WO1994009821A1 - Recombinant vaccine for porcine pleuropneumoniae - Google Patents

Recombinant vaccine for porcine pleuropneumoniae Download PDF

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WO1994009821A1
WO1994009821A1 PCT/US1993/010500 US9310500W WO9409821A1 WO 1994009821 A1 WO1994009821 A1 WO 1994009821A1 US 9310500 W US9310500 W US 9310500W WO 9409821 A1 WO9409821 A1 WO 9409821A1
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dna
leukotoxin
leu
gly
sequence
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PCT/US1993/010500
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French (fr)
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Yung-Fu Chang
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Cornell Research Foundation, Inc.
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    • 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
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • 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 the bacterium Actinobacillus pleuropneumonia (A. pleuropneumonia) , which causes porcine pleuropneumonia in pigs. More particularly, the invention relates to a gene(s) or gene fragment (s) encoding a novel leukotoxin secreted from A ⁇ . pleuropneumonia, serotype 2, recombinant DNA sequences and expression systems for directing expression of the gene(s), a method of using the gene(s) or gene fragment (s) as immunogens in vaccine formulations to protect pigs against porcine pleuropneumonia and as reagents in diagnostic assays.
  • A. pleuropneumonia Actinobacillus pleuropneumonia
  • the invention relates to a gene(s) or gene fragment (s) encoding a novel leukotoxin secreted from A ⁇ . pleuropneumonia, serotype 2, re
  • Actinobacillus pneumonia (porcine pleuropneumonia) of swine is a highly contagious respiratory disease caused by the gram-negative bacterium A. pleuropneumoniae (Pearson et al., Improved Tools for Biological Sequence Comparison, 1988, Proc. Natl. Acad. Sci., vol. 5, pp. 2444-2448).
  • pleuropneumoniae Pieris pleuropneumoniae
  • RTX Repeat of Toxin
  • McWhinney et al . Separable Domains Define Target Cell Specificities of an RTX Hemolysin from Actinobacillus pleuropneumoniae, 1992, J.
  • the genetic determinants for the secreted RTX toxins consist of four genes: " A”, the structural gene for toxin protein; "C”, which is required for "activation” of the toxin prior to secretion; and "B” and “D” , which are essential for the process of secretion.
  • the four RTX genes are typically found in a single transcriptional unit, "CABD” , and are expressed from a common promoter located upstream of the "C” gene (Felmleel et al. , Nucleotide Sequence of an Escherichia coli Chromosomal Hemolysin, 1895, J. Bacteriol., vol. 163, pp. 94-105; Highlander et al . , Secretion and Expression of Pasteurella haemolytica
  • the AppII gene has weak hemolytic activity and moderate cytotoxic activity.
  • the Appl and AppII polypeptides can be used as a protective immunogen for swine against porcine pneumonia.
  • the homology between the different peptides is only about 50%.
  • the different peptides are not cross-reactive against different serotypes of A. pleuropneumoniae. This is significant because a swine with pneumonia typically has been infected by more than one A ⁇ _ pleuropneumoniae serotype.
  • the present inventions discloses gene or gene fragments encoding a novel leukotoxin secreted from A. pleuropneumoniae serotypes 2, 3, 4, 6 and 8.
  • the novel genes of the invention or fragments thereof, alone, or in combination with the Appl and AppII genes can be effective immunogens for pigs against porcine pleuropneumonia.
  • the present invention is directed to a novel leukotoxin (cytotoxic protein) secreted from A ⁇ . pleuropneumoniae, serotype 2, as well as the molecularly cloned gene(s) or gene fragment (s) which encodes for this cytotoxic protein.
  • the leukotoxin (cytotoxic protein) of the present invention and related peptides or proteins can be used as immunogens in vaccine formulations for swine against porcine pleuropneumoniae.
  • the leukotoxin (cytotoxic protein) of the invention can be purified from A. pleuropneumoniae or produced using recombinant DNA techniques in any vector-host system, or synthesized by chemical methods.
  • the invention is also directed to novel DNA sequences (Seq. ID No. 1) and corresponding amino acid sequences (Seq. ID No. 2) and vectors including plasmid
  • DNA, and viral DNA such as human viruses, animal viruses, insect viruses or bacteriophages which can be used to direct the expression of the leukotoxin (cytotoxic protein) and related peptides and/or proteins in appropriate host cells from which the peptides and/or proteins can be purified.
  • the present invention discloses the cloning and sequencing of novel genes designated AppIIIC and AppIIIA (AppIIICA) which encode for the leukotoxin of the invention.
  • the novel leukotoxin is a 120 kDa protein secreted from A. pleuropneumoniae serotypes 2, 3, 4, 6 and 8.
  • Fig. 1(A) shows a Western blot analysis of culture supernatants from different serotypes of A ⁇ _ pleuropneumoniae using swine anti-leukotoxin serum as the first antibody. The lanes are labeled to the serotype number.
  • Fig. 1(B) shows a Western blot analysis of antigenic proteins expressed from recombinant bacteriophages. Lysates were from E. coli LE392 infected with ⁇ -Dash (lane 1) , ⁇ yfc26 (lane 2) , ⁇ yfc27 (lane 3) , and ⁇ yfc28 (lane 4) . The first antibody was swine anti-leukotoxin serum.
  • Fig. 2. shows a restriction map of clones of the A. pleuropneumoniae leukotoxin of the invention. EcoRI sites derived from the vector flank the inserts of each clone. The locations of the two open reading designated
  • Fig. 3. shows the nucleotide sequence (Seq. ID
  • Fig. 5 shows a Western blot analysis of aliquots of E. coli TB1 carrying pYFCH7 (lane 1) and the culture supernatant from A ⁇ _ pieuropneumoniae (lane 2) .
  • the first antibody was swine anti-leukotoxin serum.
  • the apparent molecular weights of prestained standards are shown in kilodaltons (kDa) .
  • the present invention is directed to a novel 120 kDa leukotoxin (cytotoxic protein) secreted from A. pleuropneumoniae serotypes 2, 3, 4, 6 and 8. Also disclosed are novel gene(s) or gene fragment (s) designated AppIIIC and AppIIIA, which encode the leukotoxin of the invention.
