EP0948528A2 - Laktoferrin bindendes protein aus streptococcus uberis - Google Patents

Laktoferrin bindendes protein aus streptococcus uberis

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Publication number
EP0948528A2
EP0948528A2 EP97913041A EP97913041A EP0948528A2 EP 0948528 A2 EP0948528 A2 EP 0948528A2 EP 97913041 A EP97913041 A EP 97913041A EP 97913041 A EP97913041 A EP 97913041A EP 0948528 A2 EP0948528 A2 EP 0948528A2
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European Patent Office
Prior art keywords
uberis
binding
binding protein
protein
blf
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EP97913041A
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English (en)
French (fr)
Inventor
Min Jiang
Andrew A. Potter
Philip Ronald Maclachlan
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University of Saskatchewan
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University of Saskatchewan
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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 present invention relates generally to bacterial antigens. More particularly, the present invention pertains to the characterization and recombinant production of a bovine lactoferrin-binding protein from Streptococcus uberis ( S . uberis) and the use of the same. The invention also relates to the characterization of a regulatory region, mga, located upstream of the lactoferrin-binding protein gene.
  • Streptococcus uberis S . uberis
  • S . uberis is an environmental pathogen responsible for a high proportion of cases of mastitis in lactating cows and is the predominant organism isolated from mammary glands during the nonlactating period (Bramley, A.J. (1984) Br. Vet . J. 140:328-335; Bramley and Dodd (1984) J. Dairy Res . 51:481-512; Oliver, S.P. (1988) Am . J. Vet . Res . 4.9:1789-1793) . Mastitis resulting from infection with S .
  • S . uberis mastitis may also take the form of an acute clinical condition, with obvious signs of disease such as clots or discoloration of the milk and swelling or hardness of the mammary gland. Some cases of the clinical disease can be severe and pyrexia may be present.
  • S . uberis mastitis see, Bramley (1991) Mastitis: physiology or pathology, p.
  • S . uberis infection The pathogenesis of S . uberis infection is poorly understood. Furthermore, the influence of S . uberis virulence factors on host defense mechanisms and mammary gland physiology is not well defined. Known virulence factors associated with S . uberis include a hyaluronic acid capsule, hyaluronidase, R- like protein, plasminogen activator and CAMP factor. However, very little is known of their roles in pathogenicity.
  • Lactoferrin is a mammalian iron-binding glycoprotein secreted by polymorphonuclear leukocytes (PM ⁇ s) and various exocrine glands (Baggiolini et al . (1970) J " . Exp . Med . 131: 559-570 ; Masson et al . (1966) Clin . Chim . Acta 14:735-739) . This protein is found at high concentrations in milk and at mucosal surfaces (Masson et al . , supra; Reiter and Oram (1967) Nature 216:328-330) .
  • bovine lactoferrin (bLf) concentrations in lacteal secretions can increase up to 30 -fold during acute bovine mastitis, depending on the severity of infection (Harmon et al . (1976) Infect . Immun . 13:533-542) .
  • Lf A regulatory function for Lf in various physiological pathways, including the adhesion of PM ⁇ s to the endothelial surface, feedback inhibition of the granulocyte-monocyte colony-stimulating factor, and the regulation of antibody production, has been suggested.
  • Specific interaction of Lf with certain mammalian cells seems to be involved in the above pathways and specific receptors for Lf have been identified on macrophages, monocytes, B lymphocytes, PMNs, activated T lymphocytes, and hepatocytes (Bennet and Davis (1981) J " . Immunol . 127:1211-1216; Dehanne et al. (1985) Am . J. Physiol . 248:463-469: Maneva et al . (1983) Int .
  • Lf inhibits the growth of E. coli and certain other microorganisms in vi tro (Bullen et al . (1972) Br. Med . J. 1:69-75) .
  • This Lf-mediated antimicrobial action has mainly been attributed to its iron deprivation capacity with bacteria (Arnold et al . (1977) Science 197:263-265; Law and Reiter (1977) J. Dairy Res . 44:595-599; Oram and Reiter (1968) Biochim . Biophys . Acta . 170:351-365) .
  • iron is essential for microbial growth ( einberg, E.D. (1978) Microbiol . Rev.
  • bacterial pathogens have developed specific iron uptake mechanisms.
  • these mechanisms involve the synthesis and secretion of small compounds called siderophores which display high affinity for ferric iron (Felll) .
  • Siderophores are capable of removing TF- or Lf-bound iron to form ferrisiderophore complexes which in turn are recognized by specific iron-repressible membrane receptors and internalized into the bacterium where the iron is released (Crosa, J.H. (1989) Microbiol . Rev. . 53:517-530) .
  • This iron uptake mechanism has been described for many gram-negative bacterial species.
  • Some gram-negative bacteria do not secrete detectable siderophores when grown in an iron-deficient environment but produce outer membrane proteins that bind directly and specifically to Tf or Lf, thereby allowing iron transport into the bacterial cell .
  • Tf binding appears to be mediated by the activity of two proteins present in bacterial outer membranes, transferrin-binding protein 1 and 2 (Tbpl and Tbp2) (Gonzalez et al . (1990) Mol . Microbiol . 4 . : 1173-1179; Ogunnariwo and Schryvers (1990) Infect . Immun . 58 . : 2091-2097 ; Schryvers, A.B. (1989) J. Med . Microbiol . 29.: 121-130) ; Schryvers and Lee (1989) Can . J. Microbiol . 35:409-415; Schryvers and Morris (1988) Mol . Microbiol . 2:467-472) . Transferrin binding proteins tend to be highly specific for the transferrin of their natural host.
  • Lbpl has been shown to be 46% identical to Tbpl (Comelissen et al . (1992) J. Bacteriol . 174:5788-5797) of the same gonococcal strain but only 18% identical to Tbp2 (Anderson et al . (1994) J. Bacteriol . 176:3162-3170) .
  • the group A streptococcal M protein is considered to be one of the major virulence factors of this organism by virtue of its ability to impede attack by human phagocytes (Lancefield, R.C. (1962) J. Immunol . 89:307-313).
  • the bacteria persist in the infected tissue until antibodies are produced against the M molecule.
  • Type-specific antibodies to the M protein are able to reverse the antiphagocytic effect of the molecule and allow efficient clearance of the invading organism.
  • M proteins are one of the key virulence factors of S . pyogenes, due to their involvement in mediating resistance to phagocytosis (Kehoe, M.A.
  • group A streptococci the genes for M protein (emm) as well as a peptidase ⁇ scpA) and, if present, genes encoding M protein-related IgG- and IgA-binding proteins ( fcrA and enn, respectively) are clustered on the chromosome (Haanes et al . (1992) J " . Bacteriol . 174:4967-4976; Hollingshead et al . (1993) Mol . Microbiol . 8:707-717; Podbielski, A. (1993) Mol . Gen . Genet . 237:287-300). Expression of these virulence-associated surface proteins is co-regulated at the level of transcription by the protein Mga (which stands for multigene regulator of group A
  • Mga is a part of a crucial regulatory system in GAS, possibly functioning as a second component in a two-component regulatory system.
  • Vaccination is one approach to enhance resistance of the mammary gland to new infection and reduce clinical severity of the disease.
  • Previous studies have shown that primary infection with S . uberis can considerably reduce the rate of infection following a second challenge with the same strain (Hill, A.W. (1988) Res . Vet . Sci . 44:386-387).
  • Local vaccination with killed S . uberis protects the bovine mammary gland against intramammary challenge with the homologous strain (Finch et al . (1994) Jnfect. Immun . 62.:3599-3603) .
  • subcutaneous vaccination with live S . uberis has been shown to cause a dramatic modification of the pathogenesis of mastitis with the same strain (Hill et al .
  • the present invention is based on the discovery of a bovine lactoferrin (bLF) binding protein (bLbp) from S . uberis, and the characterization thereof.
  • bLF-binding protein, immunogenic fragments and analogs thereof, and/or chimeric proteins including the same can be used, either alone or in combination with other antigens, in novel subunit vaccines to provide protection from bacterial infection in mammalian subj ects .
  • the subject invention is directed to an isolated, immunogenic S . uberis bLF-binding protein, as well as a nucleic acid molecule comprising a coding sequence for an immunogenic S. uberis bLF-binding protein.
  • the invention is directed to recombinant vectors including the same, host cells transformed with these vectors and methods of recombinantly producing S. uberis bLF-binding proteins .
  • the subject invention is directed to vaccine compositions comprising a pharmaceutically acceptable vehicle and an immunogenic S . uberis bLF-binding protein.
