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WO1996010583A1 - Vaccin contenant une hemoproteine - Google Patents

Vaccin contenant une hemoproteine Download PDF

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Publication number
WO1996010583A1
WO1996010583A1 PCT/GB1995/002350 GB9502350W WO9610583A1 WO 1996010583 A1 WO1996010583 A1 WO 1996010583A1 GB 9502350 W GB9502350 W GB 9502350W WO 9610583 A1 WO9610583 A1 WO 9610583A1
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WO
WIPO (PCT)
Prior art keywords
haemoprotein
vaccine
antigenic
antigenic material
mammal
Prior art date
Application number
PCT/GB1995/002350
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English (en)
Inventor
John Pius Dalton
Stuart John Andrews
Original Assignee
Mallinckrodt Veterinary, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mallinckrodt Veterinary, Inc. filed Critical Mallinckrodt Veterinary, Inc.
Priority to AU35747/95A priority Critical patent/AU3574795A/en
Publication of WO1996010583A1 publication Critical patent/WO1996010583A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/43559—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from trematodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention relates to the use of a class of haemoproteins as protective antigens against helminth parasites.
  • Each species of domestic animal can be parasitised by a number of different species of helminths, a process which usually causes disease.
  • the parasitic trematode Fasciola hepatica is known to be one cause of the economically important disease fascioliasis in ruminants, such as cattle and sheep.
  • the parasite enters the mammalian host by penetrating the gut wall and spends approximately seven weeks feeding on and burrowing through the liver mass before migrating into the bile duct. Following infection, development of immunity in the host can be poor and resistance to reinfection in already infected hosts may be only partial or non-existent.
  • Other parasitic flukes include Fasciola ⁇ i ⁇ antica and Dicrpcoeljurn spp. , Pa arnphjgtPmwn spp. and also Schistosoma spp., eg S.bovis and £_». mangpni.
  • nematodes such as hookworms (e.g. Necator. Ancylostom . Uncinaria and Bunosto um spp.) .
  • parasitic worms of economic importance include the various species of the following helminth genera:- Trichpgtrpncrylus, ematpd,iru,s, Dictvocaiiliis. Cooperia. ⁇ ca j , Djrpfilarja, Trichuri ⁇ and StroncryliiR.
  • helminth genera - Trichpgtrpncrylus, ematpd,iru,s, Dictvocaiiliis. Cooperia. ⁇ ca j , Djrpfilarja, Trichuri ⁇ and StroncryliiR.
  • Control of helminth parasites of grazing livestock currently relies primarily on the use of anthelmintic drugs combined with pasture management. Such techniques are often unsatisfactory, firstly because anthelmintic drugs may have to be administered frequently, secondly because resistance against anthelmintic drugs is becoming increasingly widespread and thirdly because appropriate pasture management is often not possible on some farms and even where it is, it can place constraints on the best use of available grazing.
  • a vaccine against F.hepatica has been proposed in W090/08819 comprising a glutathione-S-transferase from F.hepatica as antigenic material. Further vaccines against F.hepatica have been proposed in WO94/09142 and W094/28925 comprising respectively a Cathepsin and a dipeptidyl peptidase from F.hepatica as antigenic material.
  • Bennett UK Patent No. 2169606B extracted various antigens from Fasciola organisms by a process which separates antigens specific to the juvenile stage from antigens present throughout the juvenile and adult stages. Furthermore crude in vitro excretory/secretory (E/S) products can under some circumstances confer immunity on rats (Rajasekariah et al, Parasitol. 22. (1979), p. 393- 400) .
  • F.hepatica excretory/ secretory products contain a novel haem-binding protein (haemoprotein) described in more detail hereinafter.
  • haemoprotein haem-binding protein
  • a first aspect of the present invention provides a vaccine for use in combating a parasitic infestation of helminths in a mammal wherein the antigenic material comprises a haemoprotein, in at least partially purified form, or an antigenic fragment or epitope thereof, together with a carrier and/or adjuvant.