  • leukotoxin refers to the novel 120 kDa cytotoxic protein, mutations and recombinants thereof, secreted from A. pleuropneumoniae, serotype 2. This protein is encoded by novel genes or gene fragments designated AppIIIC and AppIIIA. Because this cytotoxic protein is not hemolytic to erythrocytes, but rather leukotoxic to leukocytes and macrophages, it has been termed “leukotoxin”. As used herein, the terms “cytotoxic protein” and “exotoxin” are analogous to "leukotoxin”.
  • the apparant molecular weight of the leukotoxin as determined using SDS-PAGE reflects the total molecular weights of the mature (i.e., proteolytically processed) forms, including any post translational modifications.
  • the leukotoxin of the invention can be produced using recombinant DNA methods, by chemical synthesis or can be obtained in substantially pure form from cultures of A. pleuropneumoniae using methods of isolation and purification.
  • the leukotoxin and/or epitopes thereof can be used as immunogens in various vaccine formulations to protect porcine against A. pleuropneumoniae, an etiological agent of porcine pleuropneumoniae.
  • the vaccine formulations can be effective against A.
  • the leukotoxin of the invention can be combined in a vaccine formulation with the Appl gene as described by Frey et al. , Nucleotide Sequence of the Hemolysin I gene from Actinobacillus pleuropneumoniae, 1991, Infect. Immun., vol. 59, pp. 3026-2032; and the AppII gene as described by Chang et al. , Cloning and Characterization of a Hemolysin Gene from
  • Actinobacillus (Haemophilus) pleuropneumoniae DNA, 1989, vol. 8, pp. 635-647, which disclosures are hereby incorporated by reference, thereby creating a vaccine which confers resistance to swine against porcine pleuropneumoniae caused by serotypes of A. pleuropneumoniae.
  • the present invention also relates to the DNA sequence (s) (Seq. ID No. 1) of the genes AppIIIA and AppIIIC genes which encode for the leukotoxin, as well as the amino acid sequences (Seq. ID No. 2) encoded by the DNA sequences. More particularly, the DNA sequences (Seq. ID No. 1)
  • AppIIIC and AppIIIA These genes, AppIIIC and AppIIIA, collectively AppIIICA, encode for the leukotoxin of the invention.
  • the AppIIIC gene encodes a polypeptide of 162 amino acid residues and the AppIIIA gene encodes a polypeptide of 1048 amnio acid residues.
  • the DNA (Seq. ID No. 1) and corresponding amino acid sequences (Seq. ID No. 2) of the present invention are shown in Figure 3.
  • any modifications i.e., insertions, deletions, mutations, recombinants, etc., of the DNA sequence (s) are within the scope of the invention provided that the modified sequence (s) encode for a gene producing a protein, its homologs or a fragment thereof having substantially the same physical, immunological or functional properties as the leukotoxin of the invention.
  • recombinant DNA techniques are used to insert the DNA sequence (s) (Seq. ID No. 1) of the AppIIIC and
  • AppIIIA genes encoding the leukotoxin into an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • vector systems known in the art can be used, such as, plasmids, bacteriophage virus or other modified viruses.
  • Suitable vectors include, but are not limited to the following viral vectors such as lambda vector system gtll, gtWES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl and other similar systems.
  • the DNA sequences are cloned into the vector using standard cloning proceedures in the art, as described by Maniatis et al. , 1982, Molecular Cloning: A
  • the vector system must be compatable with the host cell used.
  • the recombinant vectors can be introduced into the host cells via transformation, transfection or infection using standard techniques in the art.
  • a variety of host cell systems can be used to express the protein-encoding sequence (s) .
  • host cell systems include, but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA such as E. coli JM103, E. coli C600, E. coli C04, E. coli DH20 and E.
  • coli TB1 coli TB1
  • micororganisms such as yeast containing yeast vectors
  • mammalian cell systems infected with virus vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus baculovirus
  • RNA polymerase normally binds to the promotor and initiates transcription of a gene or a group of linked genes and regulatory elements (operon) .
  • Promotors vary in their strenth, i.e., ability to promote transcription.
  • any one of a number of suitable promotors can be used. For examle, when cloning in E.
  • coli its bacteriophages or plasmids
  • promotors such as the lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the P R and P L promotors of coliphage lambda and others including but not limited to lacUV5, ompF, bla, lpp and the like, can be used to direct high levels of transcription of adjacent DNA segments.
  • a hybrid trp-lacUV5 (tac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques can be used to provide for transcription of the inserted gene(s) .
  • Bacterial host cell strains and expression vectors can be chosen which inhibit the action of the promotor unless specifically induced.
  • the addition of specific inducers is necessary for efficient transcription of the inserted DNA; for example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside) .
  • IPTG isopropylthio-beta-D-galactoside
  • trp is induced when tryptophan is absent in the growth media
  • the P L promotor of lambda can be induced by an increase in temperature in host cells containing a temperature sensitive lambda represor, e.g., cl857. In this way, greater than 95% of the promotor-directed transcription may be inhibited in uninduced cells.
  • cytotoxin the genetically engineered cytotoxin protein
  • transformants may be cultured under conditions such that the promotor is not induced, and when the cells reach a suitable density in the growth medium, the promotor can be induced for production of the protein.
  • promotor/operator system is the so-called “tac” or trp-lac promotor/operator system (Russell and Bennett, 1982, Gene vol 20, pp.231-243, which disclosure is hereby incorporated by reference) .
  • This hybrid promotor is constructed by combining the -35 b.p.
  • tac is also controlled by the lac represor.
  • enhancer sequences When cloning in a eucaryotic host cell, enhancer sequences (e.g., the 72 bp tandem repeat of SV40 DNA or the retroviral long terminal repeats of LTRs, etc.) may be inserted to increase transcriptional efficiency.
  • Enhancer sequences are a set of eucaryotic DNA elements that appear to increase transcriptional efficiency in a manner relatively independent of their position and orientation with respect to a nearby gene.
