WO1986002839A1

WO1986002839A1 - Anthelmintic vaccines comprising a nematode species

Info

Publication number: WO1986002839A1
Application number: PCT/AU1985/000282
Authority: WO
Inventors: Gary Stewart Cobon; David Bruce Adams
Original assignee: Biotechnology Australia Pty. Ltd.; Commonwealth Scientific And Industrial Research Or
Priority date: 1984-11-15
Filing date: 1985-11-15
Publication date: 1986-05-22
Also published as: EP0201568A1; NZ214215A; IL77061A0; ES548889A0; ES8705231A1; ZA858789B; GR852768B

Abstract

A vaccine preparation comprising a vaccine preparation comprising a suspension, homogenate or extract of a nematode species said species being non-parasitic to mammals or birds, and a pharmaceutically acceptable adjuvant and/or diluent. The vaccine is useful for immunisation of mammals and birds against other nematode species which are parasitic to such mammals and birds.

Description

ANTHELMINTIC VACCINES COMPRISING A NEMATODE SPECIES

The present invention relates to vaccines, particularly anthelmlnthic vaccines.

BACKGROUND ART Helminth infections of mammals or birds, particularly by parasitic nematodes, are a source of great economic loss especially of sheep and cattle, but also of horses, pigs, goats, dogs, cats and poultry. These animals must regularly be treated with anthelmlnthic chemicals in order to keep the infections under control. If the helminth infections get out of control, infected animals will become diseased which may result in anaemia, diarrhoea, dehydration, loss of appetite and other symptoms and, in some cases the animals may die. Man may also suffer helminth infection. The only available means for controlling helminth infections are anthelmlnthic chemicals. Anthelmlnthic chemicals are only effective against resident worms present at the time of treatment. Treatments with chemicals must therefore be regular as the animals are constantly being re-infected. In some cases, sheep may be treated each three weeks. In the case of dogs and cats the treatments are more frequent (each day or every second day) in order to control Dlrofllarla immitls or heart worm. This is an expensive and labour intensive procedure. Due to the widespread use of anthelmlnthic chemicals, the worms may develop resistance and so hew and more potent classes of chemicals must be developed. An alternative approach is clearly desirable.

The development of vaccines against parasitic nematodes would overcome many of the problems associated with chemical treatments. The protection afforded by vaccination would presumably last for a prolonged period of time - perhaps more than 12 months. Only the vaccinated animal would be affected and there would not be any problems associated with toxicity and persistence of residues. Several vaccination attempts have been made by workers in this area but with little success and where some success has been observed, as in the use of irradiated larval vaccines, practical problems associated with availability of material and stability of the vaccines, have been factors precluding the large scale commercial development of the vaccines. These attempts have all used a nematode parasitic to the mammal.

Failure of killed vaccine preparations to afford" protection has been thought to be due to a number of factors. For example it has been considered that parasitic nematodes may have evolved mechanisms by which they can secrete products which immunosuppress or immunomodulate the host so that an effective immune response is not mounted in a natural infection. These iπ-munomodulato s may be present in the crude preparations of parasitic nematodes used in the killed vaccines.

Secondly, it has been considered that parasitic nematodes may have altered their antigen profile to one which resembles that of the host so that in a natural infection, vigorous immunological reactions are not provoked by important (protective) parasite antigens. This would also occur following vaccination with killed preparations or extracts of the nematodes. A third possibility is that the readily abundant L3 developmental stage of parasites which have been used as a source of antigens in the majority of these studies may not contain the potentially protective antigens associated with the parasitic L4, L5 and adult stages of the nematodes. Limited availability of the parasitic stages precludes their use in large scale vaccines even if they were protective as these parasitic nematodes cannot be readily cultivated In vitro beyond the L3 stage.

DISCLOSURE OF INVENTION The present invention results from the discovery by the present inventors that nematode species which are non parasitic to mammals or birds contain antigens which 'cross react with antibodies produced by mammals or birds which have been infected with or vaccinated with extracts of parasitic nematodes. Therefore it is considered that vaccine preparations derived from a readily cultivated species of nematode non-parasitic to mammals or birds may be used to vaccinate and successfully protect mammals or birds from infections by nematode species which are parasitic to said mammals or birds, even though the species used to derive the vaccine is not the same and may be classified in a - 3 - different order or even a different class from the parasitic species.

Herein there is disclosed a vaccine preparation for the vaccination of mammals or birds against infections by nematodes parasitic thereto, the said vaccine comprising an homogenate, or an extract thereof, of at least one species of nematode which is non-parasitic to mammals.

