WO1997042318A1

WO1997042318A1 - Vaccines

Info

Publication number: WO1997042318A1
Application number: PCT/GB1997/001172
Authority: WO
Inventors: Raja Varma; Ralph Martin Woodland; Kamburapola Gamarallage Indrananda Jayawardena; Agi Heller-Haupt
Original assignee: The Minister Of Agriculture Fisheries And Food
Priority date: 1996-05-02
Filing date: 1997-04-30
Publication date: 1997-11-13
Also published as: GB9609252D0; ZA973824B

Abstract

An antigenic protein which is capable of producing an immune response in an animal to which it is administered, said response including the production of antibodies which are capable of specifically binding to an antigen obtainable from P. ovis having molecular weights of 183 kDa, 143 kDa, 97 kDa, 41.8 kDa, 31.2 kDa or 12 kDa, in particular a protein obtainable from P. ovis or fragments which include antigenic determinants. These proteins are useful in diagnosis of P. ovis infestation as well as in prophylactic vaccines.

Description

,

Vacc ines

The invention relates to antigens of sheep scab mite (Psoroptes ovis) , a method for obtaining them and their use in diagnosis as well as in prophylactic treatments. In particular, some antigens may be applied in vaccines against P. ovis infestation. The invention also relates to the use of P ovi s antigens as diagnostic markers.

Sheep scab caused by the mite Psoroptes ovi s is a highly contagious disease which infects farm animals and in particular sheep and cattle as well as wild animals such as bighorn sheep and mule deer. The traditional method of preventing sheep scab is by dipping infected animals in organosphosphate insecticidal compound solutions. There is growing concern however among farmers, the public, and the veterinary and medical profession as to the toxic and polluting nature of the these insecticides. In addition dipping does not result in total eradication but a high level of control. It is an object of the invention therefore to provide a safe, non-hazardous and non-pollutive alternative way to control the disease.

Many external parasites of livestock induce immunity in their host animals resulting in protection of the animals against subsequent infestations with the particular parasites (see for example S.K. Wikel (1988) Veterinary Parasitology, 29, 235) . This has led to the concept of vaccination of animals against subsequent infection by using extracts of the parasites, which is non hazardous and environmentally friendly. Vaccination of animals against ticks (J.P. Opdebeeck et al., Immunology, (1988) 63, 363; P Willasden et al . Parasitology Today (1989) 4, 196; Y. Rechav et al . , Immunology, (1992) 75, 700; G.A.Riding et al . J.Immunololgy, (1994) 153, 5158), blow flies in particular the sheep blow fly Lucilia cuprina HI.J. East et al., International Journal of Parasitology, (1991) 38, 185) and the cattle grub Hypoderma lineatum (R.W.Baron et al . , Veterinary Parasitology (1991) 38, 185) has been extensively investigated. A genetically engineered vaccine against the cattle tick Boophilus micropl us has been developed for commercial use ( G.S.Cobon et al., New Generation Vaccines (1990), Eds Woodrow G.C. & Levine M.M. pp 901-917, Marcel Dekker, New York) .

No such studies have been reported for Psoroptes ovis .

A number of antigenic proteins which induce an immune response in sheep have apparently been isolated previously. Sera from infected animals were used for characterising the antigens of North American P. ovis from different sources and from the related P. cumculi from rabbits (W.M. Boyce et al . , Journal of Parasitology, (1991), 77, 675) . Since both species have been reported to be morphologically identical, reproductively compatible and antigenically similar, P. cunucul i mites have been used as an antigen source in serological investigations on bighorn sheep infested with P. ovis in the USA (W.M. Boyce et al . , Journal of Wildlife Diseases (1991) , 27, 10). Antigens for P. cunuculi have also been used for immunisation of rabbits and an immunogen from this species was subsequently isolated and characterised ( J. Uhlir, Folia Parasitologica (1992) 39, 375; J. Uhlir, Veterinary Parasitology, (1993), 45, 307) .

Antigenic proteins from a German strain of P. ovis and from three other mite species, Sarcoptes εuis , Choπoptes bovis and Notoedres cati , were investigated for their cross-reactivity using sera from sheep naturally infested with P. ovis (H. F. Mathes et al. International Journal of Parasitology (1996), 26, 437) .