  • promotor elements e.g., the polymerase binding site and the Goldberg-Hogness "TATA" box
  • enhancer sequences have the remarkable ability to function upstream from, within, or downstream from eucaryotic genes. Therefore, the position of the enhancer sequence with respect to the inserted gene is less critical.
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.
  • the DNA expression vector which contains a promotor, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (SD) sequence about 7-9 basis 5' to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes can be employed.
  • SD Shine-Dalgarno
  • Such combinations include but are not limited to the SD-ATG combination from the CRO gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides can be used.
  • A. pleuropneumoniae genetic sequences containing those regions coding for the cytotoxic protein (leukotoxin) can be ligated into an expression vector at a specific site in relation to the vector promotor and control elements so that when the recombinant DNA molecule is introduced into a host cell the foreign genetic sequence can be expressed (i.e., transcribed and translated) by the host cell.
  • the recombinant DNA molecule can be introduced into appropriate host cells (including but not limited to bacteria, virus, yeast, mammalian cells or the like) by transformation, infection or transfection (depending upon the vector/host cell system) .
  • Transformants are selected based upon the expression of one or more appropriate gene markers normally present in the vector, such as ampicillin resistance or tetracycline resistance in pBR322, or thymidine kinase activity in eucaryotic host systems. Expression of such marker genes should indicate that the recombinant DNA molecule is intact and is replicating.
  • Expression vectors may be derived from cloning vectors, which usually contain a marker function.
  • Such cloning vectors may include, but are not limited to the following: SV40 and adenovirus, vaccinia virus vectors, insect viruses such as baculoviruses, yeast vectors, bacteriophage vectors such as lambda gt-WES-lambda BC, lambda gt-1-lambda B, M13mp7,
  • M13mp8, M13mp9 or plasmid DNA vectors such as pBR322, pAC105, pVA51, pACYC177, pKH47, pACYC184, pUBHO, pMB9, pBR325, Col El, pSClOl, pBR313, pML21, RSF2124, pCRl, RP4, pBR328 and the like.
  • an E. coli plasmid system was chosen as the expression vector.
  • the invention is not limited to the use of an E. coli expression vector.
  • genetic engineering techniques could also be used to further characterize and/or adapt the cloned gene. For example, site directed mutagenesis of the gene encoding the leukotoxin could be used to identify regions of the protein responsible for generation of protective antibody responses. It could also be used to modify the protein in regions outside the protective domains, for example, to increase the solubility of the protein to allow easier purification.
  • the expression vectors containing the foreign gene inserts can be identified by three approaches: (1) DNA-DNA hybridization using probes comprising sequences that are homologous to the leukotoxin gene(s) ; (2) presence or absence of "marker" gene function and (3) expression of inserted sequences based on the physical, immunological or functional properties of the leukotoxin. Once a recombinant which expresses the leukotoxin is identified, the gene product should be analyzed. Immunological analysis is especially important because the ultimate goal is to use the leukotoxin or recombinant expression systems that express the leukotoxin in vaccine formulations and/or as antigens in diagnostic immunoassays.
  • the leukotoxin is cultured under conditions which facilitate growth of the cells and expression of the leukotoxin as will be apparent to one skilled in the art, then isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography) , centrifugation, differental solubility, or by any other standard techniques for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, and sizing column chromatography
  • centrifugation e.g., centrifugation, differental solubility, or by any other standard techniques for the purification of proteins.
  • the amino acid sequence is known from the DNA sequence of the invention, the protein can be synthesized by chemical methods according to the proceedure of Hunkapiller et al . , 1984, Nature, vol. 310, pp. 105-111, which disclosure is hereby incorporated by reference.
  • proteins and/or polypeptide fragments related to the leukotoxin of the invention can be used as immunogens in a vaccine formulation to protect against porcine pleuropnemoniae and other disease symptoms of A. pleuropneumoniae.
  • Vaccines comprise solely the relevant immunogenic material necessary to immunize a host.
  • Vaccines made from genetically engineered immunogens, chemically synthesized immunogens and/or immunogens comprising authentic substantially pure leukotoxin toxin, which are capable of eliciting a protective immune response are particularly advantageous because there is no risk of infection of the recipients.
  • the leukotoxin toxin related protein or fragment thereof can be pruified from recombinants that express the leukotoxin epitopes.
  • recombinants include any of the previously described bacterial transformants, yeast transformants, or cultured cells infected with recombinant viruses that express the leukotoxin toxin epitopes .
  • Suitable adjuvants include, but are not limited to: surface active substances, e.g., hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethly-dioctadecylammonium bromide,
  • the immunogen can also be incorporated into liposomes, or conjgated to polysaccharides and/or other polymers for use in a vaccine formulation.
  • the vaccine formulation can comprise live recombinant viral vaccine or an inactivated recombinant viral vaccine which is used to protect against porcine pleuropnemoniae and other disease symptoms of A. pleuropneumoniae.
  • multivalent vaccines can be prepared from a single or a few infectious recombinant viruses, proteins or polypeptides that express epitopes of organisms that cause disease in addition to the epitopes of A. pleuropneumoniae.
  • a vaccine can be engineered to include coding sequences for other epitopes, or a mixture of vaccinia or viruses each expressing a different gene encoding for different epitopes which can confer resistance to other diseases which affect swine.
  • Many methods can be used to introduce the vaccine formulations into the animal. For example, intadermal, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes of administration can be used.
  • the vaccine formulations can be used to produce antibodies for use in passive immunotherapy.
  • Another embondiment of the invention is to provide reagents for use in diagnostic assays for the detection of A. pleuropneumoniae in various fluids of animals suspected of such infection.
  • the proteins and peptides of the invention can be used in any immunoassay system known in the art including, RIA, ELISA assays, "sandwich” assays, precipitation ractions, gel diffusion precipitation reactions, agglutination assays, fluorescent immunoassays, etc.
  • the DNA sequences of the genes encoding the leukotoxin can be used in nucleic acid hybridization assays. The following examples are provide to further illustrate the present invention.
