Title:  Lymphocyte-derived antimicrobial protein (LDAP) and methods of isolating and producing and using the protein

United States Patent:  6,593,457

Issued:  July 15, 2003

Inventors:  Sordillo-Gandy; Lorraine M. (Port Matida, PA); Shafer-Weaver; Kimberly A. (Pleasant Gap, PA)

Assignee:  The Penn State Research Foundation (University Park, PA)

Appl. No.:  254064

Filed:  February 26, 1999

PCT Filed:  August 27, 1997

PCT NO:  PCT/US97/14158

PCT PUB.NO.:  WO98/08534

PCT PUB. Date:  March 5, 1998

Abstract

A purified, soluble lymphocyte-derived antimicrobial protein that has a molecular weight of 16 kD on SDS-PAGE, is inactivated by heating at 56^oC. for 30 minutes or by treatment with trypsin, is expressed by CD3-, CD2+ cytokine-stimulated cells', and is active against Gram positive or Gram-negative bacteria including Staphylococcus aureus.

SUMMARY OF THE INVENTION

The present invention is drawn to a purified soluble lymphocyte-derived antimicrobial protein (LDAP) having the following properties

a molecular weight of 16 kD or 30-50 kD when associated with bovine serum albumin (BSA),

a susceptibility to inactivation by heat treatment at 56^oC. for 30 minutes or by trypsin,

expression primarily by CD2+, CD3-, cytokine stimulated cells, and

antimicrobial activity against both Gram-positive and Gram-negative bacteria; or

a derivative of said protein which contains one or more conservative amino acid changes and

which has antimicrobial activity against both Gram-negative and Gram-positive bacteria.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, bacterial, fungal and viral infections which effect food and companion animals, are of clear and obvious concern throughout the world. For example, bovine mastitis continues to be the greatest deterrent to profitable dairy production. The incidence of mastitis increases when defense mechanisms of the mammary gland are impaired. Exposure to stressful stimuli or to some mastitis-causing pathogens can contribute to depressed immune functions within the mammary gland. If immunosuppressive changes in host immunity predispose animals to mastitis then methods of facilitating dysfunctional defense mechanisms in the mammary gland should increase disease resistance.

The periparturient period of dairy cattle is associated with diminished immune responses and increased incidence of mastitis (Nickerson, S. C. 1989. Immunological aspects of mammary involution. J. Dairy Sci. 72: 1665; Oliver, S. P. and L. M. Sordillo. 1988. Udder health in the periparturient period. J. Dairy Sci., 71: 2584). Diminished mammary gland immune function is due, in part, to alterations in neutrophil functions (Kehrli, M. E., B. J. Nonnecke, and J. A. Roth. 1989. Alterations in bovine neutrophil function during the periparturient period. Am. J. Vet. Res. 50: 207). Additionally, important lymphocyte effector functions such as proliferation as well as antibody and cytokine production have been shown to be reduced during this time (Harp, J. A. and B. J. Nonnecke. 1986. Regulation of mitogenic responses by bovine milk leukocytes. Vet. Immunol. Immunopathol. 11: 215; Kehrli, M. E., B. J. Nonnecke, and J. A. Roth. 1989. Alterations in bovine lymphocyte function during the periparturient period. Am. J. Vet. Res. 50:215; Nagahata, H., A., Ogawa, Y., Sanada, H., Noda, and S., Yamamoto. 1992. Peripartum changes in antibody producing capability of lymphocytes from dairy cows. Vet. Quarterly. 14:39; Sordillo, L. M., M. J. Redmond, M. Campos, L. Warren, and L. A. Babiuk. 1991. Cytokine activity in the bovine mammary gland secretions during the periparturient period. Can. J. Vet. Res. 55:298; Torre, P., P., Konur, and S. P. Oliver. 1992. Proliferative response of mammary gland mononuclear cells to recombinant bovine interleukin-2 (IL-2). Vet. Immunol. Immunopathol. 32:351). The role of nonspecific immune responses within the mammary gland has been well established. Conversely, considerably less is known about the protective role of resident lymphoid populations in the bovine mammary gland. Healthy mammary parenchymal tissue is infiltrated constitutively with lymphocytes, particularly during the periparturient period (Outteridge, P. M. and C. S. Lee. 1988. The defense mechanisms of the mammary gland of domestic ruminants. Prog. Vet. Microbiol. Immun. 4:165). Since lymphocytes are a predominant leukocyte type in healthy mammary tissue, these cells may act as an important first line of contact with invading microbial pathogens.

Protocols aimed at supporting important host defense mechanisms in companion or food-producing animals may be the most effective way of controlling bacterial, fungal and viral infections. Developing alternatives to chemotherapy for the prevention and control of pathogen caused infections is the purpose of this invention.