The applicants have identified a number of antigens in a British strain of P. ovis which have applications m diagnosis and/or in prophylactic vaccines.

According to the invention there is provided an antigenic protein which is capable of producing an immune response in an animal to which it is administered, said response including the production of antibodies which are capable of specifically binding to an antigen obtainable from P. ovis having molecular weights of approximately 183 kDa, 143 kDa, 97 kDa, 41.8 kDa, 31.2 kDa or 12 kDa.

Such proteins have similar antigenic specificity to the wild type P. ovis antigens of the above-mentioned molecular weights.

The molecular weights of the antigens obtainable from P. ovis referred to herein are the approximate weights which are estimated using SDS-PAGE separation followed by lmmunoblotting and densitometry scanning of the molecular weights as illustrated hereinafter.

In particular, proteins of the invention are selected from antigens which are obtainable from P. ovis and have molecular weights of the order of 183kDa, 143kDa, 97kDa, 41.8kDa, 31.2kDa and 12 kDa, or fragments thereof; or variants thereof.

Suitable proteins of the invention are selected from antigens which are obtainable from P. ovis and have molecular weights of the order of 183kDa, 143kDa, 97kDa, 41.8kDa and 31.2kDa, or fragments thereof; or variants thereof.

As used herein, the term "fragments" refers to derivatives of the proteins in which some amino acids have been deleted, usually from the N- and/or C-terminal ends of the full length protein. These fragments must still contain an antigenic determinant. Generally these fragments will be at least 6 amino acids in length. They may be conjugated or fused to other epitopic fragments either directly or spaced depending upon the nature of the epitopes and the immune response generated thereby.

The expression "variants" used herein relate to proteins which are similar to a full length antigen obtainable from P. ovis as described above or a fragment thereof, but where some amino acids in the sequence are different to those of the wild- type or native protein. These changes includes proteins or peptides comprising an amino acid sequence corresponding to native or wild-type sequences wherein some of the amino acids are replaced by conservative substitutions.

By 'conservative substitution' is meant the substitution of an amino acid by another one of the same class; the classes being as follows:

CLASS EXAMPLES OF AMINO ACID

Nonpolar: Ala, Val, Leu, lie, Pro, Met, Phe, Trp

Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gin

Acidic: Asp, Glu

Basic: Lys, Arg, His

As is well known to those skilled in the art, altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide because the side- chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation. Indeed, such changes may confer slightly advantageous properties on the peptide.

Other proteins of the invention include anti -ldiotypic antibodies which are designed to mimic the P. ovis antigens described above and which are produced by conventional methods as understood m the art and can be used, for example as surrogate vaccines.

The invention also embraces other peptides which are homologues of the native or wild type antigens. These homologues include (but are not limited to) peptides having minor artificial variations such as the deletion of one or a few residues, minor insertions of residues, or minor variations in ammo acid structure.

Also included are homologues in having a few non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical m determining its conformation may not greatly affect its activity because they do not greatly alter the peptide' s three dimensional structure.

Thus homologues which have 80%, preferably 85%, 90%, 95% or 99% homology with the wild type or native antigen sequences, provided that they produce the desire immune response, are encompassed by the present invention.

It should also be stressed that the invention also encompasses proteins or protein conjugates comprising the above described proteins, for example wherein the N or C terminus has been extended. Extension of the peptides above may confer additional desirable properties on them, for instance, other immunogenicity or a labelling function.

Preferably however, the invention provides a protein which comprises an antigen obtainable from P. ovis having molecular weights of about 183 kDa, 143 kDa, 97 kDa, 41.8 kDa, 31.2 kDa or 12 kDa.

Examples of such antigens are those having molecular weights of 183 kDa, 143 kDa, 97 kDa, 41.8 kDa or 31.2 kDa, in particular, those of molecular weights of about 183kDa, 97kDa, 41.8kDa, or 31.2kDa.

The invention extends to the use of the proteins of the invention and particularly the wild-type antigens as diagnostic markers for testing infection of P . ovis in sheep and the use of the proteins such as the antigens or a protective epitopic part thereof in vaccination of sheep against P. ovis .

