AU635349B2 - Protection vaccine against swine hemophilosis - Google Patents

Abstract

The protection vaccine against swine hemophilosis includes the toxine of the serotype 1 of Hemophilus (Actinobacillus pleuropneumoniae) which sustains a cytotoxic and hemolytic activity of H. pleuropneumoniae, in a vehicle or carrier of the type used in the production of vaccines.

Description

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 haemorrhagic pneumonia.

The germ which is responsible for this disease isHaemophilus (Actinobacillus) pleuropneumoniae which will hereafter be called H. pleuropneumoniae.

Twelve capsule serotypes have been described in the world.

Factors for the virulence of H. pleuropneumoniae are essentially three the endotoxin (LPS lipopolysaccharide), the capsule (capsule polysaccharide) and one or several toxin(s) responsible for the cytotoxic activity as well as the haemolytic activity of H. pleuropneumoniae. Said toxins are 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 contemplated. 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 macrophages, peripheral blood monocytes and testicular cells Infect. Immun., 1981, 33, 673-676) and Rosendal et al. 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 recovering 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. pleuropneumoniae', 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. pleuropneumoniae vaccination Challenge studies, 1984, 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 ,f 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 pleuropheumoniaehaemolysin, 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 serotype 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.

Object of the invention is a vaccine protecting against pig haemophilosis, characterized in that it comprises the inactivated toxin in the form of 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 adjuvant, notably aluminum hydroxide.

The anatoxin is preferably at a final concentration of 50 pg/ml.

The vaccine is preferably administered at a rate of 100 pg anatoxin per dose.

The anatoxin included in the vaccine is preferably obtained by purifying the supernatant in a culture of serotype 1 H. pleuropneumoniae 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, t 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 in the vaccine is preferably about 50 pg/ml and the dose which is administered also preferably includes an 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. pleuropneumoniae, 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, iotably with formalin.

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 obtaining 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 (BioM6rieux), 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 CaCl 2 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 0 C under stirring. 100 ml from this medium are then seeded and cultivated during 6 hours at 37 0 C, under stirring.

At this stage, the culture may be inactivated by adding 1.6 mg/ml formaldehyde, The culture is then centrifugated 7000 g during 20 minutes. The supernatant is concentrated by precipitating with 40% saturated ammonium sulphate during 30 minutes at 4 0 C with stirring.

The precipitate as obtained after centrifugation "cr "'i 7 at 10000 g during 10 minutes is redissolved in a TNC buffer (10 mM Tris HC1, 9% NaCI, 10 mM CaCI 2 The toxin may also be concentrated by ultrafiltration (Molecular Weight cut-off 10 000 Da).

The concentrate may be inactivated by heating during one hour at 56 0

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. Characteristic features of the column are 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 NaCI 10 mM CaCl 2 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 (figure 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.

8 II. Preparation of 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 and may also be made according to other assays 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 ml vaccine. The assay is made on D 21 by intraperitoneally 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/'o challenged mice Table 1 Challenge serotype Control 'Anatoxin' vaccine 1 9 0 2 4 0 3 9 3 4 8 3 5 5 0 6 5 0 7 9 2 8 7 1 9 5 0 10 10 2 11 10 3 12 10 3 4 4.2Y Th-' 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 EOPS (without specific pathogenic 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 (heterologous challenge).

Results are collected in table 2 below.

Table 2 Clinical Death rate Lung injuries symptoms 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 serotyDe 2 anatoxin, a strain of this serotype may also be cultured under conditions which are identical 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, ane may proceed as for the serotype 1 anatoxin.

Claims (12)

1. A vaccine protecting against pig haemophi- losis, characterized in that it includes the toxin, as inactivated in the form of anatoxin, of Haemophilus (Actinobacillus) ple lopneumoniae serotype 1, which is the basis of a cytotoxic and haemolytic activity by H. pleuropneumoniae, in a vehicle or excipient of a type used in the preparation of vaccines.

2. A vaccine according to claim 1, charac- terized in that the toxin is inactivated with formalin.

3. A vaccine according to claim 1 or 2, characterized in that the anatoxin is adjuvated with aluminum hydroxide. to

4. A vaccine according/any of claims 1 to 3, characterized in that the anatoxin has a final concentration of 50 pg/ml. A vaccine according to any of claims 1 to 4, characterized in that it is administered at a rate of 100 pg anatoxin per dose.

6. A vaccine according to any of claims 1 to 5, characterized in that the toxin is obtained by purifying the supernatant from a serotype 1 H. pleuropneumoniae strain culture which is a high toxin producer, in a medium to which Ca++ and NAD are added.

7. A vaccine according to any of claims 1 to 6, characterized in that it also contains an inactivated purified anatoxin from another serotype 2, 3, 4, 6, 8, and 12.