  • the present invention is directed to methods of treating or preventing S . uberis infections, such as mastitis, in a mammalian subject. The method comprises administering to the subject a therapeutically effective amount of the above vaccine compositions.
  • the invention pertains to methods of producing vaccine compositions comprising (a) providing an immunogenic S . uberis bLF- binding protein; and (b) combining the protein with a pharmaceutically acceptable vehicle.
  • the invention is directed to antibodies against the S. uberis bLF- binding proteins .
  • the invention is directed to methods of detecting S . uberi s antibodies in a biological sample comprising:
  • the invention is directed to an immunodiagnostic test kit for detecting S . uberis infection.
  • the test kit comprises a S . uberi s bLF-binding protein and instructions for conducting the immunodiagnostic test.
  • the invention is directed to an isolated S . uberis Mga protein, as well as a nucleic acid molecule comprising a coding sequence for the same .
  • Figure 1 is a restriction enzyme map of Ibp and shows progressive deletions and a summary of bLf binding data.
  • Open boxes (p region) represent 5'- sequences containing promoter and ribosome binding sites, shaded boxes (s region) represent Ibp sequences encoding the signal peptides, and hatched boxes represent the Ibp sequences coding for the mature or truncated proteins.
  • RI and R201 (R represents residue) indicate the first codon of the mature protein in pLBP5 and the truncated protein in pTP51, respectively. Other numbers indicate the last codon of each protein. Restriction sites are present on the top of the Ibp . The bLf binding ability of each clone is shown on the right.
  • Figures 2A-2C depict the nucleotide sequence and deduced amino acid sequence of S . uberis bovine Lbp . Nucleotides and amino acids are numbered on the right of the sequences. The deduced amino acid sequence is shown in the single-letter code below the nucleotide sequence. Two possible ATG start codons at positions 232 and 262, and the TAA stop codon at 1915, are shown in bold. Two putative -35 and -10 promoter sequences and Shine-Dalgarno sequences (SD) are indicated. A putative rho- independent transcription terminator (T) is underlined.
  • the double underline shows the presence of a putative signal peptide at the N-terminus of the ORF .
  • the C-terminal hydrophobic trans-membrane domain is indicated by italics and the nearby surface anchor motif is shown by italics and double underline.
  • the central repeated amino acid sequences are indicated by the letters A, B, and C.
  • Figure 3 shows profiles of the secondary structures, charged residues and hydrophobicity of Lbp.
  • the deduced amino acid sequence of Lbp was analyzed with the Novotny-Auffray algorithm. Plots marked Turn, Beta, and Alpha indicate the potential for beta turn-random coil, beta sheet, and alpha helix formation, respectively.
  • the +/- plot shows regions of the molecule with net positive (upper) and negative (lower) charges.
  • the hydrophobicity (Hydro) plot shows the hydrophobic regions of the protein. The positions of the amino acids are shown on the horizontal axis.
  • FIG. 4 depicts the construction of pMGA14F from pLBP5i and pMGA14.
  • Plasmid pMGA14F was generated by inserting the 1.5 kb Sphl -Nhel fragment of pLBP5i into the Sphl and N el sites of pMGA14.
  • Lines indicate S . uberis D ⁇ A, while the box represents the multiple cloning sites of vector pTZ18R.
  • the probe fragments used for Southern or Northern blot experiments are indicated by the hatched bars.
  • the arrows indicate the locations of the open reading frames of lbp, mga ' and mga .
  • Figures 5A-5D show the nucleotide sequence of mga and deduced amino acid sequence of Mga, as well as the ORFs downstream of mga . Nucleotides and amino acids are numbered on the right of the sequences. The deduced amino acid sequence is shown in the single-letter code below the nucleotide sequence. Possible ATG start codons are shown in bold and the stop codons are indicated by "*”. A putative -35 and -10 promoter sequence and Shine-Dalgarno sequence (SD) are indicated.
  • Figure 6 shows the probes used in hybridization analysis, as well as the structure of Lbp (on the top) , constructed from the DNA sequence analysis. The signal peptide, proline rich region and transmembrane domain are indicated by S, Pro and TM respectively. The A, B and C repeat regions are also shown. Probes used in hybridization are indicated by the hatched bars below the map.
  • Figure 7 shows the time course of 125 I-bLf binding to S . uberis . IO 9 bacteria were incubated with 6.9 nm 125 I-bLf in 0.2 ml of PBS-1% BSA. At time intervals indicated, bacteria were pelleted and the amount of cell associated 125 I-bLf was determined.
  • Figure 8 depicts the results of a competition binding assay using bLf (33% iron- saturated) as radiolabelled ligand and competitor. Percentage binding values were calculated as percentage of 125 I-bLf binding in the presence of increasing amounts of unlabelled bLf to bacteria suspended in PBS-1% BSA in the absence of unlabelled bLf.
  • Figure 9 shows the influence of iron chelators on the expression of lactoferrin-binding by S . uberis .
  • Cells grown in THB-YE with or without EDDA, dipyridyl or desferrioxamine mesylate were incubated with 6.9 nM 15 I-bLf in 0.2 ml of PBS-1% BSA at room temperature for 2 h. After three washes, cell-bound radioactivity was determined.
  • Figure 10 shows the physical map of the recombinant plasmid used to express the Lbp in Example 3.
  • the plasmid pLBP5 contains 3.7 kb of S. uberis- derived kDNA, in the vector pTZ18R.
  • the plasmid pGH- LBP was constructed by subcloning an Sphl -Rsal fragment from pLBP5 into vector pGH433.
  • P tac indicates the location of the tac promoter.
  • the Lbp gene is shown by the arrows labelled as lbp .
  • Figure 11 shows inhibition of recombinant bLf-binding protein on 125 I -labelled bLf binding to S . uberis .
  • Increasing amounts of a mixture of supernatant (sup.) and whole cell lysate (with equal volume) of E. coli pLBP5 (•) or E. coli pTZ18R (O) were mixed with IO 9 cells and incubated with 0.69 nM 125 I-bLf in 0.2 ml volumes. Inhibition values were calculated as relative percentage of bLf binding to bacteria suspended in
  • lactoferrin-binding protein intends a protein or a nucleotide sequence, respectively, which is derived from an S . uberis lbp gene.
  • the nucleotide sequence of a representative S . uberis lbp gene, and the corresponding amino acid sequence of an LF-binding protein encoded by this gene, are depicted in Figures 2A-2C (SEQ ID NOS:l-2).
  • an LF-binding protein as defined herein is not limited to the depicted sequence as several subtypes of S .
  • uberis are known and variations in LF- binding proteins will occur between strains of S . uberi s .
  • the derived protein or nucleotide sequences need not be physically derived from the gene described above, but may be generated in any manner, including for example, chemical synthesis, isolation (e.g., from S . uberis) or by recombinant production, based on the information provided herein. Additionally, the term intends proteins having amino acid sequences substantially homologous (as defined below) to contiguous amino acid sequences encoded by the genes, which display immunological and/or lactoferrin-binding activity.
  • nucleotide fragments of the gene that include at least about 8 contiguous base pairs, more preferably at least about 10-20 contiguous base pairs, and most preferably at least about 25 to 50 or more contiguous base pairs of the gene. Such fragments are useful as probes and in diagnostic methods, discussed more fully below.
  • the terms also include those forms possessing, as well as lacking, the signal sequence, as well as the nucleic acid sequences coding therefor. Additionally, the term intends forms of LF-binding protein which lack the membrane anchor region, and nucleic acid sequences encoding such deletions. Such deletions may be desirable in systems that do not provide for secretion of the protein. Furthermore, an LF-binding domain, found within about the N-terminal 200 codons, may or may not be present. Thus, for example, if the Lf binding protein will be used to purify LF, the LF-binding domain will generally be retained. If the protein is to be used in vaccine compositions, immunogenic epitopes which may or may not include the LF-binding domain, will be present.
  • the terms also include proteins in neutral form or in the form of basic or acid addition salts depending on the mode of preparation.
  • Such acid addition salts may involve free amino groups and basic salts may be formed with free carboxyls.
  • the proteins may be modified by combination with other biological materials such as lipids (both those occurring naturally with the molecule or other lipids that do not destroy immunological activity) and saccharides, or by side chain modification, such as acetylation of amino groups, phosphorylation of hydroxyl side chains, oxidation of sulfhydryl groups, glycosylation of amino acid residues, as well as other modifications of the encoded primary sequence.
  • side chain modification such as acetylation of amino groups, phosphorylation of hydroxyl side chains, oxidation of sulfhydryl groups, glycosylation of amino acid residues, as well as other modifications of the encoded primary sequence.
  • the term therefore intends deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein.