5 Thus the invention further provides protein as described above for use in the diagnosis of infection by P. ovis . Such diagnosis may be effected for example by taking a sample of body fluid such as sera from an animal suspected of having subclinical or inapparent infection with P. ovis, reacting said sample with a protein as 0 descibed above and detecting binding between the sample and said protein. Alternatively, the method may be used to confirm a sheep scab infection in an animal displaying clinical symptoms of a disease.

5 For diagnostic purposes, proteins comprising an antigen obtainable from P. ovi s having a molecular weight of about 41.8kDa or about 31.2kDa are preferred.

Alternatively the invention provides a protein as described above, for use in prophylactic vaccine. These methods may be use in the prevention of P. ovis infestation, by administering to an animal, a vaccine of the invention.

Preferably, for vaccine use, the protein used is one which has a molecular weight of at least 97kDa.

Vaccines of the invention may additionally include a pharmaceutically acceptable carrier. Suitable carriers are well known in the art and include solid and liquid diluents, for example, water, saline or 0 aqueous ethanol. The liquid carrier is suitably sterile and pyrogen free. A preferred carrier or diluent is physiological saline. A suitable adjuvant may be provided such as Quil-A saponin or Montanide Marcol or other immunopotentiators, for example an interleukm such as IL 12. The compositions may be in the form of S liquids suitable for infusion or injection, or syrups, suspensions or solutions, as well as solid forms such as capsules, tablets, or reconstitutable powders.

Vaccines using the antigens according to the invention may be administered in any appropriate fashion, e.g. orally or parenterally, (i.e. mtradermally, intravenously or intramuscularly) or in the form of slow release implants. Appropriate dosages and vaccination regimes may be determined according to the particular nature of the antigen used, and to the optimum protection conferred but will generally be in the range of from 0.3 to 100 micrograms/Kg.

In an alternative embodiment however, the vaccine may be in the form of a vector, such as a viral or bacterial vector, which is adapted to express the peptide of the invention in si tu . Such vectors form a further aspect of the invention. The vector will contain a DNA sequence which encodes a protein of the invention. In order to determine what DNA sequences will fulfill this requirement, it is necessary to further characterise the proteins of the invention, using techniques which are known in the art. Such DNA sequences form a further aspect of the invention.

The vector may contain expression control functions such as promoters, enhancers and signal sequences, as well as a selection marker in order to allow detection of successful transformants. The selection of these will depend upon the precise nature of the vector chosen and will be known to or readily determinable by a person skilled in the art.

The vaccine may alternatively be in the form of a DNA vaccine where the vector consists of a DNA plasmid which is adapted to express the peptide in situ.

Suitably the vector is a viral or bacterial vector, for example a vector derived from vaccinia , adenovirus , or herpes simplex virus (HSV) BCG or BCC. It is suitably attenuated to minimise any harmful effects associated with the virus on the host.

Preferably, the vector is derived from vaccinia virus, as it has many properties which make it a suitable vector for vaccination, including its ability to efficiently stimulate humoral as well as cell-mediated immune responses.

The invention further provides an antibody to a protein as described above. These antibodies may also be used in diagnosis or in the determination of the serological nature of the mites as understood in the art. Antibodies may be polyclonal or monoclonal and are preferably monoclonal.

The proteins of the invention may be obtained by isolation from mites. Preferably however, they are obtained by recombinant production methods.

Isolation and identification of antigens in P. ovis has been described and performed by electophoresis. However any known method for obtaining protein by molecular weight can be utilised, such as HPLC using size exclusion or ion exchange columns, and for characterisation of the antigens using lsoelectric focusing.

Although the antigens have been defined in terms of their molecular weight, the invention extends to the antigen however obtained.

In mass production of vaccines it would be impractical to produce the vaccine by obtaining the antigens by extraction from the mites themselves. The invention therefore extends to the antigens when recombinantly produced. This would entail molecular characterisation of the antibody and N terminal sequence, construction of PCR primers and screening of CDNA libraries to identify the genes responsible tor encoding the antigens. Thereafter isolation of the antigen genes and transformation of a suitable host would enable the antigens of interest to be expressed.