  • the invention also provides a method of combating a parasitic infestation of helminths in a mammal comprising administering to said mammal a vaccine according to the invention as hereinbefore defined in an amount effective to combat said infestation.
  • the mammal is preferably a ruminant, for example cattle or sheep, but the vaccine and method of the invention may also find application in humans.
  • the haemoprotein is derived from flukes such as Fasciola or Dicrocoelium. in particular from the liver fluke Fasciola hepatica.
  • the haemoprotein should be capable of stimulating an immune response which will be effective against Fasciola or Dicrocoelium.
  • F. hepatica and F. ⁇ i ⁇ antica such haemoproteins from other species as are capable of conferring a cross-protective immune response thus forming a particularly preferred aspect of the invention.
  • the F.hepatica haemoprotein shown hereinafter to possess a molecular weight of at least 200 kDa by gel filtration chromatography is particularly preferred for use in the vaccine and method of the invention and as a novel protein itself forms a further aspect of the invention.
  • the haemoprotein incorporated in the vaccine according to the invention is in at least partially purified form.
  • the haemoprotein comprises at least 75% of the total excretory/secretory proteins present in the vaccine and more preferably the haemoprotein is at least 95% pure. It will be appreciated that once haemoprotein of at least 95% purity has been obtained it can be admixed with one or more further purified antigenic proteins, including one or more further excretory/secretory proteins, to form a polyvalent vaccine.
  • a preferred form of polyvalent vaccine according to the invention will contain a haemoprotein as referred to above in combination with a Cathepsin L-type antigen as described in more detail in International Patent Application No. WO94/09142 or a dipeptidyl peptidase antigen as described in more detail in International Patent Application No. W094/28925.
  • the Cathepsin L and/or dipeptidyl peptidase are preferably derived from flukes such as Fasciola or Dicrocoelium. in particular the liver fluke F.hepatica.
  • Such a polyvalent vaccine will, by inducing immunity in the host species against two or more separate aspects of the invading helminth parasite, significantly increase the likelihood of protection against the helminth and significantly reduce the chances of infestation occurring.
  • the polyvalent vaccine comprises a haemoprotein according to the present invention together with a Cathepsin LI having molecular weight of 27 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis as disclosed in WO94/09142 and/or a Cathepsin L2 having molecular weight of 29.5 kDa by the same technique as disclosed in WO94/09142 and/or a dipeptidyl peptidase having molecular weight of 200 kDa by the same technique as disclosed in W094/28925.
  • the vaccines according to the invention may be formulated with conventional carriers and/or adjuvants and the invention also provides a process for the preparation of the vaccines comprising bringing into association purified haemoprotein or an antigenic fragment or epitope thereof and one or more adjuvants or carriers.
  • Suitable adjuvants include aluminium hydroxide, saponin (ISCOMs) , quil A and more purified forms thereof, muramyl dipeptide, mineral and vegetable oils, DEAE dextran, nonionic block copolymers or liposomes such as Novasomes (Trade Mark of Micro Vesicular Systems Inc.), in the presence of one or more pharmaceutically acceptable carriers or diluents.
  • Carriers for peptide sequences corresponding to epitopes of haemoproteins according to the invention can be proteins such as Hepatitis B core antigen multiple antigen peptide or lipopeptides such as tripalmitoyl-S- glycerylcysteinylserylserine (P 3 CSS) .
  • Suitable diluents include liquid media such as saline solution appropriate for use as vehicles. Additional components such as preservatives may be included.
  • Administration of the vaccine to the host species may be achieved by any of the conventional routes, e.g. orally or parenterally such as by intramuscular injection, optionally at intervals e.g. two injections at a 7-35 day interval.