  • the antiserum against A. pleuropneumoniae serotype 2 was prepared by intramuscular inoculation of 106 logarithmic phase live organisms into the pigs. The sera were collected before or 4-weeks after vaccination. Serum from these vaccinated pigs was shown to neutralize the leukotoxin in culture supernatants from A. pleuropneumoniae, serotype 2.
  • Immunoreactive proteins were visualized using porcine antitoxin and an anti-swine IgG second antibody conjugated to alkaline phosphatase as described by Chang et al . , 1987, Identification and Characterization of Pasteurella haemolytica. Infect. Immun., vol. 55, pp. 2348-2354; and Chang, et al . , Cloning and Characterization of a Hemolysin Gene from Actinobacillus (Haemophilus) pleuropneumoniae, 1989, DNA, vol. 8, pp. 635-647, which disclosures are hereby incorporated by reference.
  • pleuropneumoniae chromosomal DNA was isolated and partially digested with Sau3A.
  • the digested DNA was fractionated by sedimentation through a 10-40% sucrose gradient as described by Chang, et al . , Cloning and Characterization of a Hemolysin Gene from Actinobacillus
  • the vector DNA was mixed with size-selected A. pleuropneumoniae, serotype 2, DNA at a molar ratio of 1:4 and treated with T4 DNA ligase for 16 hours at 15°C.
  • the ligated DNA mixture was packaged into ⁇ particles using the commercially available in vitro packaging kit GIGAPACK plus (Stratagene Inc., La
  • the phage titers were determined and amplified on P2392.
  • Filters were hybridized in 45% formamide, 5X SSC (20X SSC containing 175.3 gm of NaCl and 88.2 gm of sodium citrate per liter, pH 7.0) , 5X Denhardt' s solution, and 100 ⁇ g/ml sheared calf thymus DNA for 12 hours at 37°C. Filters were then washed twice with 2X SSC-0.1% SDS and twice with 0.2% SSC-0.1% SDS at room temperature as described by Chang et al . , The Actinobacillus pleuropneumoniae Hemolysin Determinant : Unlinked AppCA and AppBD Loci Flanked by Pseudogenes, 1991, J.
  • DNA seguencing was performed by the dideoxy chain-termination method as described by Sanger et al . , DNA Sequencing with Chain-Termination Inhibitors, 1977, Proc. Natl. Acad. Sci., vol. 74, pp. 5463-5467, which disclosure is hereby incorporated by reference. Regions from the A. pleuropneumoniae insert in bacteriophage clone ⁇ yfc28 were subcloned into Ml3mpl8 or M13mpl9 and single stranded phage DNA was prepared by standard procedures as described by Messing, J., New 113 Vectors for Cloning, 1983, Methods Enzymol., vol. 101, pp.
  • the sequencing reactions utilized [ 35 S]dATP, T7 DNA polymerase, and the commercially available SEQUENASE kit (United States Biochemicals, Cleveland) . Certain regions of the DNA insert were sequenced directly from the recombinant bacteriophage. In these cases, 1-2 ⁇ g of ⁇ yfc28 DNA was mixed with 100 ng of an oligonucleotide primer (prepared by the Analytical and Synthetic Facility, Cornell University) in a total volume of 12 ⁇ l, boiled for 5 minutes, and then cooled rapidly on ice. The sequencing reactions were performed with reagents supplied with the SEQUENASE kit (United States Biochemicals, Cleveland) according to the manufacturer's instructions.
  • DNA sequence analysis was performed on a VAX computer using the Genetics Computer Group program package (University of Wisconsin, Madison) and programs from the PC Gene package (Intelligenetics Corp., Mountain View, CA) .
  • the amino acid sequence alignment was carried out with the GAP and LINEUP programs (Genetics Computer Group, University of Wisconsin, Madison) and similarity was calculated according to the method of Pearson et al . , Improved Tools for Biological Sequence Comparison, 1988, Proc. Natl. Acad. Sci., vol. 5, pp. 2444-2448, which disclosure is hereby incorporated as reference.
  • A. pleuropneumoniae serotypes could produce the 120 kDa leukotoxin
  • culture supernatants from A. pleuropneumoniae, 12 serotypes were analyzed by Western blot, using antiserum against the toxin, as shown in Figure 1A.
  • a cross-reacting polypeptide species with molecular weight 120 kDa was evident in serotypes 2, 3, 6 and 8.
  • BD genes secretion determinants
  • the lktBD genes derived from pYFC35 (Chang et al . , Secretion of the Pasteurella leukotoxin by Escherichia coli, 1989, FEMS Microbiol. Lett., vol. 60, pp. 169-174, which disclosure is hereby incorporated by reference) was choosen as a probe to screen an A. pleuropneumoniae genomic library constructed in the phage vector lambda-dash. Five clones were isolated and all of these clones expressed a 120 kDa polypeptide detected by Western blotting with the anti-A. pleuropneumoniae leukotoxin antibody as shown in Figure IB.
  • recombinant phage DNA were isolated from the positive recombinant clones and analyzed by restriction endonuclease mapping which overlapped with each other. Although the five clones produce the full-length leukotoxin, no cytotoxic activity could be detected in any of the phage lysates (data not shown) .
  • AppIIICA genes are more closely related to the lktCA from P. haemolytica than to other RTX gene family members. With reference to Table 1, there is shown summarizes of the similarities between AppIIICA and the other RTX CA genes for which sequence information is available.
  • the Sail fragment from ⁇ yfc32 (Fig. 2) was subcloned into vector pHG165 as described by Stewart et al . , A pBR322 Copy Number Derivation of pUC8 for Cloning and Expression, 1986, Plasmid, vol. 15, pp. 172-186, which disclosure is hereby incorporated by referencem, yielding plasmid pYFC117.
  • the AppIIICA genes of pYFC117 are likely to be expressed from an its own promoter.
  • the plasmid was transformed into the E. coli host, TB1, and the transformant was grown to early stationary phase and examined for the expression of a 120 kDa polypeptide, and hemolytic and leukocytic activities.