8. A vaccine according to claim 7, charac- terized in that the serotype 2 anatoxin, or a -variant or peptide equivalent thereof, is obtained via I I 12 genetic recombination or peptide synth,

9. A process for obtaining the toxin which is responsible for the cytotoxic and haemolytic acti- vity of serotype lHaemophilus (Actinobacillus) pleuropneumoniae for the preparation of protective vaccines according to any of claims 1 to 8 characterized in that a serotype 1 H. pleuropneumoniae strain which is a high producer of said toxin, is selected, in that the selected strain is cultivated under optimal vaccine expression conditions, in that the culture supernatant is collected, and in that the toxin is extracted and purified, and then inactivated. A process according to claim 9, characterized in that the selected strain is cultivated with a Brain-Heart Broth medium containing Ca++ and NAD.

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

12. A process according to claim 9, characterized in that the selected strain is cultivated with a Wilkins-Chalgren medium containing Ca and NAD. ABSTRACT VACCINE PROTECTING AGAINS PIG HAEMOPHILOSIS The protecting vaccine agains pig haemo- philosis includes the toxin from serotypel .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. r dBr, i".Y ~:ti~P INTERNATIONAL SEARCH REPORT International Application No PCT/FR 90/00688 I. CLASSIFICATION OF SUBJECT MATTER (it several classification symbols apply, indicate all) According to international Patent Classification (IPC) or to both National Classification and IPC IPC 5 A 61 K 39/102 II, FIELDS SEARCHED Minimum Documentation Searched 7 Classification System i Classification Symbols IPC 5 A 61 K Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched I Ill. LOCUMENTS CONSIDERED TO BE RELEVANT' Category Citation of Document, with indication, where appropriate, of the relevant passages 12 Relevant to Claim X Commonwealth Agricultural Bureaux, 1,12 abstract No. 0141079, (Castelar, Argentina), J.M. Miquet et al.:"Immunizing pigs against contagious pleuropneumonia. I. Preliminary trial" Gaceta Veterinaria 1983, Vol. No. 381, pages 626, 629-636 see abstract X Commonwealth Agricultural Bureaux, 1-12 abstract No. 0520089, (Castelar, Argentina), S. Rosendal et al.: "Protective efficacy of capsule extracts of haemophilus pleuropneumoniae in pigc and mice" Veterinary Microbiology 1986, Vol. 12, No. 3, pages 229-240 see abstract SSpecial categories of cited documents: to later document published after the international filing date document defining the general state of the art which is not or priority date and not in conflict with the application but considered to be of particular relevance cited to understand the p r inciple or theory underlying the invention earlier document but published on or after the international document of particular relevance: the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(n) or involve an inventive step which is cited to establish the publication date of another "Y document o( particular relevance: the claimed invention citation or other special reason (as specified) cnnot be considered to involve an inventive step when the document relerring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 28 November 1990 (28.11.90) 17 January 1991 (17.01.1991) International Searching Authorily Signature of Authorized Officer European Patent Office Form PCT/ISA/210 (second sheet) (January 1985) 2 'U irArnomA PCT/FR 90/00688 IU. DOCUMENTS CONUIDNMO rO S aLUVaNT rCOMWTnuaE rOm rTM SECOmD uirrm SCarry C "a D WM WImmo- -me Om rarm orf a w m-MM I WmW to he Y R. Hesse:"Haemophilus pleuropneumonia 1-12 vaccination/challenge studies", 1984, page 111, IPVS de Ghent, (BE) see the whole article (cited in the application) Y GB, A, 2197193 (OLDCASTLE VETERINARY 1-12 RESEARCH LTD.) 18 may 1988 see the whole document Y Vaccine, Vol.5, No. 4, December 1987, 1-12 (Londres, GB) Institut Pasteur Bucharest: "Attenuated haemophilus pleuropneumoniae strain is nonheamolytic and nonpathogenic intranasally or intracheally; prepara- tion from a more virulent strain", page 139 RO-90029, 30 August 1986 see the whole article Y Biological Abstract, abstract No. 84004412, -12 L. Molnar et al.:"Pleuropneumonia caused by haemophilus-pleuropneumoniae equals haemophilus-parahaemolyticus in in swine. VI. Field vaccination Trials Magy Allatory Lapja, Vol. 42, No. 1, 1987, pages 23-28 see abstract Y Chemical Abstracts, Vol. 105, No. 25, 1-12 22 December 1986, (Columbus, Ohio, US) see page 581, abstract No. 224115r W.B. Fenwick et al.:"Immune responses to the lipopolysaccharides and capsular polysaccharides of haemophilus pleuro- pneumoniae in convalescent and immuni- zed pigs" Infect. Immun. 1986, Vol. 54, No. 2 pages 575-582 Y Chemical'Abstracts, Vol.95, No. 1, 1-12 6 July 1981, (Columbus, Ohio, US) see page 365, abstract No. 12749a JP, A, 8139024 (NISSHIN FLOUR MILLING) 14 April 1981 "rm FCT-;&AM94wom memiwmawv ima PCT/FR 90/00688.. WawmwwAas" No. I1U. D0C3l HNTS CONMWSRILD TOSS9 RELEVANT f=U1MSUND P~om m& bacalso Suirn C 7. 01 OMAK woo w WI mrwat Inw o M= ont C"Wejnq0 Y Chemical Abstracts, Vol. 95, No. 4, 1-12 July 1981, (Columbus, Ohio, US) see page 339, abstract No. 30390f JP, A, 8139023 (NIPJN FLOUR MILLS) 14 April 1981 Y WO, A, 80/02113 (NORDISK DROGE KEMIKALIE) tL-12 16 October 1980 see pages 2,4,6,8-10 ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. FR 9000688 SA 40864 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 04/01/91 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date GB-A- 2197193 18-05-88 BE-A- 905715 02-03-87 WO-A- 8002113 16-10-80 BE-A- EP-A- GB-A,B NL-T- SE-A- 882619 0026209 2057882 8020132 8008500 03-10-80 08-04-81 08-04-81

