Title:  Vaccines for the protection of cattle from psoroptic scabies

United States Patent:  6,559,121

Issued:  May 6, 2003

Inventors:  Pruett; John H. (Kerrville, TX); Temeyer; Kevin B. (Boerne, TX); Kunz; Sidney E. (Kerrville, TX); Fisher; William F. (Kerrville, TX)

Assignee:  The United States of America as represented by the Secretary of Agriculture (Washington, DC)

Appl. No.:  860793

Filed:  May 18, 2001

A novel antigenic protein which is effective for stimulating antibody production in animals against the sheep scab mite, Psoroptes ovis is disclosed. In cattle, administration of the protein provides protection from infestation by P. ovis by eliciting an immediate-type hypersensitivity response and/or immunizing the animal against P. ovis. The protein is also effective for producing and/or binding antibodies to P. ovis, and may be used as an immunodiagnostic reagent.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, the nomenclature used to define the proteins is that specified by Schroder and Lubke ["The Peptides," Academic Press (1965)] wherein, in accordance with conventional representation, the N-terminal appears to the left and the C-terminal to the right. Where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented herein unless otherwise expressly indicated.

The present invention provides an antigenic protein effective for eliciting antibody production and a local immediate-type hypersensitivity reaction in an animal against the sheep scab mite, Psoroptes ovis. Elicitation of immediate-type hypersensitivity to the protein will in turn result in a grooming or scratching behavior by the treated animal localized to any site(s) of exposure to this mite, thereby partially protecting the animal from infestation. While it is envisioned that the protein is capable of eliciting these responses in a variety of animals against P. ovis, in the preferred embodiment the protein is used for eliciting antibody production in cattle, including beef or dairy cattle of the genus Bos.

The mature, native form of the protein of the invention has a molecular weight of about 16 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal amino acid sequence of the mature form of the natural protein has been determined as X₁ KVKFQDCGKGEVESLEVEGCSGDY wherein X₁ is either G, S or V. It is understood that some forms of the protein may have additional N-terminal amino acids which may occur, for example, as a result of incomplete processing or hydrolysis of the signal sequence. The protein may also be covalently bonded to a non-related fusion protein as described in greater detail hereinbelow. The invention also encompasses substantial equivalents of this protein which retain the ability to elicit antibody production in an animal against P. ovis. The practitioner of ordinary skill in the art will recognize that slight deviations of the amino acid sequences may be made without affecting the immunogenicity of the protein. Substantial equivalents of the above protein include conservative substitutions of amino acids with other amino acids, including either naturally occurring or non-conventional amino acids, which maintain substantially the same charge and hydrophobicity as the original amino acid. Conservative substitutions include for example, replacement of glycine for alanine, valine for isoleucine, leucine for isoleucine, aspartic acid for glutamic acid, lysine for arginine, asparagine for glutamine, phenylalanine for tryptophan, and tryptophan for tyrosine. Examples of conservative substitutions with non-conventional amino acids are described in Rosenberg et al. (U.S. Pat. No. 5,679,782) the contents of which are incorporated by reference herein.

The protein may be isolated from any P. ovis mite to pure or substantially pure form which is free of endogenous P. ovis material using the techniques described in Example 1 hereinbelow. In brief, P. ovis mites are ground in a suitable buffered aqueous solvent to solubilize the proteins therein. A conventional protease inhibitor such as phenylmethylsulfonylfluoride (PMSF), may be added to inhibit protein hydrolysis. Solids and cellular material may be separated, for example, by centrifugation and/or filtration, with the supernatant containing a crude mixture of soluble P. ovis proteins being retained. The soluble protein may then be fractionated by molecular size using conventional molecular sieve chromatography, and the relatively low molecular weight band(s) encompassing 16 kDa retained. Purification of the 16 kDa within this band may be accomplished by continuous elution sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

In use, it is envisioned that the isolated protein will typically be formulated in conjunction with a suitable inert carrier or vehicle as known in the art. The skilled practitioner will recognize that such carriers should of course be compatible with the protein. The concentration and amount of the protein in the final composition may vary depending upon the desired use and type of response needed, and the host animal. In any event, the protein should be employed in an amount effective to induce the preferred response as determined by routine testing.

When the protein is used to elicit antibody production against P. ovis, the proteins may be formulated with a physiologically acceptable diluent or carrier such as phosphate buffered saline. The proteins may be administered to a target animal by any convenient route, including intramuscularly, intraperitonealy or preferably subcutaneously, in a single dose or in a plurality of doses. The protein may also be administered in combination with optional stabilizers and immunopotentiating agents or adjuvants. Typical stabilizers include, for example, sucrose, an alkali metal hydrogen phosphate salt, glutamate, serum albumin, gelatin, or casein. A variety of adjuvants are suitable for use herein, although a mixture of alhydrogel and amphigen is preferred. Other conventional adjuvants which may be suitable for use herein include those described by Davis et al. (ed.) (Microbiology, second edition, Harper & Row, Hagerstown, Md., 1973, pp. 480-482), the contents of which are incorporated by reference herein. The proteins may be stored under refrigeration or in frozen or lyophilized form.