  • a Assay performed with a late log phase supernatant from a culture of A. pleuropneumoniae, serotype 2, grown in brain heart infusion with NAD.
  • One unit of toxin activity is defined as the minimal amount of toxin, as determined by serial dilution, equired to produce the morphological change in >95% of the input BL-3 cells.
  • c Assay performed with early stationary phase supernatant from the E. coli host, TBl, harboring the indicated plasmid grown in LB with ampicillin (50 ⁇ g/ml) .
  • pleuropneumoniae serotype 2,3,6 and 8 secreted a 120 kDa polypeptide (Fig. 1,A) .
  • serotype 6 secreted a 120 kDa polypeptide (Fig. 1,A, lane 6) .
  • Southern blots analysis indicated that these four serotypes have a hybridization signal at 1278 bp when a 1.2kb
  • AppIIC and AppIIA proteins from A. pleuropneumoniae, serotype 5, hemolysin determinant.
  • the AppIIIA leukotoxin does not have a classic signal sequence at its amino terminus. Instead, the amino terminus is rich in serine, threonine, and lysine (13 of the first 41 residues) and has the capability of forming a positively charged, amphiphilic alpha-helix as do the amino termini of other RTX toxins.
  • pleuropneumoniae serotypes secrete three different RTX toxin. It is speculated that the AppII and ApplII toxins are required for pathogenicity or immunogenicity in pigs against A. pleuropneumoniae, serotype 2.
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Actinobacillus - pleuropneumonia
  • ACTTTCAGGA GTAACTCGAA GTTTTGCTTT ACGGAATAAG AATGCTTCAA CAAGCACTAA 1380 AGTTGCTGCT GGCTTTGAAC TCTCAAATCA AGTAATTGGT GGTATTACGA
  • ORGANISM Actinobacillus pleuropneumoniae

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Abstract

The present invention provides a gene(s) or gene fragment(s) which encode a novel 120 kDa leukotoxin secreted from A. pleuropneumonia, serotypes 2, 3, 4, 6 and 8. Also described are recombinant DNA sequence(s) and expression systems for directing expression of the gene(s) or gene fragment(s), a method of using the gene(s) or gene fragment(s) as immunogens in vaccine formulations to protect pigs against porcine pleuropneumonia and as reagents in diagnostic assays.

Description

RECOMBINANT VACCINE FOR PORCINE PLEUROPNEUMONIAE
This is a continuation-in-part of U. S. Patent
Application Serial No. 07/972,229, filed November 5, 1992, now abandoned.
FIELD OP THE INVENTION The present invention relates generally to the bacterium Actinobacillus pleuropneumonia (A. pleuropneumonia) , which causes porcine pleuropneumonia in pigs. More particularly, the invention relates to a gene(s) or gene fragment (s) encoding a novel leukotoxin secreted from A^. pleuropneumonia, serotype 2, recombinant DNA sequences and expression systems for directing expression of the gene(s), a method of using the gene(s) or gene fragment (s) as immunogens in vaccine formulations to protect pigs against porcine pleuropneumonia and as reagents in diagnostic assays.
BACKGROUND OF THE INVENTION
Actinobacillus pneumonia (porcine pleuropneumonia) of swine is a highly contagious respiratory disease caused by the gram-negative bacterium A. pleuropneumoniae (Pearson et al., Improved Tools for Biological Sequence Comparison, 1988, Proc. Natl. Acad. Sci., vol. 5, pp. 2444-2448). In recent years, there has been a significant increase in the incidence of pneumonia in swine, and currently it is the major cause of economic loss to the swine industry. During outbreaks of the acute disease the mortality rate can reach 100% among piglets and 25% among feeder pigs. Infected pigs may develop acute local extensive pneumonia accompanied by a fibrinous pleuritis or chronic localized pulmonary necrosis with pleuritic adhesions.
In the past several years, a number of Gram-negative bacterium have been discovered which secrete high molecular weight (100-110 kDa) calcium-dependent cytotoxic proteins which are immunologically and genetically related to the alpha-hemolysin (HlyA) of E. coli . These toxins have been designated the RTX (Repeat of Toxin) family on the basis of a series of glycine/aspartic acid-rich nonapeptide repeats found in the carboxylterminal third of the toxin protein (McWhinney et al . , Separable Domains Define Target Cell Specificities of an RTX Hemolysin from Actinobacillus pleuropneumoniae, 1992, J. Bacteriol., vol. 174, pp. 291-297) . The genetic determinants for the secreted RTX toxins consist of four genes: " A", the structural gene for toxin protein; "C", which is required for "activation" of the toxin prior to secretion; and "B" and "D" , which are essential for the process of secretion. The four RTX genes are typically found in a single transcriptional unit, "CABD" , and are expressed from a common promoter located upstream of the "C" gene (Felmleel et al. , Nucleotide Sequence of an Escherichia coli Chromosomal Hemolysin, 1895, J. Bacteriol., vol. 163, pp. 94-105; Highlander et al . , Secretion and Expression of Pasteurella haemolytica
Leukotoxin, 1990, J. Bacteriol., vol. 172, pp.2343-2350; and Hackman et al . , Genetical and Functional Organization of the Escherichia coli Haemolysin Determinant 2001, 1985, Mol. Gen. Genet., vol. 201, pp.282-288) . Aj_ pleuropneumoniae strains secrete heat-labile extracellular cytotoxin proteins that differ antigenically among serotypes (Kamp et al. , Identification of Hemolytic and Cytotoxic Proteins of Actinobacillus pieuropneumoniae by Use of Monoclonal Antibodies, 1991, Infect. Immun. , vol. 59, pp. 3079-3085) . To date, only two such heat-labile exotoxins have been characterized. Frey et al. , Nucleotide Sequence of the Hemolysin I gene from Actinobacillus pleuropneumoniae, 1991, Infect. Immun., vol. 59, pp. 3026-2032, discloses A. pleuropneumoniae hemolysin I (Appl) gene, a 105 kDa polypeptide secreted from serotypes 1, 5, 9, 10 and 11. The Appl gene has strong hemolytic and cytotoxic activity.
