vaccines
of the above described type.
In this process, a strain of serotype 1 H.
_pleuropneumoniae which is a high producer of said
toxin is selected, this selected strain is cultivated
in optimal conditions for the expression of the toxin,
the culture supernatant is collected and the toxin
is extracted and purified. The toxin is then inactivated,
and this inactivation may be carried out according
to any appropriate known technique, notably with
formalin.
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According to the invention, the selected
strain may be notably cultured in any of the three
following culture media. containing Ca++ and NAD
- Brain-Heart broth medium,
- Bacto Columbia Broth Medium,
- Wilkins-Chalgren medium.
The invention will now be described in
more detail with the help of a process for
obtaining the serotype 1 toxin. and with, the help
of vaccination assay in mice and pigs.
I. _Process for obtaininq the toxin
The serotype 1 strain as described by
Shope R.E. (1964),,J. Exp. Med. 119, 357-368, was
chosen for its important toxin production.
The culture medium used must contain Ca++
and NAD. The Brain-Heart broth media (BioMerieux),
as well as Bacto Columbia Broth (Difco) and
Wilkins-Chalgren media may notably be used.
In this process, the Bacto Columbia Broth
Medium, to which are added 10 mM CaCl2 and 15 mM
NAD is used.
A freeze-dried vial from the cultured
strain is introduced in 5 ml of the medium and left
during 18 hours at 37°C under stirring. 100 ml
from this medium are then seeded and cultivated
during 6 hours at 37°C, under stirring.
At this stage, the culture may be inactivated
by adding 1.6 mg/ml formaldehyde.
The culture is then centrifugated at
7000 g during 20 minutes. The supernatant is concen-
trated by precipitating with 40~ saturated ammonium
sulphate during 30 minutes at 4°C with stirring.
The precipitate as obtained after centrifugation
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at 10000 g during 10 minutes is redissolved in a
TNC buffer (10 mM Tris HCl, 9% NaCl, 10 mM CaCl2).
The toxin may also be concentrated by
ultrafiltration (Molec'ular Weight cut-off . 10 000 Da).
The concentrate may be inactivated by heating during
one hour at 56°C.
The toxin is then purified by two consecutive
S200 HR (Pharmacia-LKB) gel filtration chromatography
runs . . The fractionating ' : , range of this gel is between
50 000 and 250 000 for globular proteins. Charac-
teristic features of thecolumn arm the following .
Gel volume 190 ml
Height . 95 cm
Section area 2 cm
Deposition
volume 10 ml
Flow rate 20 ml/h
Eluting buffer 10 mM Tris HC1
9 %o NaCl
10 mM CaCl2
The SDS-PAGE electrophoresis profile (according
to Laemli, 1970, Cleavage of structural protein during
the assembly of the head of bacteriophage T4, Nature
227, 680-685) shows only one band, which corresponds
to a molecular weight of 105 KDa, It then
seems that for serotype 1, only one toxin is responsible
for both cytotoxic and haemolytic activity. In any
case, according to the invention, the vaccine includes
the anatoxin with molecular. weight 105 KDa, even
if other antigenic components may or not, be present.
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II. Preparationof a vaccine
The vaccine is prepared from the purified anatoxin as
obtained in the above manner. The anatoxin formulation
is made starting from the haemolytic titer before
inactivation (titer about 3), and may also be made
according to other assays (eg. ELISA). The anatoxin
is adjuvated with alumina gel (0.7 mg/ml) and formalin
(1.6 mg/ml).
III. _Vaccination assay on mice
The above mentioned vaccine was tested
during a vaccination assay on mice
To the mice were given subcutaneously on D 0
0.5 ml vaccine. The assay is made on D 21 by intra-
peritoneally injecting 10 50~ lethal doses per
mouse under 0.5 ml. Results are collected in table
1 below :
Results are expressed as the number of
dead animals;' to challenged mice ':
Table 1
~~l~ge serotype Control 'Anatoxin' vaccine
1 9 0
2 4 0
g 3
g 3
5 5 0
6 5 0
7 g 2
7 1
9 5 0
10 10 2
11 ~ 10
12 10 3
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This shows that the vaccine provides an
efficient and heterologous protection against all
serotypes described in the mice model and this may
be extrapolated to pigs.
IV. _Vaccination assay on pigs
Two groups of FOPS (without specific patho-
genic organisms) piglets are raised under the same
conditions. One group is vaccinated at ages
8 weeks and 12 weeks. The other group is used as
a control.
The two groups are~intranasally(104 germs).
tested at age 14 weeks with serotype 9 (heterologou~
challenge). .
Results are collected in table 2 belos~.
Table 2
Clinical Death rate
I
symptoms Lung injuries
Vaccinated 0 0 1
pigs group (small
(for 8 pigs) abscesses)
Control 8 ! 8 1
group
(for 8 pigs) (in 48 h)
These results clearly demonstrate the
efficiency of the inventive vaccine.
In order to prepare a vaccine also containing
serotype 2 anatoxin, a strain of this serotype may
also be cultured under conditions which are identical
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or similar to those described above.
The culture supernatant is centrifugated
and concentrated by precipitating with ammonium
sulphate, the precipitate is redissolved and purified
by ion exchange chromatography. followed by gel-
filtration, or by hydroxy-apatite gel chromatography.
Inactivation of the purified toxin is preferably
made with formalin. . '
For the other serotypes such as 3, 4, 6,
8, 12, one may proceed as for the serotype 1 anatoxin.