Thus in a preferred embodiment, there is provided a method for preparing a protein as described above, which method comprises including nucleotide sequence which encodes said protein into a recombinant expression vector, transforming a host cell with said vector, and culturing said cell and recovering the peptide from the culture. The host cell may be eukaryotic or prokaryotic, but is conveniently a prokaryotic cell such as E. coli .

Test kits for use in the diagnostic method form a further aspect of the invention. These test kits will include a protein as described above. Optional additional components of the kits include label reagents such enzyme conjugated second antibodies.

The inventions will be hereinafter described by reference to the following figures in which: Figure la shows an immunoblot of P ovis soluble antigen with P. ovis g

infested serum;

Figure 1 b shows densitometry scans of the immunoblots of figure 1 a; Figure 2 shows the percentage reduction in legion size after vaccination of sheep subsequently infected with P. ovis , each line representing the results from an individual animal; and

Figure 3 shows the changes of lesion sizes from the vaccine trials over time.

Example 1 Isolation of P ovis antigens

The following is an example (a protocol) on how these antigens may be isolated. Psoroptes ovis mites were collected from infected donor sheep/cattle and stored at -70°C. They were subsequently thawed, and "cleaned up" by washing in phosphate buffered saline (PBS) containing 1% Tween 80. After two more washes in PBS, the mites were dried by placing on filter paper and transferred to a glass tissue grinder (or porcelain mortar for larger masses of mites) standing on ice. The mites were crushed to a fine powder in a small amount of liquid crushing in liquid nitrogen. This was done two more times and extraction buffer (PBS with 0.05% Tween 20 and O.lmM of the protease inhibitor PMSF) added to give an approximate 10% (w/v) suspension.

This was then sonicated on ice 4 times, 15 sec. at a time at 18μ with

1 minute intervals. The homogenate was centrifuged at 50000g for 1 hour at 4°C. The supernatant was removed, filtered sequentially through 0.45μ and 0.22μ filters and stored at -70°C. The protein content by the Bradford assay was 2.5 to 3.0mg"".

Fractionation of soluble antigens was performed by electrophoresis e.g. using the BioRad PrepCell Model 491 which combines the advantages of electrophoretic separation of antigenic proteins on gels with recovery of the eluted proteins which can be collected as timed fractions. The PrepCell containing 80ml of 8% separating gel mix was assembled according to the manufacturer's instructions and equilibrated with tris-glycine-SDS buffer. In particular 80ml of 8% separating gel mix was prepared and degassed in a vacuum chamber for 30 minutes. 250μl of 10% ammonium persulfate and 25μl if N,N<N<N< tetramethylethylenediamine (TEMED) added to the degassed solution and mixed by gentle swirling for 30 seconds and the mixture was immediately poured into the casting chamber using a 25ml plastic pipette fitted with a rubber bulb. The chamber was checked for trapped air bubbles and if there were any, they were removed by gentle tapping. Cooling system of the gel mould was connected to the cold water tap and the top of the gel mixture was overlaid with 5ml of water saturated n-butanol and left for 3 hours for polymerisation to complete. At the end of the polymerisation, top liquid layers were discarded and the surface of the gel was briefly washed three times with deionised water. An hour before the polymerisation of the separating gel was completed 20ml of 5% stacking gel mix was prepared and degassed for 30 minutes. At the end, HOμl of 10% ammonium persulfate and 22μl of TEMED added and gently mixed as before. The mixture was immediately poured on top of the separating gel and overlaid with 5ml of n-butanol and left to polymerise while the cooling system was running. Once the polymerisation was completed the top liquid layers were discarded and the surface of the gel was washed with deionised water as before. Moulding tube was detached from the casting stand and the Bio-Rad PrepCell Model 491 was assembled according to the manufacturer's instructions.

The PrepCell containing the gel was pre-run for 60 minutes at a constant power of 10W. Both upper and lower chambers contained tris- glycine-SDS buffer. The same was used as elution buffer. Prior to the application of the sample, the Prepcell apparatus was transferred to a levelled surface.