In a preferred embodiment, the objective of antibody production is the protection of cattle against P. ovis by eliciting antibody production and/or an immediate-type hypersensitivity in the animal. Generally, the proteins are administered to the target animal in an amount effective to elicit either or both of these responses in a subject animal as compared to an untreated control. The effective amount will vary with the particular target animal, its age and size, and may be readily determined by the practitioner skilled in the art. Without being limited thereto, typical doses for treatment of cattle may be greater than 5 .mu.g/animal/dose, preferably between 5 to 25 .mu.g/animal/dose administered by subcutaneous or intramuscular injection.

In a variation of this preferred embodiment, the antibodies so-produced in the host animal may be recovered for use in a diagnostic assay for the identification of P. ovis. While mites are typically identified on the basis of clinical observations and microscopic examination, identification by immunoassay with the antibodies may aid in the identification of P. ovis, particularly when a trained acarologist is unavailable. A variety of conventional immunoassay techniques are suitable for use herein, including RIA, or ELISA, or double antibody sandwich immunoassays.

In an alternative embodiment, the protein may be used as an immunodiagnostic reagent for binding and detecting antibodies in the serum of an animal. Detection of antibodies against P. ovis in the sera of animals may be used for monitoring and detecting animals which are carriers of the mites but which do not show outward signs of infestation, as well as identifying animals previously exposed or infested with P. ovis. Again, a variety of conventional immunoassays are suitable for use herein, although ELISA are preferred. For example, in such an ELISA test, the purified protein of this invention may be used as an antigen bound to the wells of a microtiter plate. Following contact of the test animal sera with the adsorbed antigen, bound anti-P. ovis antibodies may then be detected.

The invention also encompasses isolated DNA sequences, free of homologous DNA, which encode the above-described 16 kDa P. ovis protein. The DNA sequence coding for the protein was derived from cDNA synthesized from P. ovis mRNA as described in Example 1 hereinbelow and is shown in FIGS. 1 and 3. The complete predicted amino acid sequence of the encoded P. ovis protein is also shown in FIGS. 1 and 4. As shown in FIG. 1, translation starts with a 17 amino acid leader peptide (in italics) which is not present in the mature protein. The first 30 N-terminal amino acids of the mature protein are underlined and agree perfectly with the microsequence obtained from the mature protein described in Example 1 hereinbelow. The total cDNA sequence is 588 nucleotides in length which codes for 143 amino acids, of which 126 are present in the mature protein after cleavage of the leader (signal) peptide. There are post-translational modification sites present.

Because of the degeneracy of the genetic code, there exists a finite set of nucleotide sequences which can code for a given amino acid sequence. It is understood that all such equivalent sequences are operable variants of the disclosed sequence, since all give rise to the same protein (i.e., the same amino acid sequence) during in vivo transcription and translation, and are hence encompassed by the instant invention. DNA sequences which are substantially homologous to the nucleotide sequences of FIGS. 1 and 3 are also encompassed by the invention. As defined herein, two DNA sequences are substantially homologous when at least 85% (preferably at least 90% and most preferably 95%) of the nucleotides match over the defined length of the sequence. Sequences that are substantially homologous can be identified in a Southern hybridization experiment under stringent conditions as is known in the art. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, 1982, or DNA Cloning: A Practical Approach, Vol. I and II (Ed. D. N. Glover), IRL Press, Oxford, 1985.

The DNA sequences of the invention can be used to prepare recombinant DNA molecules by cloning in any suitable vector. A variety of vector-host cell expression systems may be employed in practicing the present invention. Host cells may be either procaryotic or eucaryotic, and, when the host cells are bacterial cells, they may be either gram-negative or gram-positive bacteria. Strains of Escherichia coli are generally preferred for use in procaryotic systems. However, without being limited thereto, other useful hosts include species of Salmonella (including, for example, S. typhimurium, S. enteriditis, and S. dublin) species of Mycobacterium (such as M. smegmatis and M. bovis, species of Pseudomonas (including, for example, P. aeruginosa and P. putida), Bacillus subtilis, yeasts and other fungi (for example, Saccharomyces cerevisiae), plant cells such as plant cells in culture (including, for example, both angiosperms and gymnosperms) and animal cells such as animal cells in culture.