  • a suitable dose when administered by injection might be such as to give an amount of protein within the range 10-500 ⁇ g.
  • haemoprotein for use in the vaccine according to the invention may be prepared by isolation from the excretory/secretory products of adult and/or juvenile helminths, it may also be convenient to prepare the haemoprotein polypeptide without a haem group, or a fragment or epitope of the polypeptide, by recombinant DNA techniques with the known advantages which such techniques give in terms of scaling-up of production and reproducibility.
  • the invention also provides a haemoprotein polypeptide or an antigenic fragment or epitope thereof, produced by means of recombinant DNA techniques.
  • Additional aspects of the invention related to the above include DNA molecules encoding for haemoprotein polypeptides or fragments or epitopes thereof; vectors containing one or more such DNA sequences; host cells, for example bacteria such as E. coli or yeast cells such as Saccharomyces spp., or more preferably eukaryotic cells, transformed by such vectors, for example by a baculovirus vector; and processes for preparing recombinant haemoprotein polypeptides or antigenic fragments or epitopes thereof comprising culturing such transformed host cells and isolating said haemoprotein polypeptide or fragment or epitope from the cultured cells.
  • An alternative live or inactivated vaccine formulation may comprise an attenuated or virulent virus or a host cell, e.g. a microorganism such as a bacterium, having inserted therein a nucleic acid molecule (e.g. a DNA molecule) according to the invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule.
  • a bacterial vector which elicits local gut mucosal immunity to a fluke antigen which then blocks juvenile fluke migration is particularly preferred, notably invasive species such as Salmonella species.
  • Additional antigenic materials may also be present in the vaccine thus giving an enhanced protective effect against the helminth parasite in question or a combined protective effect against one or more additional parasitic infestations.
  • a yet further aspect of the invention provides a monoclonal or polyclonal antibody capable of inducing immunity to a haemoprotein in a mammal when administered to said mammal, the antibody having an affinity for the variable region of one or more further antibodies, said further antibodies having an affinity for said haemoprotein.
  • RPMI-1640 without L-glutamine was from Gibco, Life Technologies Ltd., Paisley, Scotland. Hepes, gentamycin, Freund's Complete and Incomplete Adjuvants, sodium dodecyl sulphate (SDS) , anti-bovine IgG conjugated to alkaline phosphatase, anti-rat IgG conjugated to alkaline phosphatase, 3,3-diaminobenzidine hydrochloride (DAB) , nitro-blue tetrazolium (NBT) and 5- bromo-5-chloro-3-indoyl phosphate (BCIP) were obtained from Sigma Chemical Co., St. Louis, USA. Sephacryl S- 200 HR and diethylaminoethyl Sepharose were purchased from Pharmacia, Uppsala, Sweden.
  • Mature flukes were obtained from the infected livers of condemned cattle at a local abattoir in Ireland. The flukes were washed six times in phosphate buffered saline (PBS), pH 7.3, and then maintained in RPMI-1640, pH 7.3, containing 2% glucose, 20 mM Hepes and 25 mg/1 gentamycin at 37°C overnight. The culture medium (ES products) was removed and centrifuged at 12,000 x g for 1 hour. The ES was concentrated to 15 ml in an Amicon 8400 Ultrafiltration unit (Danvers, MA, USA) with a YM3 membrane (3,000 mw cut-off).
  • the concentrated sample was centrifuged at 12,000 x g for 30 minutes and applied to a 340 ml Sephacryl S-200 column equilibrated in 0.1M Tris-HCl, pH 7.0, at 4°C. Fractions, each of 5 ml, were collected after the void volume (110 ml) had been passed. The absorbance of the eluate was monitored at 280 nm using an Atto UV monitor. Those fractions containing haemoprotein (yellow coloured) were pooled and concentrated in an Amicon 8050 ultrafiltration unit to 5 ml.
  • This concentrate was dialysed against 0.025M Hepes, pH 6.8, and applied to a 10 ml DEAE Sepharose column equilibrated in the same buffer. Proteins were eluted using a linear gradient from 0 to 0.4M sodium chloride in 0.025M Hepes, pH 6.8. The haemoprotein containing fractions, detected by absorbance at 415 nm, were pooled, concentrated by ultrafiltration, and stored at -20C until required.