P. ovis soluble antigen extract prepared as above was concentrated in Micrσcon-30 units. The final concentration was adjusted to 0.6mg protein per lOOμl with deionised water. 600μl of the sample was mixed with 4x reducing sample buffer in 1:2 ratio and placed in a boiling bath for 5 minutes prior to transfer onto ice for a further 10 minutes. The sample was then centrifuged at 14,000g for 10 minutes and the supernatant carefully applied to the gel surface, at the end of the pre-run, by means of a 2ml plastic disposable syringe fitted with a long thin (1mm I.D.) plastic tubing.

The sample was electrophoresed at 10 W constant power for 60 minutes; the surface of the gel was flushed with 100 ml electrode buffer using a 100ml plastic syringe fited with a length of plastic tubing. Electrophoresis was continued for 60 hours. Proteins reaching the end of separating gel were eluted at the rate of 0.5ml/mm using a LKB Microperpex peristaltic pump. Fractions were collected at 6 minute ,₀

intervals and stored for at 4°C for analysis by immunoblottmg (Western blots) .

Four runs were made and over 600 fractions collected from each run. The fractions were combined into three major groups, F2 containing the 97kDa antigen, FI containing antigens below this (including the 41.8 kDa and 31.2 kDa antigens) and F3 containing antigenic bands above 97kDa (including 183 kDa and 143 kDa antigen) . Each fraction was further concentrated, the protein content determined and stored at -70°C until used for vaccination of sheep.

Example 2

Preparation of membrane bound antigens from P. ovis

After removing the supernatant containing soluble antigens membrane bound antigens were solubilised by treatment of the pellet with detergent. The pellets were taken out of -70°C thawed, made up to 4ml and whirlimixed. The suspension was centrifuged at 4000rpm for 5 minutes at 4°C and the supernatant discarded. The pellets were then resuspended m 4 ml of 1% Triton X-100 mixed on a turn-table for 30 minutes at room temperature and stored overnight at 4°C. The suspension was mixed again on a turn-table for 30 mm and centrifuged at lOOOOOg for 1 hour at 4°C. The supernatant was filtered through a 0.22μm filter and concentrated in an ultrafiltration cell fitted with a 10,000 MW cut off membrane. The preparation was further concentrated by centrifuging in Centricon 16 tube at 7500 rpm for 15 minutes at 4°C.

Example 3

Antigens as markers; Immunoblotting of separated, electro^phoresed mite antigens

The P. ovis mite soluble antigens, its three fractions and membrane bound antigens were separated on a Mini-Protean 11 system using 12% uniform gels. Each lane was loaded with 3μg of protein. One lane had standard molecular weights markers. Gels were run at 120V until the tracker dye bromophenol blue had reached the bottom of the gel.

Figures la and lb show the results of immunoblotting by probing electrophoresed crude mite extracts with sera from infested sheep. In Figure la, Lane 1 shows immunoblots of P. ovis antigens with low range molecular weight standards. Lane 2 shows antigens probed with pre infestation sheep serum and Lane 3 shows antigens probed with 6- week post infestation sheep serum. Six major antigenic bands were recognised, one in the 183 kDa, one in the 143 kDa, one in the 97kDa, two in the 41.8 kDa and the 31.2 kDa regions and the last m the 12kDa region. It was found that sera from uninfested sheep did not "see" the bands. However sheep infested with scab mites of varying virulence develop antibodies to these antigens starting from 2-3 weeks after infestation to at least 3 months after infestation and they therefore have a protective/marker role. The 31.2 kDa antigen is the most useful as a serodiagnostic tool as shown in Figure la, it is a broad deeply staining band without other bands near it to confuse it with. Additionally it is not recognised by sera from sheep infested with other ectoparasites such as sheep lice, keds and Derma tophilus congolense. The diagnostic value of the antigen is shown m the Table where serum from a contact sheep (m an infested herd) , which was negative at the time of sampling but which later developed clinical signs, recognised this band. While the 31.2kDa antigen is a marker for infestations which subsequently develop clinical manifestations, the 183kDa antigen may indicate inapparent or subclinical infestations since sera from some contact sheep which did not develop clinical signs recognised this antigen (see Table) ; it therefore will be useful in serepidemology.