It has been shown that human lymphoid cells are capable of killing both Gram-positive and Gram-negative bacteria by an extracellular mechanism (Garcia-Penarrubia, P., F. T. Koster, R. O. Kelley, T. D. McDowell, and A. D. Bankhurst. 1989. Antibacterial activity of human natural killer cells. J. Exp. Med. 169:99). Lymphocytes isolated from the mammary glands of lactating dairy cattle have been shown to exhibit a novel antibacterial property. Upon stimulation with IL-2, these lymphocytes demonstrated increased ability to kill Staphylococcus aureus in a nonspecific manner (Sordillo, L. M., M. Campos, and L. A. Babiuk. 1991. Antibacterial activity of bovine mammary gland lymphocytes following treatment with IL-2. J. Dairy Sci. 74:3370).

The present invention is based on the observation that bovine mammary gland tissues are infiltrated heavily with lymphoid cells and that stimulation of these cells with cytokines, such as IL-2, results in the generation of lymphocytes with the ability to kill bacterial targets in vitro. It is believed that this non-specific defense mechanism which is situated at strategic anatomical sites in the mammary gland that would allow prompt response to invading bacterial pathogens and contribute significantly to host defense against bacterial infections.

The present invention is drawn to a soluble factor which mediates this unique antibacterial effector function of lymphocytes.

More specifically the present invention is drawn to a soluble antimicrobial protein, produced primarily by CD2³⁰ CD3³¹ lymphocytes, having a molecular weight of approximately 30-50 kD when complexed with BSA and a purified molecular weight of approximately 16 kD, as determined by SDS-PAGE and having bactericidal activity against Gram-negative and Gram-positive pathogens which cause bacterial infections without adversely affecting host cells. This protein has been designated as LDAP and such designation in the present application will be used to refer to the protein.

The LDAP protein of the present invention has been shown to be active against both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus, Streptoccus uberis and Escherichia coil (Table 7).

The presently claimed protein may be isolated from immune or non-immune lymphocytes which have been activated with cytokines, such IL-2 or interferon, or bacteria. The antimicrobial activity of the protein results in non-Major Histocompatibility Complex (MHC) restricted lysis of the pathogen.

The present invention encompasses both LDAP isolated from natural sources and recombinant LDAP protein. Recombinant LDAP may be expressed using conventional recombinant techniques and expression systems such as those presented in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press.

It is contemplated that with such expression systems, nucleic acid and nucleic acid analogues, including single and double DNA and RNA, encoding LDAP may be, for example, inserted into a suitable plasmid vector. The nucleic acid and nucleic acid analogues encoding LDAP may be obtained from either a lymphocyte cDNA library or synthetically synthesized. The vector containing the nucleic acid and nucleic acid analogues encoding LDAP may then be expressed in suitable host cells. Suitable host cells include bacterial expression systems such as E. Coli, or eurkaryotic expression systems such as baculovirus systems, mammalian transformed cells and fungal systems such as yeast.

The present invention is further drawn to nucleic acid and nucleic acid analogues sequences, including DNA and RNA, encoding LDAP, as well as nucleic acid and nucleic acid analogue sequences which hybridize under stringent conditions to a complementary strand of a nucleic acid and nucleic acid analogue sequence encoding LDAP. Stringent hybrization conditions may be as presented in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press.

Nucleic acid analogues encompass any naturally occurring nucleic acid which has been modified or derivatized. Included as nucleic acid analogues are substituted nucleotide units. Such substituted nucleotide units include nucleotide units which confer resistance to nucleases and which are also compatible with polymerase chain reaction amplification. Examples of nucleic acid analogues include substituted pyrimidines, such as 2'fluoro-pyrimidines including 2'-fluoro-2'deoxycytidine or 2'-fluoro-2'-deoxyuridine residues. The substituted pyrimidines may also comprise 2'-amino-pyrimidine such as 2'-amino-2'-deoxycytidine or 2'-amino-2'deoxyuridine residues. The nucleotide units may also comprise substituted purines such as 2'-fluoro-purine including 2'-fluoro-2'-deoxyadenine or 2'-fluoro-2'deoxyguanidine residues. Also included are substituted purines comprising 2'-amino-purine and including 2'-amino-2'deoxyadenine or 2'amino-2'-deoxyguanidine residues. Nucleic acid analogues further encompass nucleic acids which have been modified to contain a chemical, fluorescent or radioactive marker.

Also encompassed by the present invention are LDAP proteins containing one or more conservative amino acid changes from the native protein. It is well understood in the art that conservative changes may be made to a protein while retaining function of the protein. Such conservative amino acid changes are those wherein amino acid substitutions are made which maintain charge identity of amino acid residues and steric configurations of amino acids. For example, acidic amino acids may be substituted for other acidic amino acids and one sterically bulky amino acid may be substituted for another. Conservative amino acid changes also include the deletion or addition of one or more amino acids, particularly from the amino or carboxy terminal end of the protein such that the antimicrobial activity of the protein is retained.

Given the small size of LDAP, derivatives which encompass conservative amino acid changes may be made using conventional techniques as presented in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press. Such derivatives may be readily assayed for activity as described in the Examples, below. For example, it will be readily apparent to one practicing the present invention to determine portions of LDAP protein which may be deleted from the amino or carboxy terminal end of the protein while retaining activity. Such proteins which contain conservative amino acid changes are considered to be encompassed by the present invention.