  • the Sephacryl S200 column was calibrated with the following standards; moi e immunoglobulin, 150 kDa; albumin, 68 kDa,- ovalbumin, 45 kDa; ⁇ -lactoglobulin, 18.5 kDa.
  • a large protein peak elutes at the void volume of the gel filtration column, followed by a second peak which contains the haemoprotein.
  • the fractions containing the haemoprotein were easily identified by their yellowish colour due to the presence of haem (Fig. 1A) .
  • These fractions were pooled, concentrated and dialysed before being applied to the DEAE Sepharose ion exchange column.
  • the haemoprotein elutes at approximately 200 mM NaCl (Fig. IB) .
  • Analysis of the pooled haemoprotein- containing fraction by non-denaturing PAGE revealed the presence of a single protein with a minor slower- migrating protein (some protein is also seen at the top of the separating gel) (Fig. 1C, lane 2) .
  • the predominant protein co-migrates with a major component of mature fluke ES products (Fig. 1C lane 1) .
  • the haemoprotein elutes on the Sephacryl S200 column before the protein mouse IgG (150 kDa) , and was estimated to have a molecular weight of at least 200 kDa.
  • Mature liver flukes were removed from the medium at the end of the culture period and washed three times with PBS. An equal volume of PBS was then added to the flukes and the parasites homogenised on ice with a teflon homogeniser. Following centrifugation of the homogenate at 10,000 x g for 30 minutes, the supernatant was decanted and stored at -20°C.
  • Liver flukes were extracted in the presence and absence of the following proteinase inhibitors, phenylmethylsulfonylfluoride (PMSF) , lOmM; iodoacetamide, lO M; leupeptin, 5 ⁇ g/ml; and L-transepoxysuccinyl-leucylamido- (4-guanidino) - butane (E 64) , 5 ⁇ g/ml.
  • PMSF phenylmethylsulfonylfluoride
  • E 64 L-transepoxysuccinyl-leucylamido- (4-guanidino) - butane
  • Non-denaturing 10% polyacrylamide gel electrophoresis was performed on the extracts described under (3) using the buffer system of Laemmli (1970, Cleavage of structural proteins during the assembly of the head of Bacteriophage T4, Nature 227, 680-685). Gels were stained for protein with Coomassie brilliant blue R (Fig. 2A) , and for haemoproteins using 3,3- diaminobenzidine hydrochloride (DAB) and hydrogen peroxide as described by McDonnel and Staehelin (1981, Analytical Biochemistry 117, 40-44) (Fig. 2B) . Bovine haemoprotein, used as a positive control in these gels (lane 1 in each of Figs. 2A asnd 2B) , was obtained by lysing bovine erythrocytes in water followed by centrifugation at 10,000 x g for 30 minutes in order to remove cell debris.
  • DAB 3,3- diaminobenzidine hydrochloride
  • Fig. 2B
  • the mature fluke extracts contain multiple proteins; however, the extract prepared in the absence of inhibitors contained more faster-migrating proteins than the extract prepared in the presence of inhibitors (Fig. 2A, lanes 2 & 3 respectively) .
  • a single protein which is also the most predominant protein in both of these extracts, stains positive for the presence of a haem group.
  • the haem-staining protein in the homogenates prepared in the absence of proteinase inhibitors migrates faster than that in homogenates prepared in the presence of proteinase inhibitors (Fig 2B, lanes 2 & 3 respectively) . This observation suggests that the haemoprotein is susceptible to proteinase digestion.
  • Bovine haemoprotein which appears as a doublet when stained for haem, migrates much further into the gel than the liver fluke haemoprotein. Coomassie staining reveals the presence of many contaminating bands in the bovine haemoprotein preparation (Fig. 2A & B, lane 1) . This observation confirms that the haemoprotein identified in mature fluke extracts and ES products did not originate from bovine erythrocytes.
  • transfer buffer 25 mM Tris-HCl, 190 mM glycine and 10% methanol
  • the membrane was stained with Coomassie brilliant blue R and the bands corresponding to the liver fluke haemoprotein sequenced at the Protein Sequencing Facility, Department of Biochemistry, Tennis Court Rd., Cambridge, CB2 1QW using an Applied Biosys erns 477A protein sequencer.