Vectors used in practicing the present invention are selected to be operable as cloning vectors or expression vectors in the selected host cell. Numerous vectors are known to practitioners skilled in the art, and selection of an appropriate vector and host cell is a matter of choice. The vectors may, for example, be bacteriophage, plasmids, viruses, or hybrids thereof. A number of procaryotic expression vectors are described in U.S. Pat. Nos. 4,652,525, 4,440,859, 4,436,815, and 4,342,832, and a number of eucaryotic expression vectors have also been described in U.S. Pat. Nos. 4,546,082, 4,510,245, 4,446,235, and 4,443,540. Further, the vectors may be non-fusion vectors (i.e., those producing the antigenic protein of the invention not fused to any heterologous polypeptide), or alternatively, fusion vectors (i.e., those producing the antigenic protein fused to a vector encoded polypeptide). The fusion proteins would of course vary with the particular vector chosen. Suitable non-fusion plasmid vectors for use with E. coli include but are not limited to pTrc99 for use with E. coli JM 105, or pANK-12, pANH-1 or pPL2 for use with E. coli MZ 1. Conversely, suitable fusion plasmid vectors include PGEX and pMC1871 for use with E. coli JM 105, pMAL with E. coli PR 722, pVB2 with E. coli LA5709, pTrcHis with E. coli INV F', pCO5 with E. coli N6405, and pRIT2T or pEZZ 18 with E. coli N4830-1. Other, non-E. coli expression systems which may also he employed include pAc360 or pBluescript for use with SP2 or High 5 insect cells, pYesHis with the yeast S. cerevisiae INVSc1 or INVSc2, pLS405 with Salmonella dublin SL598, and pYUB12 with Mycobacterium smegmatis or M. bovis. Still other suitable vector-host combinations that may be used in practicing the instant invention are described, for example, in U.S. Pat. Nos. 5,122,471 and 5,670,339 the contents of each of which are incorporated by reference herein.

Within each specific vector various sites may be selected for insertion of the isolated DNA sequence. These sites are usually designated by the restriction enzyme or endonuclease that cuts them. For example, in pBR322 the Pst I site is located in the gene for penicillinase between the nucleotide triplets that code for amino acids 181 and 182 of the penicillinase protein.

The particular site chosen for insertion of the selected DNA fragment into the vector to form a recombinant vector is determined by a variety of factors. These include size and structure of the polypeptide to be expressed, susceptibility of the desired polypeptide to enzymatic degradation by the host cell components and contamination by its proteins, expression characteristics such as the location of start and stop codons, and other factors recognized by those of skill in the art. None of these factors alone absolutely controls the choice of insertion site for a particular polypeptide. Rather, the site chosen reflects a balance of these factors, and not all sites may be equally effective for a given protein.

The DNA sequences of the invention may be inserted into the desired vector by known techniques. If, however, the vector is to serve as an expression vector, the vector should have a promoter, and the DNA sequence should be inserted in the vector downstream of the promoter and operationally associated therewith. While control sequences may be ligated to the coding sequence prior to insertion into the vector, preferably, the vector should be selected so as to have a promoter operable in the host cell into which the vector is to be inserted (that is, the promoter should be recognized by the RNA polymerase of the host cell). In addition, the vector should have a region which codes for a ribosome binding site positioned between the promoter and the site at which the DNA sequence is inserted so as to be operatively associated with the DNA sequence of the invention once inserted (in correct translational reading frame therewith). The vector should be selected to provide a region which codes for a ribosomal binding site recognized by the ribosomes of the host cell into which the vector is to be inserted.

The antigenic proteins of the invention are produced by growing host cells transformed by the expression vectors described above under conditions whereby the antigen is produced. The antigens are then isolated from the host cells. Depending on the host cell used, transformation is done using standard techniques. For example, the calcium treatment employing calcium chloride, as described by Cohen (1972, Proc Natl Acad Sci USA, 69:2110), or the RbC1 method described in Maniatis et al. (ibid, p. 254) may be used for procaryotes or other cells which contain substantial cell wall barriers. Infection with Agrobacterium tumefaciens such as described by Shaw (1983, Gene, 23:315) may be used for certain plant cells. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology, 52:546) may be used. Transformations into yeast may be conducted, for example, according to the method of Van Solingen, et al., (1977, J. Bacter., 130:946), and Hsiao et al. (1979, Proc Natl Acad Sci USA, 76:3829).

In general, after construction of a suitable expression system, the system is transfected into the appropriate host and successful transformants may be selected by markers contained on the expression vectors. Successfully transformed colonies are then cultured in order to produce the protein. Optionally, a promoter which can be controlled by regulating conditions in the environment may be used such that the cells can be grown under conditions where the gene encoding the desired protein of the invention is not expressed, but production of the protein may be induced by appropriate manipulation of conditions, as described in U.S. Pat. No. 5,670,339. This protocol may be used to prevent premature accumulation of the protein which may be harmful to the growth of the cell.

The protein may be produced intracellularly, or in secreted form by construction of vectors wherein the peptide is preceded by a signal peptide workable in the appropriate host. The recombinant protein may then be recovered from the medium or from the cells using suitable techniques generally known in the art, and purified by, for example, ion exchange chromatography, ammonium sulfate precipitation, or gel permeation chromatography.

Claim 1 of 3 Claims

We claim:

1. A method for protecting bovine against Psoroptes ovis comprising administering an isolated protein to said bovine in an amount effective to elicit a local immediate-type hypersensitivity response against Psoroptes ovis therein, wherein said protein comprises the amino acid sequence:

X₁ KVKFQDCGKGEVESLEVEGCSGDYCVIHKGKKLDLAI SVTSNQDSANLKLDIVADING VQIEVPGVDHDGCHYVKCPIKKGQHFDVKYTYSIPAILPTTKAKIIAKIIGDKGLGGCIV INGEIQD (SEQ ID No. 1)

wherein said X₁ is selected from the group consisting of G, S, and V.
 

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