  • amino acids are generally divided into four families: (1) acidic -- aspartate and glutamate; (2) basic -- lysine, arginine, histidine;
  • mastitis is meant an inflammation of the mammary gland in mammals, including in cows, ewes, goats, sows, mares, and the like, caused by the presence of S . uberis .
  • the infection manifests itself by the infiltration of phagocytic cells in the gland.
  • mastitis 4 clinical types are recognized: (1) peracute, associated with swelling, heat, pain, and abnormal secretion in the gland and accompanied by fever and other signs of systemic disturbance, such as marked depression, rapid weak pulse, sunken eyes, weakness and complete anorexia; (2) acute, with changes in the gland similar to those above but where fever, anorexia and depression are slight to moderate; (3) subacute, where no systemic changes are displayed and the changes in the gland and its secretion are less marked: and (4) subclinical, where the inflammatory reaction is detectable only by standard tests for mastitis.
  • Standard tests for the detection of mastitis include but are not limited to, the California Mastitis Test, the Wisconsin Mastitis Test, the Nagase test, the electronic cell count and somatic cell counts used to detect a persistently high white blood cell content in milk.
  • a somatic cell count of about 300,000 to about 500,000 cells per ml or higher, in milk will indicate the presence of infection.
  • a vaccine is considered effective in the treatment and/or prevention of mastitis when, for example, the somatic cell count in milk is retained below about 500,000 cells per ml.
  • nucleic acid molecule is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith.
  • subunit vaccine composition is meant a composition containing at least one immunogenic polypeptide, but not all antigens, derived from or homologous to an antigen from a pathogen of interest. Such a composition is substantially free of intact pathogen cells or particles, or the lysate of such cells or particles.
  • a “subunit antigen composition” is prepared from at least partially purified (preferably substantially purified) immunogenic polypeptides from the pathogen, or recombinant analogs thereof.
  • a subunit antigen composition can comprise the subunit antigen or antigens of interest substantially free of other antigens or polypeptides from the pathogen.
  • epitope refers to the site on an antigen or hapten to which specific B cells and/or T cells respond.
  • the term is also used interchangeably with "antigenic determinant” or "antigenic determinant site.”
  • Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
  • an "immunological response" to a composition or vaccine is the development in the host of a cellular and/ or antibody-mediated immune response to the composition or vaccine of interest.
  • an "immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or ⁇ T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.
  • the host will display either a therapeutic or protective immunological response such that resistance of the mammary gland to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered somatic cell count in milk from the infected quarter.
  • immunogenic protein or polypeptide refer to an amino acid sequence which elicits an immunological response as described above.
  • An "immunogenic" protein or polypeptide, as used herein, includes the full-length sequence of the LF- binding protein, with or without the signal sequence, membrane anchor domain and/or LF-binding domain, analogs thereof, or immunogenic fragments thereof.
  • immunogenic fragment is meant a fragment of an LF- binding protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epi tope Mapping Protocols in Methods in Molecular Biology, Vol.
  • linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports.
  • Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871; Geysen et al . (1984) Proc . Natl . Acad . Sci . USA 81:3998-4002; Geysen et al . (1986) Molec . Immunol .
  • conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2 -dimensional nuclear magnetic resonance. See, e.g., Epi tope Mapping Protocols, supra .
  • Immunogenic fragments for purposes of the present invention, will usually include at least about 3 amino acids, preferably at least about 5 amino acids, more preferably at least about 10-15 amino acids, and most preferably 25 or more amino acids, of the Lbp molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or even a fusion protein comprising two or more epitopes of Lbp.
  • Native proteins or polypeptides refer to proteins or polypeptides isolated from the source in which the proteins naturally occur.
  • Recombinant polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide.
  • Synthetic polypeptides are those prepared by chemical synthesis.
  • a “vector” is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a DNA "coding sequence” or a "nucleotide sequence encoding" a particular protein is a DNA sequence which is transcribed and translated into a polypeptide in vi tro or in vivo when placed under the control of appropriate regulatory elements. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3 ' to the coding sequence .
  • control elements refers collectively to promoters, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. Not all of these control sequences need always be present in a recombinant vector so long as the desired gene is capable of being transcribed and translated.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter and the coding sequence and the promoter can still be considered “operably linked" to the coding sequence.
  • RNA polymerase directs the transcription of a coding sequence in a cell when RNA polymerase will bind the promoter and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • a "host cell” is a cell which has been transformed, or is capable of transformation, by an exogenous nucleic acid molecule.
  • a cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the exogenous DNA may be maintained on an episomal element, such as a plasmid.
  • a stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.
  • homology refers to the percent identity between two polynucleotide or two polypeptide moieties.
  • the correspondence between the sequence from one moiety to another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs such as ALIGN, Dayhoff, M.O. (1978) in Atlas of Protein Sequence and Structure 5:Suppl. 3, National biomedical Research Foundation, Washington, DC. Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al . , supra ; DNA Cloning, supra ; Nucleic Acid Hybridization, supra .
  • the term "functionally equivalent” intends that the amino acid sequence of an LF-binding protein is one that will elicit a substantially equivalent or enhanced immunological response, as defined above, as compared to the response elicited by an LF-binding protein having identity with the reference LF-binding protein, or an immunogenic portion thereof.
  • a "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature.
  • the heterologous region encodes a bacterial gene
  • the gene will usually be flanked by DNA that does not flank the bacterial gene in the genome of the source bacteria.
  • Another example of the heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene) . Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.
  • a "biological sample” refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vi tro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components .
  • label and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores , dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.
  • fluorescer refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range.
  • labels which may be used under the invention include fluorescein, rhodamine, dansyl , umbelliferone, Texas red, luminol , NADPH and - ⁇ - galactosidase .
  • a single ORF of 1683 bp depicted as residues 232-1914, inclusive of Figures 2A-2C (SEQ ID N0S:l-2), encoding 561 amino acid residues, gives rise to two protein species able to bind bovine lactoferrin, having molecular weights of 76 kDa and 165 kDa, respectively.
  • the 165 kDa protein is likely a dimer of the 76 kDa protein since urea treatment results in a single band and Northern blot analysis shows only one major transcript in S . uberis, as well as in recombinant E. coli transformed with a construct encoding the LF-binding protein.
  • S. uberis bovine LF-binding protein includes a putative N-terminal signal peptide of about 50 amino acids (if translation starts at the first ATG codon shown in Figure 2A) .
  • the full-length bovine LF- binding protein depicted, including the signal sequence is found at amino acid positions 1-561, inclusive, (encoded by nucleotide positions 232-1914, inclusive) of Figures 2A-2C (SEQ ID NOS: 1-2).
  • the mature protein, lacking the signal peptide is found at amino acid positions 52-561, inclusive, (nucleotide positions 445-1914, inclusive) of Figures 2A-2C.
  • a membrane anchor motif at the C-terminus is also present, as indicated in Figures 2A-2C (SEQ ID N0S:1- 2) .
  • a bovine Lf binding domain is present in a 200 codon N-terminal region of the molecule. The protein appears to lack disulfide bridges.
  • the binding of 125 1 -bLf to S. uberis was time-dependent and displaceable by unlabelled bLf.
  • Apo-bLf inhibits 125 i- bLf binding as effectively as iron-saturated bLf.
  • Bovine transferrin, human lactoferrin and human transferrin do not interfere with bLf binding.
  • the Scatchard plot is linear and approximately 7800 binding sites are expressed by each bacterial cell, with an affinity of 1.0 x IO "7 M. Reduced iron availability does not significantly modify the saturation of S . uberis by bLf.
  • the bLf binding protein described herein is lactoferrin species- specific, in that human Lf does not appear to effectively block the binding of bovine Lf .
  • the Lf binding protein of S . uberis differs from the transferrin receptors of Haemophilus and Neisseria sp . , which consist of two distinct transferrin-binding proteins, termed Tbpl and Tbp2 , which range in molecular weight from 68 to 105 kDa depending on the strain.
  • Tbpl and Tbp2 transferrin-binding proteins
  • the bovine Lf receptor of S . aureus consists of two distinct bLf binding proteins with estimated molecular weights of 92 and 67 kDa (Naidu et al . (1991) J " . Dairy Sci . 74:1218-1226) and therefore appears to be different from the receptor described herein.
  • streptococcal LF-binding protein described herein appears to be different from the S. aureus human Lf binding protein, an approximately 450 kDa protein which, under reducing SDS-PAGE gel conditions, resolves into two components of 67 and 62 kDa.
  • S . uberis bLf binding protein Analysis of the primary and secondary structure of the S . uberis bLf binding protein suggests that it is an M-like protein.