30-01-81 03-12-80 7 For more details about this annex see Official Journal of the European Patent Office, No. 12/82 RAPPORT DE RECHERCHE IN17ERNATIONALE Demands Internationals N- PCT /FR 9 0 '00 68 8 1. CLASSEMENT DE LINVENTION (si Plusiours symboles de classification sont applicable%, la iniforO k'ous, Selon In classification internatiofl des brevets (CIB) Ou A Ia fois salon In claistfication ,latioriale at Is CIB CIB' A 61 K 39/102 I1. DOMAINES SUR LESQUELS LA RECHERCH4E A PORTE Documentation minimal. consult~e Syst~me de clasairication Symboles da classification cn.. A 61 K Documentation consultde auite que In documentation minimal. dana Is mesurs oii de tels documents font Paflie des domaines sur losquels Is recherche a port: 1l1. DOCUMENTS CONSIOERtS COMME PERTINENTS to Cat~gorleIdentifiction desdocuments cit~s,11 avec indication, si n~cessairs.-F e eclcto Catgoie Ientfiatin ea des passage% pertinents is v i d e 1 ::at X Cornm.nwealth Agricultural Bureaux, 1,12 abr~g6 no. 0141079, (Castelar, Argentina), J.M. Miquet et al.: "Immunizing pigs against contagious pleuropneumonia. I. Preliminary trial" Gaceta Veterinaria 1983, volume no. 381, pages 626,629-636 voir l'abrdg6 X Commonwealth Agricultural Bureaux, 1-12 abr~g6 no. 0520089, (Castelar, Argentina), S. Rosendal et al.: 'Protective efficacy of capsule extracts of haemophilus pleuropneumoniae in pigs and mice" veterinary Microbiology 1986, volume 12, no. 3, pages 229-240* voir l'abr~g6 *Catilgorios spilcialos do documents diil:" a T a document ultdriour publii Postiou~ement kto date do dhp6t a document d~finissant l'dtat gilndral do as technique, non International ou A Ia date do priorith at riappartenant 042 o ni A6 com aiuiroetprinn 'tat dotI& technique pertinent, mats citt pour comprendre consdir coma prliuliremet prtientI. principo ou I& thdorio constituent I& bass do linvention Ea document anl~riour, maim Publl A I& date do d~p6t Interna. a X a document carticulitremant pertinent: IInventlon revendl- tional ou spree carte date quils no pout itre considdrie comma oouvelle ou comma o L a document Pouvant later un dout. aur une revondilcation do impliquant une activild inventive prioritd ou citi pour d~torminer Is date do publication d'une aYa document particulitirament pertinent: IlInvention revon- autro citation ou pour une raison spidiate (tells ou'Indiqluit) dlqui. no pout 6tra considilrke comma Impliquant une a01 Os document so ritirant A uns dlvulgatlon orals, A un usage, A activitt inventive tongue Is document ost associd a un ou unt exposition ou tous autres moyana plusiours autres documents do m~ine nature, cato cornbi- a P document PublI6 avant I& date do d~p6t International, mai naison itant ivldente pout une parsonne du m~tier. poatiriouremont ii a& date do priorlth roendil'As a Asa document ut fit partie do I& m~ma famille do brevets IV. CERTIFICATION Data h saquall. In recherche Internationas a 06A etectlvoment Date doexc~ditlon du pr~sent rapport do recherche Internationals achov~e 1 7, o, 91 28 novembre 1990 Administration cttsrgile do Ia recherche Internationale Signature du lorictlonnaIre autortal OFFICE EUROPEEN DES 13DEVETS -175~3 FTTS7 Formulaire PCTIISA1210 (deauxisime Iuille) (jenvwe 1095) Demande internationale N, PCT/FP. 90/00688 (SUITE DES RENSEIGNEMENTS INDIQUES SUR LA III, DOCUMENTS CONSID9R95 COMME PERTINENTS DEUXIIME FEUILLE) Cat~gorjo *ide ixt~ation an oocumonta cites. avoc indication. si n.cssaire. W 0 fts reyencdica:,ons Caoal es passage$ pertlnerits 01144 Y Hesse: "Haemophilus pleuropneumonia 1-12 vaccination challenge studies", 1984, page 111, IPVS de Ghent, (BE) voir !'article en entier (cit6 dans la demande) Y GB, A, 2197193 (OLDCASTLE VETERINARYL 1-12 RESEARCH LTD.) 18 mai 1933 voir le document en entier Y Vaccine, volume 5, no. 4, d~cembre 1987, 1-12 (Londres, GB), Institut-Pasteur Bucharest: "'Attenuated haemophilus pleuropneumoniJae strain is nonheamolytic and nonpathogrenic intranasally or intracheally; prepara- tion from a more virulent strain", page 319 RO-90029, 30 aodt 1936 voir l'article en entier Y Biological Abstract, abr~g6 no. 84004412, 1-12 L. Molnar et al.: "Pleuropneumonia caused by haemophilus-pleuropneumoniae equals haemophilus-parahaemolyticus in in swine. VI. Field vaccination trials Magy Allatory Lapja, volume 42, no. 1, 1987, pages 23-23 voir l'abr~g6 Y Chemical Abstracts, volume 105, no. 25, 1-12 22 d6cembre 1986, (Columbus, Ohio, US) voir page 531, abr~g6 no. 224115r W.B. Fenwick et al.: "Immune responses to the lipopolysaccharides and capsular 11 polysaccharides of haemophilus pleuro- pneumoniae in convalescent and imnanized pigs" Infect. Immun. 1986, volume 54, no. 2, pages 575-582 Y Chemical Abstracts, volume 95, no. 1, 1-12 6 juillet 1981, (Columbus, Ohio, US) voir page 365, abrdgd no. 12749a JP, A, 8139024 (NISSHIN FLOUR MILLI NG) 14 avril 1981 Formulaire PCTIISA/210 (fioullie addltlonnsle) (Janvww 1995) Demande internationae N, PCTI/FR 90 /00688 (SUITE DES RENSEIGNEMENTS INDIQUES SUR LA