In another form of the present invention, there is provided a method of vaccinating mammals or birds against parasitic nematode Infections, said method comprising vaccinating the mammal or bird with an effective amount of a suspension, homogenate or extract of a nematode species which is not parasitic to said mammals or birds.

In an alternative form of the invention the vaccine preparation comprises a suspension of at least one species of a nematode, non-parasitic to mammals or birds, wherein the nematodes have been killed or inactivated.

The vaccine preparations of the present invention are adapted for introduction into the mammalian o ^*avian system preferably by injection. Therefore, preferably the homogenate and/or extract is sterilised by, e.g. filtration through a small pore filter, the inclusion of antibacterial compounds, formalinisation or other methods known in the art^'. When killed nematode suspensions are used, the nematodes are killed by methods known to those skilled in the art, such as by formalinisation.

The active ingredients in the vaccine preparation may be delivered in the presence of any of the adjusments known in the art of the vaccine preparation, such as aluminium salt precipitates or oily based adjuvants such as Freund's adjuvants.

The amount of active ingredients incorporated into the vaccines will vary depending on the size of the animal or bird being vaccinated, the adjuvant used and the purity of the active ingredient. Thus doses from as little as 10 μg to 500 mg of dry weight of material may be required. Preferably dosage will be in the range of 50 μg to lmg.

The species of nematode used for incorporation into the vaccine will preferably be readily cultivated in vitro. This enables the availability of sufficient quantities of all stages of development for incorporation into a commercial vaccine. More mature developmental stages may quantitatively and/or qualitatively be a superior source than juvenile forms.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the immunological cross reactivity between parasitic and free living nematodes.

Figure 1A demonstrates that antiserum from sheep vaccinated with homogenates of Haemonchous contortus adults, a parasitic nematode, reacted with antigens from homogenates of free living nematodes.

Figure IB demonstrates that antiserum from sheep vaccinated with homogenates of Haemonchous contortus larvae reacted with antigens from homogenates of free living nematodes.

Figure 1C demonstrates that antiserum from sheep vaccinated with homogenates of Stelnernema feltiae, a free living nematode, exhibits cross-reactivity with homogenates of parasitic nematodes.

MODES OF CARRYING OUT THE INVENTION As will be shown in the example below the vaccinating nematode need not be a member of the same family, superfamily, suborder, order or possibly even class as the parasitic species. Thus a member of the order Rhabditida, (Stelnernema feltiae Mexican) can provide protection of sheep against infection by a member of the order Strongylida (Haemonchus contortus) . Therefore a large number of species would be suitable for incorporation into a vaccine but would preferably be readily cultivated in vitro such as are members of the

Stelnernema (e.g. S_. feltiae) , Caenorhabditis (e.g. C.elegans) and Heterorhabditis (e.g. H. heltiothldis) species. US Patents 4,178,366 and 4.334,498 disclose methods for large scale iri vitro cultivation of such nematodes. The parasitic nematode species against which vaccination would be expected. to be effective are similarly numerous. In the example presented, protection is observed against a member of the order Strongylida (Haemonchus contortus) by vaccination with extract from a member of the order Rhabditia (Stelnernema feltlae Mexican) . Thus it is obvious that extracts f om readily cultivated nematode species would be expected to provide an effective vaccine against any of the large number of species of nematodes which are parasitic to man and domestic animals. For example, Trichinella spiralls or Ancylostoma canlnum infections of man, Strongylus vulgarls infections of horses, Trlchostronqylus colubrlformls infections of sheep, Haemonchus contortus infections of goats, Ostertagla ostertaαi infections of cattle, Ascaris suum or

Trichinella spiralls infections of pigs, Toxascaris leonina or Uncinarla stenocephala infections of cats and Ancylostoma caninum or Trichurls vulpls infections of dogs would be expected to be effectively vaccinated against. Furthermore, the vaccine would be expected to be effective against nematodes which parasitise tissues other than gastro-intestinal tract. For example, the vaccine would be expected to be effective against infections of the circulatory system of man by larvae of Texocara spp and of the circulatory system of dogs by Dirofilaria immitls as well as infections of the circulatory system, urogenital system, respiratory system, skin and subcutaneous tissues of these and other species of animal. It should be noted that this list is by no means complete. The invention will be further described with reference to the following examples. Example 1

Homogenates of Caenorhabdltls elegans (lane 1), Stelnerna e feltlae (lane 2), Trlchostronqylus colubriformls infective larvae (lane 3), Haemonchus contortus infective larvae (lane 4), and Haemonchus contortus adults (lane 5) were prepared and solubilized by boiling for 5 minutes in a solution of sodium dodecyl sulphate (2%), B-mercaptoethanol (2%), glycerol (30%) and 65 mM Tris pH 6.8. Insoluble material was pelleted by centrifugation for 2 minutes at