  • a gene homologous to the group A streptococcal mga a positive regulator of M and M-like proteins, has been found in the upstream adjacent region of lbp .
  • Southern blot analysis reveals that mga is present in all S. uberis strains tested that contained the lbp .
  • the sequence of S . uberis mga and the protein product therefrom is presented in Figures 5A- 5D (SEQ ID NOS:3-12) .
  • the deduced gene product, Mga is comprised of 499 amino acid residues with a calculated molecular weight of 58,454 Da.
  • the N-terminus of Mga lacks the features of a signal peptide, suggesting that it is a cytoplasmic protein.
  • Preceding the start codon of mga is a putative ribosome binding site AGGAGA. Sequences resembling the -35 and -10 promoter motifs have also been identified, as shown in Figures 5A-5D (SEQ ID NOS: 3-12) .
  • S . uberis LF-binding protein immunogenic fragments thereof or chimeric proteins including the same
  • S . uberis LF-binding protein can be provided in subunit vaccine compositions to treat or prevent bacterial infections caused by S . uberis, including mastitis in mammals, such as in bovine, equine, ovine and goat species.
  • the proteins and fragments thereof, antibodies thereto, and genes coding therefor can be used as diagnostic reagents to detect the presence of infection in a mammalian subject.
  • the genes encoding the proteins can be cloned and used to design probes to detect and isolate homologous genes in other bacterial strains.
  • fragments comprising at least about 15-20 nucleotides, more preferably at least about 20-50 nucleotides, and most preferably about 60-100 or more nucleotides, will find use in these embodiments.
  • the S . uberis LF-binding proteins also find use in purifying bovine LFs from streptococcal species and from recombinant host cells expressing the same.
  • S . uberis Lf binding proteins can be used in vaccine compositions either alone or in combination with other bacterial, fungal, viral or protozoal antigens. These antigens can be provided separately or even as fusion proteins comprising one or more epitopes of an LF-binding protein fused to one or more of these antigens.
  • other immunogenic proteins from S . uberis such as the CAMP factor, hyaluronic acid capsule, hyaluronidase, R-like protein and plasminogen activator, can be administered with the LF-binding protein.
  • immunogenic proteins from other organisms involved in mastitis such as from the genera Staphylococcus , Corynebacterium, Pseudomonas, Nocardia, Clostridium, Mycobacterium, Mycoplasma, Pasteurella, Prototheca, other streptococci, coliform bacteria, as well as yeast, can be administered along with the bLF-binding proteins described herein to provide a broad spectrum of protection.
  • zooepidemicus Corynebacterium pyogenes, Pseudomonas aeruginosa, Nocardia asteroides, Clostridium perfringens, Escherichia coli , Enterobacter aerogenes and Klebsiella spp. can be provided along with the bLF-binding proteins of the present invention.
  • LF-binding proteins can be isolated directly from bacteria which express the same. This is generally accomplished by first preparing a crude extract which lacks cellular components and several extraneous proteins. The desired proteins can then be further purified i.e. by column chromatography, HPLC, immunoadsorbent techniques or other conventional methods well known in the art .
  • the proteins can be recombinantly produced as described herein.
  • these recombinant products can take the form of partial protein sequences, full-length sequences, precursor forms that include signal sequences, mature forms without signals, or even fusion proteins (e.g., with an appropriate leader for the recombinant host, or with another subunit antigen sequence for Streptococcus or another pathogen) .
  • the lbp genes of the present invention can be isolated based on the ability of the protein products to bind LF, using LF-binding assays as described below.
  • gene libraries can be constructed and the resulting clones used to transform an appropriate host cell. Colonies can be pooled and screened for clones having LF-binding activity. Colonies can also be screened using polyclonal serum or monoclonal antibodies to the LF-binding protein.
  • oligonucleotide probes which contain the codons for a portion of the determined amino acid sequences can be prepared and used to screen genomic or cDNA libraries for genes encoding the subject proteins.
  • the basic strategies for preparing oligonucleotide probes and DNA libraries, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. See, e.g., DNA Cloning: Vol. I, supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis, supra; Sambrook et al . , supra .
  • a clone from the screened library has been identified by positive hybridization, it can be confirmed by restriction enzyme analysis and DNA sequencing that the particular library insert contains an LF-binding protein gene or a homolog thereof.
  • the genes can then be further isolated using standard techniques and, if desired, PCR approaches or restriction enzymes employed to delete portions of the full-length sequence.
  • genes can be isolated directly from bacteria using known techniques, such as phenol extraction and the sequence further manipulated to produce any desired alterations. See, e.g., Sambrook et al . , supra, for a description of techniques used to obtain and isolate DNA.
  • DNA sequences encoding the proteins of interest can be prepared synthetically rather than cloned.
  • the DNA sequences can be designed with the appropriate codons for the particular amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al . (1984) Science 223 :1299; Jay et al. (1984) J. Biol . Chem . 259:6311.
  • coding sequences for the desired proteins can be cloned into any suitable vector or replicon.
  • Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice.
  • Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage ⁇ (E. coli) , pBR322 (E. coli ) , pACYC177 (E. coli) , pKT230 (gram-negative bacteria) , pGV1106 (gram-negative bacteria) , pLAFRl (gram-negative bacteria) , pME290
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements) , so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence. If a signal sequence is included, it can either be the native, homologous sequence, or a heterologous sequence.
  • the signal sequence for S. uberis LF-binding protein shown in Figure 2A
  • Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397. Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • the control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are described in, e.g., Sambrook et al . , supra; DNA Cloning, supra ; Nucleic Acid Hybridization, supra . The expression vector is then used to transform an appropriate host cell.
  • a number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC) , such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS) , human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”) cells, as well as others.
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • baby hamster kidney (BHK) cells baby hamster kidney (BHK) cells
  • COS monkey kidney cells
  • MDBK Madin-Darby bovine kidney
  • bacterial hosts such as E. coli , Bacillus subtilis, and Streptococcus spp . , will find use with the present expression constructs.
  • Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisi
  • Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti , Autographa californica, Bombyx ori , Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni .
  • the proteins of the present invention are produced by culturing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The protein is then isolated from the host cells and purified. If the expression system secretes the protein into the growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • the proteins of the present invention may also be produced by chemical synthesis such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest . Such methods are known to those skilled in the art. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide
  • LF-binding proteins of the present invention can be used to produce antibodies, both polyclonal and monoclonal.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • Serum from the immunized animal is collected and treated according to known procedures. See, e.g., Jurgens et al . (1985) J " . Chrom . 348 :363-370.
  • serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures .
  • Monoclonal antibodies to the LF-binding proteins and to the fragments thereof can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by using hybridoma technology is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al . , Hybridoma Techniques (1980);
  • Panels of monoclonal antibodies produced against the LF-binding protein, or fragment thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc.
  • Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens which they are directed against . Both polyclonal and monoclonal antibodies can also be used for passive immunization or can be combined with subunit vaccine preparations to enhance the immune response. Polyclonal and monoclonal antibodies are also useful for diagnostic purposes .
  • LF-binding proteins of the present invention can be formulated into vaccine compositions, either alone or in combination with other antigens, for use in immunizing subjects as described below. Methods of preparing such formulations are described in, e.g., Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 18 Edition, 1990.
  • the vaccines of the present invention are prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in or suspension in liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.
  • the active immunogenic ingredient is generally mixed with a compatible pharmaceutical vehicle, such as, for example, water, saline, dextrose, glycerol , ethanol, or the like, and combinations thereof.
  • a compatible pharmaceutical vehicle such as, for example, water, saline, dextrose, glycerol , ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents.
  • Adjuvants which enhance the effectiveness of the vaccine may also be added to the formulation.
  • Adjuvants may include for example, muramyl dipeptides, avridine, aluminum hydroxide, dimethyldioctadecyl ammonium bromide (DDA) , oils, oil-in-water emulsions, saponins, cytokines, and other substances known in the art .
  • DDA dimethyldioctadecyl ammonium bromide
  • the Lf binding protein may be linked to a carrier in order to increase the immunogenicity thereof.
  • Suitable carriers include large, slowly metabolized macromolecules such as proteins, including serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles .
  • proteins including serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art
  • polysaccharides such as sepharose, agarose, cellulose, cellulose beads and the like
  • polymeric amino acids such as polyglutamic acid, polylysine, and the like
  • amino acid copolymers amino acid copo
  • the LF-binding proteins may be used in their native form or their functional group content may be modified by, for example, succinylation of lysine residues or reaction with Cys-thiolactone .