Table 1 shows results of serological tests on sheep during a outbreak of sheep scab

Y = Yes

N = No 'Presence of antigenic band in immunoblot assays 'Staining intensity; + (weak) to ++++(strong) 'Normal at time of sampling but later developed clinical illness

Example 4

The use of the antigens in vaccines

The inventors have also determined that the antigens of P. ovis are effective in provoking an immune response in sheep such that the use of the antigens as vaccine agents has been demonstrated.

The following describes a trial in which sheep were vaccinated using the P. ovis antigens Forty two sheep in six groups were used in the trial. One group was retained as vaccinated controls. The other five were vaccinated intramuscularly with the antigens mixed with Montanide-Marcol; each animal was inoculated three times at two week intervals. One group was inoculated with soluble antigen, each animal receiving a total of 4.5 mg antigen. A second group was inoculated with membrane bound antigen, each animal receiving a total of 4.5 mg. Each animal in the third group had a total of 1.1 mg of Fraction 1; animals in the fourth group each received a total of O.Olβmg of Fraction 2 and in the fifth group a total of 0.344mg of Fraction 3. Two weeks after the last inoculation, all animals were challenged with mites by plucking wool from the withers and placing twenty five ovigerous females of P ovis placed on the bare abraded skin. The area of lesion present was calculated as well as the number of live ovigerous female mites.

Figures 2 and 3 show the results of vaccination trial. Sheep immunised with crude soluble antigen and the F2 and F3 fractions containing antigens at and above 97kDa gave measurable protection against mite infestation compared to unvaccinated controls; lesion size was reduced, these are protective antigens which can be used as protective antigens in sheep to provoke an immune response. FI containing the 41.8kDa and 31.2kDa antigen appeared not to give any protection; these antigens are however still useful as marker antigens .

Claims

H Claims

1. An antigenic protein which is capable of producing an immune response in an animal to which it is administered, said response including the production of antibodies which are capable of specifically binding to an antigen obtainable from P. ovis having molecular weights of 183 kDa, 143 kDa, 97 kDa, 41.8 kDa, 31.2 kDa or 12 kDa.

2. A protein according to claim 1 which is selected from antigens which are obtainable from P. ovis and have molecular weights of the order of 183kDa, 143kDa, 97kDa, 41.8kDa, 31.2kDa and 12 kDa, or fragments thereof; or variants thereof.

3. A protein according to claim 1 or claim 2 which comprises an antigen obtainable from P. ovis having molecular weights of about 183 kDa, 143 kDa, 97 kDa, 41.8 kDa, 31.2 kDa or 12 kDa.

4. A protein as claimed in any one of claim 1 to 3 for use in the diagnosis of infection by P. ovis.

5. A protein according to claim 4 which has a molecular weight of the order of 41.8kDa or 31.2kDa.

6. A method of diagnosis of P. ovis infestation, which method comprises taking a sample of body fluid such as sera from an animal suspected or at risk of being infected with P. ovis , reacting said sample with a protein as claimed in claim 4 or claim 5 and detecting binding between at least one component of the sample and said protein.

7. A protein as claimed in any one of claims 1 to 4 for use in a prophylactic vaccine.

8. A protein as claimed in claim 7 wherein the protein has a molecular weight of 97kDa or greater.

9. A pharmaceutical composition comprising a protein as claimed in any one of claims 1 to 4 , or a vector or nucleic acid capable of expressing said protein when administered to an animal in combination with a pharmaceutically acceptable carrier.

10. A nucleotide sequence which encodes a protein according to any one of claims 1 to 4.

11. An isolated antibody which specifically binds to a protein as claimed in any one of claims 1 to 4.

12. A method for producing a protein according to any one of claims 1 to 4 which method comprises either

(A) (i) extracting soluble protein from P. ovi s mites

(ii) fractionating a solution of soluble protein obtained by step (i)

(iii) collecting fractions having molecular weights of about 183kDa, 143kDa, 97kDa, 41.8kDa, 31.2kDa and/or 12kDa; or

(B) incorporating a nucleotide sequence which encodes said protein into a recombinant expression vector, transforming a host cell with said vector, and culturing said cell and recovering the peptide from the culture.

13. A test kit for use in the diagnosis of P. ovis infestation, said kit comprising a protein according to any one of claims 1 to 4 or an antibody according to claim 11.