Chang et al . , Cloning and Characterization of a Hemolysin Gene from Actinobacillus (Haemophilus) pleuropneumoniae, DNA, 1989, vol. 8, pp. 635-647, discloses
A. pleuropneumoniae hemolysin II (AppII) gene, a 105 kDa polypeptide secreted from serotypes 1, 2, 3, 4, 6, 7, 8, 9, lip and 12. The AppII gene has weak hemolytic activity and moderate cytotoxic activity.
It is believed that the Appl and AppII polypeptides can be used as a protective immunogen for swine against porcine pneumonia. However, it has been determined that the homology between the different peptides is only about 50%. Moreover, it has been determined that the different peptides are not cross-reactive against different serotypes of A. pleuropneumoniae. This is significant because a swine with pneumonia typically has been infected by more than one A^_ pleuropneumoniae serotype. The present inventions discloses gene or gene fragments encoding a novel leukotoxin secreted from A. pleuropneumoniae serotypes 2, 3, 4, 6 and 8. The novel genes of the invention or fragments thereof, alone, or in combination with the Appl and AppII genes, can be effective immunogens for pigs against porcine pleuropneumonia.
SUMMARY OF THE INVENTION
The present invention is directed to a novel leukotoxin (cytotoxic protein) secreted from A^. pleuropneumoniae, serotype 2, as well as the molecularly cloned gene(s) or gene fragment (s) which encodes for this cytotoxic protein. The leukotoxin (cytotoxic protein) of the present invention and related peptides or proteins can be used as immunogens in vaccine formulations for swine against porcine pleuropneumoniae.
The leukotoxin (cytotoxic protein) of the invention can be purified from A. pleuropneumoniae or produced using recombinant DNA techniques in any vector-host system, or synthesized by chemical methods.
Accordingly, the invention is also directed to novel DNA sequences (Seq. ID No. 1) and corresponding amino acid sequences (Seq. ID No. 2) and vectors including plasmid
DNA, and viral DNA such as human viruses, animal viruses, insect viruses or bacteriophages which can be used to direct the expression of the leukotoxin (cytotoxic protein) and related peptides and/or proteins in appropriate host cells from which the peptides and/or proteins can be purified. More particularly, the present invention discloses the cloning and sequencing of novel genes designated AppIIIC and AppIIIA (AppIIICA) which encode for the leukotoxin of the invention. The novel leukotoxin is a 120 kDa protein secreted from A. pleuropneumoniae serotypes 2, 3, 4, 6 and 8.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1(A) shows a Western blot analysis of culture supernatants from different serotypes of A^_ pleuropneumoniae using swine anti-leukotoxin serum as the first antibody. The lanes are labeled to the serotype number.
Fig. 1(B) shows a Western blot analysis of antigenic proteins expressed from recombinant bacteriophages. Lysates were from E. coli LE392 infected with λ-Dash (lane 1) , λyfc26 (lane 2) , λyfc27 (lane 3) , and λyfc28 (lane 4) . The first antibody was swine anti-leukotoxin serum.
Fig. 2. shows a restriction map of clones of the A. pleuropneumoniae leukotoxin of the invention. EcoRI sites derived from the vector flank the inserts of each clone. The locations of the two open reading designated
AppIIIC and AppIIIA found by sequence analysis are indicated (E = EcoRI; H = Hindlll; P = Pstl; X = Xbal) .
Fig. 3. shows the nucleotide sequence (Seq. ID
No. 1) of the AppIIICA region and the predicted amino acid sequences (Seq. ID No. 2) of the AppIIIC and AppIIIA proteins. Promoter-like regions proximal to the AppIIIC are indicated by the symbol Λ directed beneath the nucleotide sequence. Potential ribosome binding sequences preceding AppIIIC, AppIIIA and immediately after AppIIIA are indicated by underlining. A potential rho-independent transcription terminator and polyT track distal AppIIIA are indicated by < > and ***, respectively. The three transmembrane segments are double underlined within the AppIIIA amino acid sequence. The glycine-rich repeated sequences are underlined within the AppIIIA sequence. Fig. 4 shows a Southern blot analysis of A^_ pleuropneumoniae 12 serotype reference strains. The lanes are labeled according to the serotype number.
Fig. 5 shows a Western blot analysis of aliquots of E. coli TB1 carrying pYFCH7 (lane 1) and the culture supernatant from A^_ pieuropneumoniae (lane 2) . The first antibody was swine anti-leukotoxin serum. The apparent molecular weights of prestained standards are shown in kilodaltons (kDa) .
DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a novel 120 kDa leukotoxin (cytotoxic protein) secreted from A. pleuropneumoniae serotypes 2, 3, 4, 6 and 8. Also disclosed are novel gene(s) or gene fragment (s) designated AppIIIC and AppIIIA, which encode the leukotoxin of the invention.
As used herein, "leukotoxin" refers to the novel 120 kDa cytotoxic protein, mutations and recombinants thereof, secreted from A. pleuropneumoniae, serotype 2. This protein is encoded by novel genes or gene fragments designated AppIIIC and AppIIIA. Because this cytotoxic protein is not hemolytic to erythrocytes, but rather leukotoxic to leukocytes and macrophages, it has been termed "leukotoxin". As used herein, the terms "cytotoxic protein" and "exotoxin" are analogous to "leukotoxin".
The apparant molecular weight of the leukotoxin as determined using SDS-PAGE reflects the total molecular weights of the mature (i.e., proteolytically processed) forms, including any post translational modifications. The leukotoxin of the invention can be produced using recombinant DNA methods, by chemical synthesis or can be obtained in substantially pure form from cultures of A. pleuropneumoniae using methods of isolation and purification. The leukotoxin and/or epitopes thereof can be used as immunogens in various vaccine formulations to protect porcine against A. pleuropneumoniae, an etiological agent of porcine pleuropneumoniae. The vaccine formulations can be effective against A. pleuropneumoniae, serotypes 2, 3, 6 and 8. More preferably, the leukotoxin of the invention can be combined in a vaccine formulation with the Appl gene as described by Frey et al. , Nucleotide Sequence of the Hemolysin I gene from Actinobacillus pleuropneumoniae, 1991, Infect. Immun., vol. 59, pp. 3026-2032; and the AppII gene as described by Chang et al. , Cloning and Characterization of a Hemolysin Gene from
Actinobacillus (Haemophilus) pleuropneumoniae, DNA, 1989, vol. 8, pp. 635-647, which disclosures are hereby incorporated by reference, thereby creating a vaccine which confers resistance to swine against porcine pleuropneumoniae caused by serotypes of A. pleuropneumoniae.