  • a sulfhydryl group may also be incorporated into the carrier (or antigen) by, for example, reaction of amino functions with 2-iminothiolane or the N-hydroxysuccinimide ester of 3- (4-dithiopyridyl propionate.
  • Suitable carriers may also be modified to incorporate spacer arms (such as hexamethylene diamine or other bifunctional molecules of similar size) for attachment of peptides.
  • Suitable carriers for the LF-binding proteins of the present invention include VP6 polypeptides of rotaviruses, or functional fragments thereof, as disclosed in U.S. Patent No. 5,071,651. Also useful is a fusion product of a viral protein and the subject immunogens made by methods disclosed in
  • Still other suitable carriers include cells, such as lymphocytes, since presentation in this form mimics the natural mode of presentation in the subject, which gives rise to the immunized state.
  • the proteins of the present invention may be coupled to erythrocytes, preferably the subject's own erythrocytes. Methods of coupling peptides to proteins or cells are known to those of skill in the art.
  • the LF-binding proteins (or complexes thereof) may be formulated into vaccine compositions in either neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic
  • Vaccine formulations will contain a "therapeutically effective amount" of the active ingredient, that is, an amount capable of eliciting an immune response in a subject to which the composition is administered.
  • a "therapeutically effective amount” would preferably be an amount that enhances resistance of the mammary gland to new infection and/or reduces the clinical severity of the disease. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered somatic cell count in milk from the infected quarter.
  • the ability of the composition to retain or bring the somatic cell count (SCC) in milk below about 500,000 cells per ml, the threshold value set by the International Dairy Federation, above which, animals are considered to have clinical mastitis, will be indicative of a therapeutic effect.
  • the exact amount is readily determined by one skilled in the art using standard tests.
  • the LF- binding protein concentration will typically range from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate.
  • 20 to 500 ⁇ g of active ingredient per ml of injected solution should be adequate to raise an immunological response when a dose of 1 to 3 ml per animal is administered.
  • the vaccine is generally administered parenterally, usually by intramuscular injection. Other modes of administration, however, such as subcutaneous, intraperitoneal and intravenous injection, are also acceptable.
  • the quantity to be administered depends on the animal to be treated, the capacity of the animal's immune system to synthesize antibodies, and the degree of protection desired.
  • Effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.
  • the subject is immunized by administration of the vaccine in at least one dose, and preferably two doses.
  • the animal may be administered as many doses as is required to maintain a state of immunity to infection.
  • Additional vaccine formulations which are suitable for other modes of administration include suppositories and, in some cases, aerosol, intranasal, oral formulations, and sustained release formulations.
  • the vehicle composition will include traditional binders and carriers, such as, polyalkaline glycols, or triglycerides .
  • binders and carriers such as, polyalkaline glycols, or triglycerides .
  • Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w) , preferably about 1% to about 2%.
  • Oral vehicles include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate, and the like.
  • oral vaccine compositions may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.
  • Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function.
  • Diluents such as water, aqueous saline or other known substances can be employed with the subject invention.
  • the nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride.
  • a surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
  • Controlled or sustained release formulations are made by incorporating the protein into carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel ® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures.
  • carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel ® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures.
  • the LF-binding proteins can also be delivered using implanted mini-pumps, well known in the art.
  • the LF-binding proteins of the instant invention can also be administered via a carrier virus which expresses the same.
  • Carrier viruses which will find use with the instant invention include but are not limited to the vaccinia and other pox viruses, adenovirus, and herpes virus.
  • vaccinia virus recombinants expressing the novel proteins can be constructed as follows. The DNA encoding the particular protein is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK) . This vector is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the instant protein into the viral genome.
  • the resulting TK " recombinant can be selected by culturing the cells in the presence of 5- bromodeoxyuridine and picking viral plaques resistant thereto .
  • An alternative route of administration involves gene therapy or nucleic acid immunization.
  • nucleotide sequences (and accompanying regulatory elements) encoding the subject LF-binding proteins can be administered directly to a subject for in vivo translation thereof.
  • gene transfer can be accomplished by transfecting the subject's cells or tissues ex vivo and reintroducing the transformed material into the host.
  • DNA can be directly introduced into the host organism, i.e., by injection (see International Publication No.
  • Liposome-mediated gene transfer can also be accomplished using known methods. See, e.g., Hazinski et al. (1991) Am. J. Respir. Cell Mol . Biol . 4:206- 209; Brigham et al . (1989) Am. J. Med . Sci . 298:278- 281; Canonico et al . (1991) Clin . Res . 39:219A; and Nabel et al . (1990) Science 249:1285-1288.
  • Targeting agents such as antibodies directed against surface antigens expressed on specific cell types, can be covalently conjugated to the liposomal surface so that the nucleic acid can be delivered to specific tissues and cells susceptible to infection.
  • the LF-binding proteins of the present invention may also be used as diagnostics to detect the presence of reactive antibodies of S . uberis in a biological sample in order to determine the presence of S . uberis infection.
  • the presence of antibodies reactive with LF-binding proteins can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis ; immunoprecipitation, etc.
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve separation of unbound antibody in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates) ; polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • a solid support is first reacted with a solid phase component (e.g., one or more LF- binding proteins) under suitable binding conditions such that the component is sufficiently immobilized to the support.
  • a solid phase component e.g., one or more LF- binding proteins
  • immobilization of the antigen to the support can be enhanced by first coupling the antigen to a protein with better binding properties.
  • Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA) , keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art.
  • molecules that can be used to bind the antigens to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like.
  • Such molecules and methods of coupling these molecules to the antigens are well known to those of ordinary skill in the art. See, e.g., Brinkley, M.A. Bioconjugate Chem . (1992) 3:2-13; Hashida et al . , J. Appl . Biochem. (1984) 6.: 56-63; and Anjaneyulu and Staros, International J. of Peptide and Protein Res . (1987) 30:117-124.
  • any non- immobilized solid-phase components are removed from the support by washing, and the support -bound component is then contacted with a biological sample suspected of containing ligand moieties (e.g., antibodies toward the immobilized antigens) under suitable binding conditions.
  • a biological sample suspected of containing ligand moieties e.g., antibodies toward the immobilized antigens
  • a secondary binder moiety is added under suitable binding conditions, wherein the secondary binder is capable of associating selectively with the bound ligand.
  • the presence of the secondary binder can then be detected using techniques well known in the art. More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with an LF-binding protein.
  • a biological sample containing or suspected of containing anti-LF- binding protein immunoglobulin molecules is then added to the coated wells. After a period of incubation sufficient to allow antibody binding to the immobilized antigen, the plate (s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample antibodies, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
  • the presence of bound anti-LF-binding antigen ligands from a biological sample can be readily detected using a secondary binder comprising an antibody directed against the antibody ligands.
  • a secondary binder comprising an antibody directed against the antibody ligands.
  • a number of anti-bovine immunoglobulin (Ig) molecules are known in the art which can be readily conjugated to a detectable enzyme label, such as horseradish peroxidase, alkaline phosphatase or urease, using methods known to those of skill in the art.
  • An appropriate enzyme substrate is then used to generate a detectable signal .
  • competitive-type ELISA techniques can be practiced using methods known to those skilled in the art .
  • Assays can also be conducted in solution, such that the LF-binding proteins and antibodies specific for those proteins form complexes under precipitating conditions.
  • LF-binding proteins can be attached to a solid phase particle (e.g., an agarose bead or the like) using coupling techniques known in the art, such as by direct chemical or indirect coupling.
  • the antigen-coated particle is then contacted under suitable binding conditions with a biological s ' ample suspected of containing antibodies for the LF-binding proteins.
  • Cross-linking between bound antibodies causes the formation of particle-antigen-antibody complex aggregates which can be precipitated and separated from the sample using washing and/or centrifugation.
  • the reaction mixture can be analyzed to determine the presence or absence of antibody- antigen complexes using any of a number of standard methods, such as those immunodiagnostic methods described above.
  • an immunoaffinity matrix can be provided, wherein a polyclonal population of antibodies from a biological sample suspected of containing anti-LF-binding molecules is immobilized to a substrate.
  • an initial affinity purification of the sample can be carried out using immobilized antigens.
  • the resultant sample preparation will thus only contain anti-S. uberis moieties, avoiding potential nonspecific binding properties in the affinity support.
  • a number of methods of immobilizing immunoglobulins (either intact or in specific fragments) at high yield and good retention of antigen binding activity are known in the art . Not being limited by any particular method, immobilized protein A or protein G can be used to immobilize immunoglobulins .
  • labeled LF-binding proteins are contacted with the bound antibodies under suitable binding conditions. After any non-specifically bound antigen has been washed from the immunoaffinity support, the presence of bound antigen can be determined by assaying for label using methods known in the art .