  • the major and minor bands in the purified haemoprotein preparation were transferred to PVDF membrane and subjected to N-terminal sequencing. No sequence was obtained for the minor component. The following sequence was obtained for the major component:
  • the absorption spectra of mature F. hepatica homogenate haemoprotein and the purified haemoprotein were measured with a recording spectrophotometer (Shimadzu UV-160A) .
  • Derivatives of liver fluke haemoprotein were prepared as described by Tsuneshige et al. (1989, Journal of Biochemistry 106, 406-417) .
  • Oxyhaemoprotein in the mature fluke extracts was reduced to deoxyhaemoprotein by adding five milligrams of solid sodium dithionite to a 200 mg extract.
  • an excess of potassium ferricyanide crystals was added to the liver fluke extract.
  • the cyanomet derivative was obtained by including ImM potassium cyanide in the above oxidation reaction.
  • the absorption spectra of the haemoprotein in mature fluke extracts and its deoxy, met, and cyanomet derivatives were examined over the visible light range, 325-650 nm (Fig. 3A) .
  • the haemoprotein in the fluke extract showed a spectrum with maxima at 578 nm and 540 nm which is characteristic of the ⁇ and ⁇ peaks, respectively, of oxyhaemoproteins.
  • a Soret peak maximum 415 nm
  • the ⁇ and ⁇ were peaks no longer evident; however a flattened peak between 550 and 560 nm was seen. Furthermore, the Soret peak showed a maximum at 430 nm in the de-oxyhaemoprotein (Fig. 3A) .
  • the cyanomet derivative of the haemoprotein also lacked the a and ⁇ peaks but showed a peak at 540 nm and a Soret band at 420 nm (Fig. 3A) .
  • the absorption spectra obtained confirmed that the liver fluke extract contains a haemoprotein with characteristics of a haemoprotein (Tsuneshige, et al. , 1989).
  • the purified haemoprotein shows an adsorption spectrum with a slight peak around 500 nm, a second broad peak between 630 and 640 nm and a Soret band at 400 nm (Fig. 3B, MET-ES) .
  • This spectra was similar to that obtained for the haemoprotein in mature fluke extracts when it was oxidised to met-haemoprotein (Fig. 3B, MET-EXT) .
  • the oxidation of the purified haemoprotein to met- haemoprotein could not be reversed with the reducing agents sodium dithionite and iron (II) sulphate (data not shown) .
  • Polyclonal antisera against the purified haemoprotein were prepared by immunising male Wistar rats subcutaneously with 20 ⁇ g purified protein. The initial injection was prepared in Freund's Complete Adjuvant and the four subsequent injections, given at three week intervals, in Freund's Incomplete Adjuvant. Sera were also obtained weekly, for 11 weeks, from four cattle (a- d in Figure 4) experimentally infected with £a. 500 metacercariae of F. hepatica.
  • liver fluke homogenates extracted in the presence and absence of proteinase inhibitors
  • purified haemoprotein were electrophoresed under non-denaturing conditions and transferred to nitrocellulose paper using an Atto semi- dry blotting system. Non-specific binding sites were blocked with 1% BSA and 0.1% Tween 20 in PBS. Nitrocellulose filters were then incubated in rat anti- haemoprotein serum or normal rat serum (1/400 dilution) . Bound antibody was visualised using alkaline phosphatase-conjugated anti-rat IgG. Nitro-blue tetrazolium and 5-bromo-5-chloro-3-indoyl phosphate were used as substrates for alkaline phosphatase.
  • ELISA enzyme linked immunosorbant assays
  • 50 ⁇ l (1 ⁇ g) of purified haemoprotein was dispensed into wells of a 96-well plate and incubated overnight at 37°C.
  • the plates were then blocked with 1% BSA and 0.1% Tween 20 in PBS for one hour before the antisera were added.
  • Bound antibodies were detected using alkaline phosphatase rabbit anti-bovine serum (Fig. 4) .
  • Anti- aemoprotein antibodies could be detected in all animals as early as one week after infection using serum dilutions of 1:100.
  • the immune responses to the haemoprotein were highly variable between the four animals examined.
  • Antibody titres rose in the first four weeks of infection and then remained steady until the final bleeding date of 11 weeks after infection.
  • the titre of antibodies in the serum from week four to eleven after infection were >8,000 (data not shown).
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant

