CA2035474C - Vaccine protecting against pig haemophilosis - Google Patents

Abstract

The protecting vaccine agains pig haemo-philosis includes the toxin from serotype1 Haemophilus (Actinobacillus) pleuropneumoniae, which is the basis for the cytotoxic and haemolytic activity of H. pleuropneumoniae, in a vehicle or excipient belon-ging to the types used in the preparation of vaccines.

Description

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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 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 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.

e:~l ~,: :! t~, it 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, ~;~;~ ~~ ~~E~
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 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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as .' ~'x 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 g 3 g 3 7 g 2 11 ~ 10 4 i ~..~ I'j ,!~

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 1 ~ l .~
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.

Claims (13)

CLAIMS:

1. A protective vaccine against porcine haemophilosis, comprising a purified and inactivated toxin of serotype 1 of Haemophilus (Actinobacillus) pleuropneumoniae, which has been inactivated by formalin, which appears in the form of 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.

2. The vaccine according to claim 1, characterized in that it further comprises an adjuvant.

3. The vaccine according to claim 2, characterized in that aluminium hydroxide is used as adjuvant.

4. The vaccine according to any one of claims 1 to 3, characterized in that the toxin is purified from a supernatant H. pleuropneumoniae culture of serotype 1, wherein the culture is a strong toxin producer and wherein the culture was grown in a medium to which Ca++ and NAD have been added.

5. The vaccine according to any one of claims 1 to 4, characterized in that it also contains a purified anatoxin obtained from a toxin of any one of serotypes 2, 3, 4, 6, 8 and 12.

6. The vaccine according to claim 5, characterized in that the purified anatoxin is inactivated 120 kDa toxin of H.
pleuropneumoniae serotype 2.

7. The vaccine according to any one of claims 1 to 4, characterized in that it also contains a purified anatoxin of serotype 6.

8. A process for preparing an inactivated toxin of serotype 1 as defined in claim 1, 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.

9. The process according to claim 8, characterized in that the strain is cultivated with a Bacto Columbia Broth medium containing Ca++ and NAD.

10. A process for preparing a protective vaccine according to claim 1 comprising admixing the purified and inactivated toxin of serotype 1 and the vehicle or excipient.

11. The process of claim 10 further comprising the step of adjuvating the toxin.

12. The process of claim 11 wherein aluminium hydroxide is used as an adjuvant in the adjuvating step.

13. The process of any one of claims 10 to 12 characterized in that the toxin is purified from a supernatant H. pleuropneumoniae culture of serotype 1, wherein the culture is a strong toxin producer and wherein the culture was grown in a medium to which Ca++ and NAD have been added.