  • antibodies raised to the LF- binding proteins can be used in the above-described assays in order to detect the presence of antibodies to the proteins in a given sample. These assays are performed essentially as described above and are well known to those of skill in the art.
  • kits can be provided in kits, with suitable instructions and other necessary reagents, in order to conduct immunoassays as described above.
  • the kit can also contain, depending on the particular immunoassay used, suitable labels and other packaged reagents and materials (i.e. wash buffers and the like) . Standard immunoassays, such as those described above, can be conducted using these kits.
  • S . uberis strain (su-1) from the American Type Culture Collection (ATCC 9927) was used for study. Bacteria were grown on base #2 sheep blood agar plates (PML Microbiologicals) at 37°C for 18 h. Iron- restricted conditions were achieved in Todd-Hewitt broth supplemented with 0.3% yeast extract (THB-YE) by the addition of 800 ⁇ M EDDA, 800 ⁇ M dipyridyl or 100 ⁇ M desferrioxamine mesylate. All of the iron chelators were obtained from Sigma.
  • iron-binding proteins including bLf (from bovine milk) , bovine transferrin (bTf) , human lactoferrin (hLf) and human transferrin (hTf) , were purchased from Sigma in the most iron-free form available. Iron-saturated proteins and the apoproteins were prepared by methods described previously (Mazurier and Spik (1980) Biochim . Biophys . Acta 629:399-408) .
  • Bovine Lf was iodinated by the lactoperoxidase method of Thorell and Johansson (1971) Biochim . Biophys . Acta 251: 363-368. Approximately 70 ⁇ g of bLf (33% iron-saturated) was used for iodination; 12S I-labelled protein was separated from free Na 125 I by chromatography on a Sephadex G-25 column. The labelled protein was aliquoted and stored at -70°C until use. Lactoperoxidase was purchased from Boehringer-Mannheim; and Na 125 I from Amersham.
  • the binding assays were performed as described (Naidu et al . (1990) J. din . Microbiol .
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • IO 9 bacteria in 0.1 ml of PBS-1% BSA were mixed and incubated with 0.1 ml of 125 I-bLf solution (6.9 nM in PBS-1 % BSA) for periods of 5, 10, 15, 20, 25, 30, 60, 90, 120, 150 min at room temperature. Bacteria were pelleted and washed three times with 1 ml of ice- cold PBS containing 0.1% Tween 20. Radioactivity bound to the bacterial pellet was measured in a ⁇ - counter. In competitive binding experiments, IO 9 bacteria were mixed with 2 x 10 s cpm 125 I-bLf in the presence of serially-diluted unlabelled bLf and incubated at room temperature for 2 h.
  • Proteinase K (Boehringer mannheim) treatment was carried out in 40 mM potassium phosphate buffer (pH 7.5), and the digestion was inhibited by the addition of phenylmethylsulfonyl fluoride (500 ⁇ g/ml) .
  • the bacterial suspension (10 10 cell/ml) was incubated in a water bath for 1 h at each of the following temperatures: 50°C, 80°C and 100°C. Both enzyme and heat-treated cells were washed once in PBS and resuspended in PBS-1% BSA prior to the binding experiments .
  • SDS-polyacrylamide gel electrophoresis of proteins was performed using the method described by Laemmli (Laemmli, U.K. (1970) Nature 227 : 680-685) . Samples were solubilized in sample buffer at 37°C for 30 min in the absence of 2- mercaptoethanol (non-reducing conditions) or at 100°C for 5 min in the presence of 1% 2-mercaptoethanol (reducing conditions) . Proteins were electrophoretically transferred to nitrocellulose membranes as recommended by the supplier (Bio-Rad) and blocked with TBS-1% BSA.
  • 125 I-bLf was added to the membrane to a final concentration of 80 ng/ml in TBS-1% BSA and incubated at room temperature for 2 h. After three washes with TBS containing 0.05% Tween 20, the membrane was exposed to X-ray film for 24 h at room temperature. To compete the 125 I-bLf binding, the transferred membrane was incubated with 35 ⁇ g/ml of unlabelled bLf for 2 h before incubation with 125 I-bLf. Results Time-dependent binding of bLF to S . uberis .
  • Inhibition values were calculated as relative percentage of bLf binding to bacteria suspended in PBS in the absence of any inhibitor.
  • Bovine Lf binding to S. uberis was time and concentration dependent (demonstrated by the ability of the unlabelled ligand to compete for binding with 125 i- bLf) , indicating the existence of a limited number of binding receptors on the cell surface. Scatchard plot analysis estimated that there were 7800 bLf-binding sites/cell.
  • S. aureus Lf receptors have been shown to bind lactoferrins from both human and bovine sources ( ⁇ aidu et al. (1990) J “ . Clin . Microbiol . 28: 2312-2319 ; ⁇ aidu et al. (1991) J “ . Med . Micrbiol . 34:323-328; ⁇ aidu et al. (1991) J “ . Dairy Sci . 74:1218-1226; ⁇ aidu et al . (1992) J “ . Med. Microbiol . 36:177-183).
  • S uberis is exclusively a bovine pathogen
  • aureus could not be blocked by human or bovine transferrin. Although the expression of most of the transferrin and lactoferrin receptors of Gram-negative bacteria such as N. meningi tidis and H. influenza is iron regulated, S . uberis receptor activity was not regulated by growth medium iron availability. The results are consistent with the findings of Modun et al . (1994) Infect . Immun . 62:3850-3858 and Rainard, P. (1992) FEMS Microbiol . Lett . 98:235-240 who showed that the binding of S. aureus to Tf and S. agalactiae to Lf was not regulated by iron.
  • the lactoferrin-binding protein of S. uberis differs from the transferrin receptors of Haemophilus and Neisseria spp., which consist of two distinct transferrin-binding proteins, termed Tbpl and Tbp2 , which range in molecular weight from 68 to 105 kDa depending on the strain.
  • Tbpl and Tbp2 two distinct transferrin-binding proteins, termed Tbpl and Tbp2 , which range in molecular weight from 68 to 105 kDa depending on the strain.
  • the S . uberis Lbp is also different from the lactoferrin receptors of the mentioned two species.
  • the bovine lactoferrin receptor of S. aureus consists of two distinct bLf-binding proteins with estimated molecular weights of 92 and 67 kDa (Naidu et al . (1991) J. Dairy Sci . 74: 1218-1226) and therefore appears to be different than the receptor described herein.
  • streptococcal Lbp described here appears to be different from the S. aureus human lactoferrin-binding protein which is as an approximately 450 kDa protein which under reducing SDS-PAGE gel conditions resolves into two components of 67 and 62 kDa.
  • Bacterial strains Bacterial strains, plasmids and media.
  • S . uberis strains used are listed in Table 2 below. Bacteria were grown on base #2 sheep blood agar plates (PML Microbiologicals) at 37°C for 18 h, or in Todd-Hewitt broth supplemented with 0.3% yeast extract (THB-YE) at 37°C overnight. E. coli cells were grown in Luria broth or on Luria broth-agar plates. Ampicillin was used at 50 ⁇ g/ml for the growth of E. coli strains containing recombinant plasmids. The cloning vector used was pTZ18R (Mead et al. (1986) Protein Eng. 1:67-74).
  • the 100 fold-concentrated culture supernatant of the recombinant E. coli was obtained by precipitation with 10% trichloroacetic acid (TCA) .
  • PBS-1% BSA bovine serum albumin
  • Serum against the recombinant Lbp of S . uberis was raised in rabbits by subcutaneous injection of 0.5 ml of TCA-precipitated culture supernatant of recombinant E. coli in complete Freund's adjuvant. Two subcutaneous boosts with the same amount of sample in incomplete Freund's adjuvant were given to each animal .
  • Nonspecific binding was blocked by incubation in TBS (10 mM Tris-HCl, pH 7.5, 140 mM NaCl) -1% bovine serum albumin (BSA) . Blots were incubated with antibody diluted 1:200 in TBS-1% BSA at room temperature for 1 h. After three washes in TBS containing 0.05% Tween 20, seroreactive proteins were detected with goat anti-mouse (or rabbit) IgG coupled to alkaline phosphatase (Kirkegaard & Perry Laboratories, Inc.) at 1:5,000 in TBS-1% BSA. Alkaline phosphatase activity was detected using the Nitro Blue Tetrazolium-5-bromo- 4-chloro-3 -indolylphosphate toluidinium system as described by the supplier (Promega) .
  • Plasmid DNA was purified as described by
  • DNA fragments were isolated from agarose gels using a Gene Clean kit (Bio/can Scientific) .