111. DOCUMENTS CONSIDgRgS COMME PERTINENTS DEUXItME FEUILLE) Ia~ol o.,ntrticaton Oe" oauments al1so. avec indcir-at si rc.sairs, N 0 c~ r.oifndmations Y Chemical Abstracts, volume 95, no. 4, 1-12 juillet 1981, (Columbus, Ohio, us) voir page 339, abr~g6 no. 30390f JP, A, 8139023 (NIPPON FLOUR M 1 ILLS) 14 avril 1981 Y WO, A, 80/02113 (NORDISK DROGE KEMIKALIE) 1-12 16 octobre 1980 voir pages.2,4,6,8-10 Formulaire PCTIISA,1210 (feugIle adtionnelt.) (JSatvwe 1995) ANNEXE AU RAPPORT DE RECHERCHE INTERNATIONALE RELATIF A LA DENIANDE INTERNATIONALE NO. FR 9000688 SA 40864 La prksente anneoxe indiquL los memhres de la famille do brevets relatifs aux documents brevets cites dans le rapport de recherche intornationalo vise ci-dessus. Lesdits membres sont contenus au fichier informatiquc de l'Office curopien des brevet,; A la date dui 04/01/91 Los rcnseigncruents fou.rnis sont donnes a titre indicatif et n'engagent pas la responsabilit6 dc l'Qllice europeen aes brevets. Document brevet cit6 Date de \lembre(s) de la Date de au rapport do rchorche p ub lication famille de brevet(s) publication GB-A- 2197193 18-05-88 BE-A- 905715 02-03-87 WO-A- 8002113 16-10-80 BE-A- 882619 03-10-80 EP-A- 0026209 08-04-81 GB-A,B 2057882 08-04-81 NL-T- 8020132 30-01-81 SE-A- 8008500 03-12-80 Pour tout renseignement concernant ceete annexe voir Journal Oficiel do I'Ofice europ~en des brevets, No.12/82