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  • Tropical Medicine & Parasitology (AREA)
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Abstract

L'invention concerne l'utilisation d'hémoprotéines dans la formulation de vaccins destinés à combattre des parasites, les helminthes. Ces hémoprotéines sont de préférence dérivées d'une douve telle que Fasciola hepatica.
PCT/GB1995/002350 1994-10-04 1995-10-04 Vaccin contenant une hemoproteine WO1996010583A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU35747/95A AU3574795A (en) 1994-10-04 1995-10-04 Vaccine containing a haemoprotein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9419969A GB9419969D0 (en) 1994-10-04 1994-10-04 Vaccine
GB9419969.2 1994-10-04

Publications (1)

Publication Number Publication Date
WO1996010583A1 true WO1996010583A1 (fr) 1996-04-11

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PCT/GB1995/002350 WO1996010583A1 (fr) 1994-10-04 1995-10-04 Vaccin contenant une hemoproteine

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GB (1) GB9419969D0 (fr)
WO (1) WO1996010583A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997047740A3 (fr) * 1996-06-11 1998-03-26 Mallinckrodt Veterinary Inc VACCIN CONTENANT UNE PEROXIREDOXINE ET/OU UNE β-TUBULINE

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025697A1 (fr) * 1992-06-15 1993-12-23 California Institute Of Technology Renforcement de la croissance des cellules par expression de proteines clonees fixatrices d'oxygene
WO1994009142A1 (fr) * 1992-10-21 1994-04-28 Mallinckrodt Veterinary, Inc. Vaccin contenant une protease de thiol
WO1995009182A1 (fr) * 1993-09-28 1995-04-06 The University Of Melbourne Antigenes de protection contre les parasites

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025697A1 (fr) * 1992-06-15 1993-12-23 California Institute Of Technology Renforcement de la croissance des cellules par expression de proteines clonees fixatrices d'oxygene
WO1994009142A1 (fr) * 1992-10-21 1994-04-28 Mallinckrodt Veterinary, Inc. Vaccin contenant une protease de thiol
WO1995009182A1 (fr) * 1993-09-28 1995-04-06 The University Of Melbourne Antigenes de protection contre les parasites

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A.TSUNESHIGE E.A.: "Spectrophotometric,EPR and oxygen binding studies on the hemoglobin from the marine polychatePerinereis aibuhitensis", JOURNAL OF BIOCHEMISTRY, vol. 106, TOKYO JP, pages 406 - 417 *
G.R.RAJASEKARIAH E.A.: "Fasciola hepatica:attempts tonduce protection against infection in rats and mice by injection of excretory/secretory products of immature worms", PARASITOLOGY, vol. 79, pages 393 - 400 *
S.MCGONIGLE E.A.: "Isolation of Fasciola hepatica haemoglobin", PARASITOLOGY, vol. 111, pages 209 - 215 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997047740A3 (fr) * 1996-06-11 1998-03-26 Mallinckrodt Veterinary Inc VACCIN CONTENANT UNE PEROXIREDOXINE ET/OU UNE β-TUBULINE
AU732807B2 (en) * 1996-06-11 2001-05-03 John Pius Dalton Vaccine containing a peroxiredoxin and/or a beta-tubulin
US6676944B2 (en) 1996-06-11 2004-01-13 John P. Dalton Vaccine containing a peroxiredoxin and/or a β-tubulin

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Publication number Publication date
GB9419969D0 (en) 1994-11-16
AU3574795A (en) 1996-04-26

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