  • chromosomal DNA was prepared as previously described (Caparon and Scott (1987) Proc . Natl . Acad . Sci . USA 84.: 8677-8681) and partially digested with Sau3AI . Fragments of 2,000 to 5,000 bp were recovered following sucrose density gradient centrifugation (Sambrook et al . , supra) . The ends of these fragments were partially filled in with dGTP and dATP and ligated into pTZ18R which was cut with Sail and partially filled in with dTTP and dCTP . Transformation of E.
  • coli DH5 ⁇ competent cells was carried out as recommended by the supplier (GIBCO BRL, Gaithersburg, Md) .
  • transformants were replica-plated onto nitrocellulose discs (Schleicher & Schuell, Keene, NH) and lysed in chloroform vapor. Nonspecific binding was blocked by incubation with TBS-1% BSA. Membranes were further incubated with 125 I-bLf as described in Example 1.
  • DNA sequences were determined by the dideoxy-chain termination method of Sanger et al . (1977) Proc . Natl . Acad . Sci . USA 74:5463-5467 on double-stranded plasmid templates by using a T7 Sequencing kit (Pharmacia Canada Ltd.). RNA analyses .
  • RNA from E. coli strains was isolated as described previously (Lloubes et al . (1986) Nuclei c Acid Res . 14:2621-2636) with an additional RNase-free DNase I digestion.
  • a gene library was constructed in pTZ18R with chromosomal DNA from S . uberis (su-1) . About 5000 transformants were initially screened for expression of bLf binding protein (Lbp) by colony blotting with 125 I-bLf . One colony with the strongest signal and six with weaker signals were selected and used to make whole cell lysates, which were further tested for their ability to bind 125 I-bLF under non- reducing conditions. The clone with the strongest signal, E. coli pLBP5, generated three major bands; two of them had molecular weights of 165 and 76 kDa which are quite close in size to those of S . uberi s .
  • E. coli pLBP5 The presence of free and functionally active recombinant Lbp in the cell lysate and supernatant of E. coli pLBP5 was also detected by performing competitive inhibition assays.
  • the E. coli pLBP5 cell lysate and supernatant mixture effectively inhibited 125 I-bLf binding to S . uberis cells in a dose-dependent manner, while samples from E. coli pTZl ⁇ R did not ( Figure 11) .
  • the dimer could have been disassociated by urea to monomers which could be further denatured and unfolded, resulting in the apparent molecular weight increase from 76 kDa to 105 kDa.
  • a significant increase in the apparent molecular weight of Tbp2 following urea-treatment has been observed in Neisseria meningi tidis by other researchers (Vonder haar et al . (1994) J. Bacteriol . 176:6207-6213).
  • the above data also indicates that disulfide bonds are not essential for the formation of the oligomer.
  • the lbp sequence contained two potential translation start codons (ATG) at positions 232 and 262 of the DNA sequence. Both are associated with putative Shine-Dalgarno sequences ( Figures 2A-2C) . These start points would give proteins with predicted sizes of 62.857 and 61.454, respectively. The predicted sizes are comparable to 76 KDa molecular weight protein. The reason for the discrepancy between the observed and calculated molecular weights of this protein is not clear. Posttranslational modification, such as lipid modification might have occurred and increased the apparent molecular mass in the SDS-PAGE determination. A similar difference observed in Gram-negative bacteria has been demonstrated to be caused by protein lipid modification (Theisen et al . (1992) Infect . Immun . £0: 826-831; Theisen et al . (1993) Infect . Immun . 61:1793-1798) . The DNA sequence shows two putative -10 and
  • the plasmid pLBP5 contained an incomplete gene, mga' , in addition to lbp .
  • the pLBP5 molecule was inverted to pLBP5i ( Figure 4) for convenience of description.
  • a 991 bp StyI fragment from mga ' was radioactively ( 32 P) labelled as a probe (VP1 in Figure 4) and used to screen an S. uberis gene library.
  • a positive E. coli DH5 ⁇ clone was obtained and the restriction enzyme map of the plasmid pMGA14 is shown in Figure 4.
  • each of the Bam ⁇ l , Hindi , Hindi11 and Kpnl restriction fragments of pMGA14 ( Figure 4) was individually cloned into the corresponding site of pTZ18R and sequenced from both orientations using universal and reverse primers.
  • Plasmid pMGA14 contained the complete ORF of Mga. However, the associated promoter region was not present. Since the 1.5 kb Sphl -Nhel fragment of pLBP5i contained the majority of the 5' region of the mga ' and the complete promoter region, it was inserted into the Sphl and N el sites of pMGA14 to generate pMGA14F which then contained the complete mga gene including the promoter ( Figure 4) . The sequence of a 3558 bp DNA fragment of pMGA14F is presented in Figures 5A-5D.
  • Mga the deduced gene product
  • Mga is comprised of 499 amino acid residues with a calculated molecular weight of 58,454 Da.
  • the N- terminus of Mga lacks the features of signal peptides as described (Simonen and Palva (1993) Microbiol . Rev. 57:109-137), suggesting that it is a cytoplasmic protein.
  • Preceding the start codon of mga is a putative ribosome binding site AGGAGA. Sequences resembling the -35 and -10 promoter motifs have also been identified.
  • RNA was prepared from S. uberis (su-1) , E. coli DH5 ⁇ (pLBP5) , E. coli DH5 ⁇ (pLBP5L) , E. coli DH5 ⁇ (pMGA14F) and E. coli DH5 ⁇ (pTZ18R) , and used for two Northern blots.
  • One blot was probed with the 1.5 kb Hindlll-Hpal internal fragment of lbp (LP1 in Figure 6) .
  • a 2.0 kb band was seen in lanes containing RNA from S. uberis, E. coli DH5 ⁇ (pLBP5) and E.
  • DNA was prepared and digested with the restriction endonuclease Hindlll and separated on agarose gels.
  • uberis strains while the region encoding the N-terminal portion varies greatly.
  • the diversity in sizes of chromosomal restriction fragments from the different strains that hybridized with the lbp probes indicates some degree of restriction site heterogeneity (a different location for Hindlll site) among these strains.
  • the Lbp gene of S. uberis was cloned in E . coli after screening a gene library by colony blotting with 125 I-bLf.
  • E . coli transformants produced functionally active bLf-binding proteins with molecular weights of 76 kDa and 165 kDa, similar to the native proteins produced by S. uberi s .
  • bLf with S. uberis Example 1
  • Lbp The complex nature of the S. uberis Lbp was further confirmed from the nucleotide sequence analysis. As expected, the two Lbps were encoded by a single ORF of 1,683 bp . It is quite possible that two copies of the translated product interact with each other to form a homodimer. Interactions between subunits could be extensive and extend throughout the length of the molecule due to the existence of the high ⁇ -helical content. Like M proteins (Fischetti, V.A. (1989) Clin . Microbiol . Rev. 2:285-314), Lbp shares significant sequence homology with a number of ⁇ -helical coiled structure-containing mammalian fibrillar proteins such as human myosin heavy chain and kinesin heavy chain.
  • the deduced amino acid sequence of Lbp indicated the existence of a signal peptide of 50 amino acids (if translation started at the first ATG start codon) at the N-terminus, as expected for a protein that appears on the outside of a bacterial cell.
  • the structure of this signal peptide is comparable to the consensus structures for signal peptides in proteins from Gram positive bacteria described previously (Simonen and Palva (1993) Microbiol . Rev. 57:109-137; Goward et al . (1993)
  • VirR49 of an OF + GAS showed less homology (76%) to Mry or VirR of OF " GAS (Podbielski et al . (1995) Infect . Immun . 63.: 9-20) .
  • the cytoplasmic location of Mga was suggested by the absence of a signal peptide at the N- terminus of the deduced protein. A potential -10 and -35 promoter was found.
  • mry is autoregulated and environmentally regulated in response to the level of C0 2 (Okada et al . (1993) Mol . Mi crobiol . 7:893-903) .
  • Expression of mry was stimulated by increased concentrations of C0 2 .
  • S. uberi s cells were cultured under conditions without an additional supplementation of C0 2 .
  • the absence of a stimulating environment could have resulted in a very low level of mga expression. This could be the reason that Northern blot analysis did not detect any mga transcript from S. uberi s . It would be expected that in recombinant E . coli , mga would not likely be regulated by environmental signals due to the absence of other regulatory components such as the sensing protein.