The present invention also relates to the DNA sequence (s) (Seq. ID No. 1) of the genes AppIIIA and AppIIIC genes which encode for the leukotoxin, as well as the amino acid sequences (Seq. ID No. 2) encoded by the DNA sequences. More particularly, the DNA sequences (Seq. ID
No. 1) of the invention encode for two genes, designated
AppIIIC and AppIIIA. These genes, AppIIIC and AppIIIA, collectively AppIIICA, encode for the leukotoxin of the invention. The AppIIIC gene encodes a polypeptide of 162 amino acid residues and the AppIIIA gene encodes a polypeptide of 1048 amnio acid residues. The DNA (Seq. ID No. 1) and corresponding amino acid sequences (Seq. ID No. 2) of the present invention are shown in Figure 3. It is understood that any modifications i.e., insertions, deletions, mutations, recombinants, etc., of the DNA sequence (s) are within the scope of the invention provided that the modified sequence (s) encode for a gene producing a protein, its homologs or a fragment thereof having substantially the same physical, immunological or functional properties as the leukotoxin of the invention.
In accordance with one embodiment of the invention, recombinant DNA techniques are used to insert the DNA sequence (s) (Seq. ID No. 1) of the AppIIIC and
AppIIIA genes encoding the leukotoxin into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequences. A large number of vector systems known in the art can be used, such as, plasmids, bacteriophage virus or other modified viruses. Suitable vectors include, but are not limited to the following viral vectors such as lambda vector system gtll, gtWES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl and other similar systems. The DNA sequences are cloned into the vector using standard cloning proceedures in the art, as described by Maniatis et al. , 1982, Molecular Cloning: A
Laboratory Manual, Cold Springs Laboratory, Cold Springs
Harbor, New York, which disclosure is hereby incorporated by reference.
The vector system must be compatable with the host cell used. The recombinant vectors can be introduced into the host cells via transformation, transfection or infection using standard techniques in the art. A variety of host cell systems can be used to express the protein-encoding sequence (s) . For example, host cell systems include, but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA such as E. coli JM103, E. coli C600, E. coli C04, E. coli DH20 and E. coli TB1; micororganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (vaccinia virus, adenovirus, etc.) ; insect cell systems infected with virus (baculovirus) .
In order to obtain efficient expression of the gene(s) or gene fragment (s) , a pro otor must be present in the expression vector, RNA polymerase normally binds to the promotor and initiates transcription of a gene or a group of linked genes and regulatory elements (operon) . Promotors vary in their strenth, i.e., ability to promote transcription. For the purpose of expressing the cloned gene(s) of the invention, it is desirable to use strong promotors in order to obtain a high level of transcription and, hence, expression of the gene(s) . Depending upon the host cell system utilized, any one of a number of suitable promotors can be used. For examle, when cloning in E. coli, its bacteriophages or plasmids, promotors such as the lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the PR and PL promotors of coliphage lambda and others including but not limited to lacUV5, ompF, bla, lpp and the like, can be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques can be used to provide for transcription of the inserted gene(s) . Bacterial host cell strains and expression vectors can be chosen which inhibit the action of the promotor unless specifically induced. In certain operons the addition of specific inducers is necessary for efficient transcription of the inserted DNA; for example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside) . A variety of other operons, such as trp, pro, etc., are under different controls. The trp operon is induced when tryptophan is absent in the growth media; and the PL promotor of lambda can be induced by an increase in temperature in host cells containing a temperature sensitive lambda represor, e.g., cl857. In this way, greater than 95% of the promotor-directed transcription may be inhibited in uninduced cells. Thus, expression of the genetically engineered cytotoxin protein (leukotoxin) can be controlled. This is important if the protein product of the cloned gene is lethal or detrimental to host cells. In such cases, transformants may be cultured under conditions such that the promotor is not induced, and when the cells reach a suitable density in the growth medium, the promotor can be induced for production of the protein.
One such promotor/operator system is the so-called "tac" or trp-lac promotor/operator system (Russell and Bennett, 1982, Gene vol 20, pp.231-243, which disclosure is hereby incorporated by reference) . This hybrid promotor is constructed by combining the -35 b.p.
(-35 region) of the trp promotor and the -10 b.p. (-10 region or Pribnow box) of the lac promotor (the sequences of DNA which are the RNA polymerase binding site) . In addition to maintaining the strong promotor characteristics of the tryptophan promotor, tac is also controlled by the lac represor.
When cloning in a eucaryotic host cell, enhancer sequences (e.g., the 72 bp tandem repeat of SV40 DNA or the retroviral long terminal repeats of LTRs, etc.) may be inserted to increase transcriptional efficiency. Enhancer sequences are a set of eucaryotic DNA elements that appear to increase transcriptional efficiency in a manner relatively independent of their position and orientation with respect to a nearby gene. Unlike the classic promotor elements (e.g., the polymerase binding site and the Goldberg-Hogness "TATA" box) which must be located immediately 5' to the gene, enhancer sequences have the remarkable ability to function upstream from, within, or downstream from eucaryotic genes. Therefore, the position of the enhancer sequence with respect to the inserted gene is less critical.
Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promotor, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (SD) sequence about 7-9 basis 5' to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes can be employed. Such combinations include but are not limited to the SD-ATG combination from the CRO gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides can be used.