12,000 x g. Aliquots of the soluble material were applied to a 5-15% polyacrylamide gel (Laemmli, U.K. 1970 Nature 227 680-685) and electrophoresed at 100 ma (500 V maximum) until the broraophenol blue dye neared the bottom of the gel. The separated components were transferred electrophoretically to nitrocellulose paper (Schleicher and Schull 0.2 uM Cat. # 401396) as described (Towbin et, al.. , 1979 Proc. Natl. Acad. Sci. US Vol 76 4350-4354). The strips of nitrocellulose paper were treated for 1 h at 4°C with a solution of 0.5% Tween 20, 10 mM Tris/150 mM NaCl pH 8 and incubated in a 1:250 dilution of serum from sheep which had been vaccinated with homogenates of Haemonchus contortus adults (Fig 1A), Haemonchous contortus infective larvae (Fig IB) or Stelnernema feltlae larvae (Fig

1C) . The strips of nitrocellulose were then incubated for 3 h at room temperature with a rabbit anti-sheep immunoglobulin which was conjugated to horse radish peroxidase. The strips of nitrocellulose were then washed three times with a solution of 0.05% Tween 20 in Tris/Saline and incubated in a solution containing 0.4 mg/ml 4-chloro-l-naphthol in Tris/saline pH 7.5 with 0.03% H-O- . Bands of precipitation of the substrate appeared in the areas where sheep antibodies had bound to components on the nitrocellulose. Molecular weight markers are as shown in kilo daltons. Control reactions in which serum was used from sheep which had not been vaccinated with Haemonchus contortus extracts showed no detectable reaction with nematode extracts in this assay. Example 2 Three sheep were vaccinated with an homogenate obtained by ultrasonication of Stelnernema feltlae (Mexican) . The sheep received two vaccinations subcutaneously in the absence of adjuvant four weeks apart, each vaccination being the equivalent of 83mg wet weight of nematodes per kg body weight of sheep. Three weeks after the second vaccination the sheep and five non vaccinated infection controls were challenged with 10,000 infective larvae of Haemonchus contortus. On days 23, 27, 29, 33, 36 and 40 post infection faecal egg counts were performed on all sheep. The result (eggs/g faeces) are presented in the Table 1, below. - 7 -

^TABLE 1

SHEEP Faecal egg counts on days post infection NUMBER (eggs/g faeces)

23 27 29 33 36 40 TOTAL

Group 1 - Infection Controls

1 1433 4300 2800 6467 4267 5900 25167

2 700 3600 3400 7367 7267 8600 30934

3 600 4467 4767 4033 4367 7850 26084

4 667 10200 11400 18333 16533 6100 63233

5 3200 5333 5133 7367 4433 6700 32166

Group 2 - Vaccinates

6 0 333 733 900 1100 2100 5166

7 200 5867 5333 8667 9267 11100 40434

8 0 267 500 333 1200 500 2800

It is clear that two vaccinated animals were well protected from infection (animals 6 and 8). It is also clear that some cross species protection has been afforded by vaccination and as the purity of the antigens used, the dose of vaccine, route of vaccination, infection timing, infection dose and other parameters are optimised, the significance of the protection observed should improve.

Claims

1. A vaccine preparation comprising a suspension, homogenate or extract of a nematode species said species being non-parasitic to mammals or birds, and a pharmaceutically acceptable adjuvant and/or diluent.

2. A vaccine preparation according to claim 1 wherein the nematodes have been killed or inactivated.

3. A vaccine preparation according to either of claims 1 or 2 wherein the vaccine is adapted for introduction into the mammalian system by injection.

4. A vaccine preparation according to any one of the preceding claims wherein the suspension, homogenate or extract of a nematode species is chosen from a species readily cultivated in vitro.

5. A vaccine preparation according to any one of the preceding claims wherein the suspension, homogenate or extract of a nematode species is chosen from the order Rhabditida.

6. A vaccine preparation according to claim 5 wherein the nematodes are chosen from one of Stelnernema, Caenorhabditis or Heterorhabditis.

7. A vaccine preparation according to any one of the preceding claims wherein the nematode species is selected from one or more of the following: Stelnernema feltlae, Caenorhabditis elegans, Heterorhabditis heltiothldls.

8. A method of protecting mammals or birds from infection by nematode species which are parasitic to said mammals or birds comprising vaccinating said mammals or birds with an effective amount of a vaccine preparation according to any one of the preceding claims.