  • the N-terminal region is distal to the streptococcal cell surface, and thus would be expected to be the region of the molecule most exposed to immunological selective pressure. Therefore it is not surprising that the N-terminal region varies in sequence among different serological types of M protein. In contrast, the sequence of the C-terminal region of the molecule should be evolutionarily conserved to assure attachment to the streptococcal surface. Since Lbp has a structure similar to that of M protein, it is not surprising to find that its C- terminal sequence is conserved among strains, whereas the N-terminal region shows variation.
  • Tbp2 transferrin binding proteins
  • pleuropneumoniae serotypes contain a variable N-terminal half and a conserved C-terminal half (Bunka et al . (1995) Cloning and sequencing of the transferrin-binding protein genes of
  • Bacterial strains Bacterial strains, vectors and media.
  • S. uberis strain su-1 (ATCC 9927) was obtained from a clinical case of bovine mastitis.
  • E. coli strain DH5 ⁇ was used in all transformation experiments.
  • E . coli cells were cultured in Luria medium, and the medium for the growth of transformants was supplemented with 50 ⁇ g/ml of ampicillin.
  • the plasmid pGH433 (Anderson et al . (1991) Infect . Immun . 59:4110-4116) was used to express the recombinant proteins under the control of an IPTG (isopropyl 3-D-thiogalacto-pyranoside) - inducible promoter.
  • Lbp inclusion bodies were dissolved in sample buffer in the presence of 4 M urea and run on an SDS-PAGE gel containing 4 M urea.
  • Western blotting was done using convalescent serum from an su-1- infected cow using a 1:75 dilution of cow convalescent serum and a 1:5000 dilution of goat anti-bovine IgG coupled to alkaline phosphatase. Protein purification.
  • a culture of E. coli transformants (1 L) was grown to an absorbance at 660 nm of 0.5 and induced with 2 mM IPTG. After 2 h of continuous, vigorous shaking at 37°C, the cells were harvested and the protein inclusion bodies were prepared as described by Gerlach et al . (1992) Infect . Immun . 60:892-898.
  • a mid-log phase broth culture (1 L) grown at 30°C was cultured at 42 °C for 2 h with vigorous shaking for protein induction. Cells were harvested by centrifugation, resuspended in 5 ml of 25% sucrose-50 mM Tris-HCl buffer (pH 8.0), and frozen at -70°C.
  • Lysis was achieved by the addition of 1 mg of lysozyme in 250 mM Tris-HCl buffer (pH 8.0), 10 min of incubation on ice, addition of 25 ml of a detergent mix (5 parts of 20 mM Tris-HCl buffer, pH 7.4, 300 mM NaCl, 2% deoxycholic acid, 2% Nonidet P-40 and 4 parts of 100 mM Tris-HCl buffer, pH 8.0 , 50 mM EDTA, 2%
  • Inclusion bodies were harvested by centrifugation for 30 min at 15,000 x g and resuspended in H 2 0 to a concentration of 5 to 10 mg/ml .
  • Antigens for ELISA were purified from SDS-PAGE gels or 4 M urea SDS-PAGE gels by elution.
  • Protein purity was estimated by SDS-PAGE and subsequent Coomassie blue staining.
  • the protein concentration was determined using Bio-Rad DC protein assay as described by the supplier. Bovine serum albumin (Pierce Chemical Co., Rockford, IL) was used as a standard.
  • the amount of target protein vs. total protein was determined after scanning the Coomassie blue stained SDS-PAGE with a Bio-Rad 620 Densitometer .
  • the vaccines consisted of proteins emulsified in the adjuvant VSA3 which had been diluted with 0.1 M PBS, pH 7.2. Each 2 ml dose of vaccine contained VSA (0.67 ml), PBS (1.33 ml), and 50 ⁇ g of protein antigen dissolved in 5-10 ⁇ l of 4 M guanidine hydrochloride . Fifteen healthy lactating dairy cows from the Pennsylvania State University Mastitis Research Herd were vaccinated intramuscularly at drying off and again 28 days later. Groups of five cows were given Lbp or adjuvant only.
  • the bacterial challenge culture was prepared by rolling the stock bead cultures onto esculin blood agar plates containing 5% whole blood. After 24 hours incubation at 37°C, a single colony was used to inoculate 100 ml of Ultra High Temperature pasteurized (UHT) milk and incubated for 12 hours at 37°C. The 24 hour culture was mixed well and a 100 ⁇ l aliquot was removed to inoculate a second 100 ml of UHT milk. After a second 9 hour incubation at 37°C, the culture was serially diluted in 10 -fold increments using sterile saline.
  • UHT Ultra High Temperature pasteurized
  • CFU colony forming units
  • Total Ig titers for the Lbp antigen were determined by an indirect ELISA.
  • Nunc Immunlon-2 plates were coated with antigen in carbonate buffer. Prior to use, the plates were blocked with TBST (100 mM Tris-Cl, pH 8.0; 150 mM NaCl ; 0.05% Tween-20) and 3% BSA for 1 hour. After blocking, the plates were washed with distilled water. Serum and milk samples were serially diluted in 3 -fold increments using TBST containing 1% BSA. Rabbit antisera for the Lbp antigen was also diluted and served as a positive control. Negative control samples contained TBST with 1% BSA.
  • the diluted samples and controls were transferred to the coated plates and were incubated for 1 hour at room temperature.
  • the plates were washed thoroughly with distilled water and all wells were incubated with a horse radish peroxidase conjugate of goat anti-IgG diluted 1:2000 in TBST containing 1% BSA. Following a 1 hour incubation at room temperature, the plates were washed with distilled water.
  • the amount of antibody present in samples was visualized using ABTS substrate.
  • the titers of each sample were based on the absorbance reading at 405 nm with a reference wavelength of 495 nm.
  • a positive reading for samples was one in which the absorbance was two times the absorbance of the blank (negative control) . Titers were determined by taking the reciprocal of the last dilution giving a positive reading. Consistency among assay plates was monitored by the absorbance reading of positive controls.
  • Plasmid pGH-LBP was constructed by inserting the 1.8 kb Sphl -Rsal fragment from pLBP5 into the BamKI site of pGH433 (Fig. 10) which provides a 12 amino acid leader peptide and an IPTG-inducible tac promoter. Before ligation, the insert was treated with mung bean nuclease to remove the 3' overhang from the end generated by Sptil , and the BamHI-cut vector was filled in by the Klenow fragment to produce blunt ends.
  • the pGH-LBP contained the carboxy-terminal 96% of the Lbp gene ( lbp) , which was preceded by a 12 amino acid leader peptide and the tac promoter provided by vector pGH433. Analysis of the nucleotide sequence at the fusion site revealed identity to the sequence of the lbp presented in Figure 1.
  • Lbp of S. uberis was expressed in E. coli DH5 ⁇ using pGH-LBP. In this system, expression of the recombinant protein was repressed under normal growth conditions. Upon IPTG-induction, the recombinant protein was produced in an aggregated form. The 82 and 90 kD Lbp made up 36% of the total protein. Isolated protein aggregates were dissolved in 4 M guanidine hydrochloride and used for vaccine formulation. Lbp was purified from a 4 M urea SDS- PAGE gel and used as the antigen for ELISA. Both aggregated and purified Lbp were demonstrated to be antigenically active by Western blotting using convalescent serum from S. uberis infected cow. Somatic cell counts in milk following experimental bacterial challenge.
  • the mean somatic cell count in milk from challenged quarters of nonvaccinated control animals increased up to 3,000,000 cell/ml 3 days following challenge.
  • the challenged quarters of animals vaccinated with Lbp showed a rapid increase in the mean SCC which was close to that in the control group.
  • S. uberis Lbp The surface exposure of S. uberis Lbp makes it a potential vaccine candidate by increasing the levels of opsonic antibody in milk and potentiating the speed of PMN recruitment into the mammary gland. Furthermore, the above example shows that Lbp is recognized by bovine serum from an animal which recovered from an S. uberis infection, indicating that the protein is expressed in vivo and cows respond to it immunologically . Based on these considerations, the protective capacity of recombinant Lbp was tested in lactating dairy cows against challenge by S. uberi s .
  • Lbp was produced as inclusion bodies in the expression system used, facilitating the purification of large quantities of pure recombinant proteins.
  • Lbp did not induce high levels of specific antibodies in the immunized cows, likely due to the formulation parameters.
  • bacterial challenge resulted in high somatic cell counts, indicative of mastitis. Therefore, vaccination with Lbp did not prevent the occurrence of mastitis.
  • the serological response to vaccination was very poor and therefore it is not possible to draw any conclusions regarding the protective capacity of Lbp from this study.
  • Lbp elicited an antibody response in immunized cows. Protection studies are on-going and preliminary results indicate that Lbp is effective in protecting subjects from mastitis.

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