It is contemplated based on accepted knowledge regarding the biochemistry of proteins that significant portions of a protein's amino acid sequence may be altered either synthetically or as naturally occurs between different species, wherein the protein maintains its biological activity. As such, the present invention further encompasses a functionally active protein which has at least 60% homology, preferably at least 80% homology, more preferably at least 95% homology to LDAP isolated from bovine peripheral blood lymphocytes.

The present invention is further drawn to pharmaceutical compositions containing LDAP. Such pharmaceutical compositions may be used in both the prophylaxis and treatment of pathogen caused infections in food and companion animals. Administration of LDAP to other food and companion animals during periods of diminished immune response will help prevent bacterially induced infectious diseases. For example, the periparturient period of dairy cattle is associated with diminished immune responses and an increased incidence of mastitis. Administration of exogenous LDAP to the dairy cattle during the periparturient period with help prevent the occurrence of mastitis.

LDAP protein is also useful in the treatment of pathogen based infections in food and companion animals. LDAP may be administered to an animal as a antimicrobial therapeutic.

Pharmaceutical compositions containing LDAP protein are administrable in the form of tablets, pills, powder mixtures, capsules, dispersions, solutions, suppositories, transdermal patches, emulsions, micelles, gels, liposomes injectables and in other suitable forms. The pharmaceutical compositions may be administered orally, parentally (including subcutaneous intradermal, intramuscular and intravenous administrations), topically or rectally. The most suitable route for administration will depend on, the identity and severity of the pathogen caused disease being treated or prevented. The pharmaceutical preparation which contains the compound is conveniently admixed with a nontoxic pharmaceutical organic or inorganic carrier, usually about 0.01 mg up to 20 mg, or higher per dosage unit, preferably 0.01 mg to 2 mg.

Typical pharmaceutically acceptable carriers are, for example, mannitol, urea, dextrans, lactose, potato and maize starches, magnesium stearate, talc, vegetable oils, polyalkylene glycols, ethyl cellulose, poly(vinylpyrrolidone), calcium carbonate, ethyl oleate, isopropyl myristate, benzyl benzoate, sodium carbonate, gelatin, potassium carbonate, silicic acid dimethylsulfoxide and other conventionally employed acceptable carriers in which proteins are stable. The pharmaceutical preparation may also contain nontoxic auxiliary substances such as emulsifying, preserving, wetting agents, excipients and the like as for example, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene monostearate, glyceryl tripalmitate, dioctyl sodium sulfosuccinate and the like.

Of particular interest are injectable forms of pharmaceutical compositions containing LDAP. LDAP may be freeze dried and, if desired, combined with other pharmaceutically acceptable excipients to prepare formulations suitable for subcutaneous parenteral, intravenous, or intramuscular injectable administration. For such administration, the formulation can be reconstituted in water (normal, saline) or a mixture of water and an organic solvent, such as propylene glycol, ethanol, and the like. Also of interest are topical pharmaceutical formulations containing LDAP. Topical formulations will be useful in the application of LDAP to localized infections, such as dermatitis. Such topical formulations include teat dips and the like. Topical formulations may further include antiseptics, analgesics, moisturizers and the like.

The dose administered may be given as a single dose, multiple doses or a daily dose in an immediate acting form or sustained release formulation using a biodegradable polymer, for example, and will, of course, vary with the chosen route of administration, size of the animal to be administered and the condition of the animal. A typical dose will be in the range of 10 .mu.g/kg body weight to 250 mg/kg body weight, preferably 10 .mu.g/kg to 25 mg/kg more preferably 10 .mu.g/kg to 2 mg/kg.

The pharmaceutical compositions of the present invention may also contain additional active components such as natural or synthetic antibiotics, antipyretics, and analgesics.

The present invention is further drawn to antibodies raised against the LDAP protein. Such antibodies may be either polyclonal or monoclonal and may be generated by conventional techniques such as those in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press. Antibodies raised against LDAP may be useful in the isolation of the protein and in diagnostic procedures in detecting the presence of the protein.

The present invention is further drawn to methods of treatment with LDAP.

In conjunction with the methods of treatment using LDAP protein the present invention is further drawn to a kit containing LDAP and a suitable carrier.

The components of the present invention may be packaged as a kit. Uses of the kit may be for the prevention or treatment of pathogen caused infections in food and companion animals with LDAP. Alternatively, the kit may be for diagnostic purposes with antibodies which recognize LDAP. Each component of the kit(s) may be individually packaged in its own suitable container. The individual containers may also be labelled in a manner which identifies the contents. Moreover, the individually packaged components may be placed in a larger container capable of holding all desired components. Associated with the kit may be instructions which explain how to use the kit. These instructions may be written on or attached to the kit.

Claim 1 of 14 Claims

What is claimed is:

1. A purified soluble lymphocyte-derived antimicrobial protein having the following properties:

a molecular weight of approximately 16 kD by SDS-PAGE,

a susceptibility to inactivation by heat treatment at 56^oC. for 30 minutes or by trypsin,

expression by CD3-, CD2+ cytokine stimulated cells,

and antibacterial activity against both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus.

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