Any of the conventional cloning methods for insertion of DNA fragments into a vector can be used to ligate the promotor and other control elements into specific sites within the vector. Accordingly, A. pleuropneumoniae genetic sequences containing those regions coding for the cytotoxic protein (leukotoxin) can be ligated into an expression vector at a specific site in relation to the vector promotor and control elements so that when the recombinant DNA molecule is introduced into a host cell the foreign genetic sequence can be expressed (i.e., transcribed and translated) by the host cell. As previously mentioned, the recombinant DNA molecule can be introduced into appropriate host cells (including but not limited to bacteria, virus, yeast, mammalian cells or the like) by transformation, infection or transfection (depending upon the vector/host cell system) . Transformants are selected based upon the expression of one or more appropriate gene markers normally present in the vector, such as ampicillin resistance or tetracycline resistance in pBR322, or thymidine kinase activity in eucaryotic host systems. Expression of such marker genes should indicate that the recombinant DNA molecule is intact and is replicating. Expression vectors may be derived from cloning vectors, which usually contain a marker function. Such cloning vectors may include, but are not limited to the following: SV40 and adenovirus, vaccinia virus vectors, insect viruses such as baculoviruses, yeast vectors, bacteriophage vectors such as lambda gt-WES-lambda BC, lambda gt-1-lambda B, M13mp7,
M13mp8, M13mp9, or plasmid DNA vectors such as pBR322, pAC105, pVA51, pACYC177, pKH47, pACYC184, pUBHO, pMB9, pBR325, Col El, pSClOl, pBR313, pML21, RSF2124, pCRl, RP4, pBR328 and the like.
In the particular embodiment in the examples of the present invention, an E. coli plasmid system was chosen as the expression vector. The invention, however, is not limited to the use of an E. coli expression vector. Furthermore, genetic engineering techniques could also be used to further characterize and/or adapt the cloned gene. For example, site directed mutagenesis of the gene encoding the leukotoxin could be used to identify regions of the protein responsible for generation of protective antibody responses. It could also be used to modify the protein in regions outside the protective domains, for example, to increase the solubility of the protein to allow easier purification.
The expression vectors containing the foreign gene inserts (e.g., DNA encoding the leukotoxin) can be identified by three approaches: (1) DNA-DNA hybridization using probes comprising sequences that are homologous to the leukotoxin gene(s) ; (2) presence or absence of "marker" gene function and (3) expression of inserted sequences based on the physical, immunological or functional properties of the leukotoxin. Once a recombinant which expresses the leukotoxin is identified, the gene product should be analyzed. Immunological analysis is especially important because the ultimate goal is to use the leukotoxin or recombinant expression systems that express the leukotoxin in vaccine formulations and/or as antigens in diagnostic immunoassays. Once the leukotoxin is identified, it is cultured under conditions which facilitate growth of the cells and expression of the leukotoxin as will be apparent to one skilled in the art, then isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography) , centrifugation, differental solubility, or by any other standard techniques for the purification of proteins. In addition, since the amino acid sequence is known from the DNA sequence of the invention, the protein can be synthesized by chemical methods according to the proceedure of Hunkapiller et al . , 1984, Nature, vol. 310, pp. 105-111, which disclosure is hereby incorporated by reference.
In another embodiment of the invention, proteins and/or polypeptide fragments related to the leukotoxin of the invention can be used as immunogens in a vaccine formulation to protect against porcine pleuropnemoniae and other disease symptoms of A. pleuropneumoniae. Vaccines comprise solely the relevant immunogenic material necessary to immunize a host. Vaccines made from genetically engineered immunogens, chemically synthesized immunogens and/or immunogens comprising authentic substantially pure leukotoxin toxin, which are capable of eliciting a protective immune response are particularly advantageous because there is no risk of infection of the recipients. Thus, the leukotoxin toxin related protein or fragment thereof can be pruified from recombinants that express the leukotoxin epitopes. Such recombinants include any of the previously described bacterial transformants, yeast transformants, or cultured cells infected with recombinant viruses that express the leukotoxin toxin epitopes .
Whether the immunogen is purified from recombinants or chemically synthesized, the final product is adjusted to an appropriate concentration and formulated with any suitable vaccine adjuvant. Suitable adjuvants include, but are not limited to: surface active substances, e.g., hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethly-dioctadecylammonium bromide,
N, N-dicoctadecyl-N-Nbis (2-hydroxyethyl-propane diamine) , methoxyhexadecylglycerol, and pluronic polyols; plyamines, e.g., pyran, dextransulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin; oil emulsions; and mineral gels, e.g., aluminum hydroxide, aluminum phosphate, etc. The immunogen can also be incorporated into liposomes, or conjgated to polysaccharides and/or other polymers for use in a vaccine formulation. In another aspect, the vaccine formulation can comprise live recombinant viral vaccine or an inactivated recombinant viral vaccine which is used to protect against porcine pleuropnemoniae and other disease symptoms of A. pleuropneumoniae. In addition, multivalent vaccines can be prepared from a single or a few infectious recombinant viruses, proteins or polypeptides that express epitopes of organisms that cause disease in addition to the epitopes of A. pleuropneumoniae. For example, a vaccine can be engineered to include coding sequences for other epitopes, or a mixture of vaccinia or viruses each expressing a different gene encoding for different epitopes which can confer resistance to other diseases which affect swine. Many methods can be used to introduce the vaccine formulations into the animal. For example, intadermal, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes of administration can be used.
Instead of actively immunizing with viral or subunit vaccines, it is possible to confer short-term protection to the host by administation of pre-formed antibody against an epitope of A. pleuropneumoniae. Thus, the vaccine formulations can be used to produce antibodies for use in passive immunotherapy.
Another embondiment of the invention is to provide reagents for use in diagnostic assays for the detection of A. pleuropneumoniae in various fluids of animals suspected of such infection. For example, the proteins and peptides of the invention can be used in any immunoassay system known in the art including, RIA, ELISA assays, "sandwich" assays, precipitation ractions, gel diffusion precipitation reactions, agglutination assays, fluorescent immunoassays, etc. In another aspect, the DNA sequences of the genes encoding the leukotoxin can be used in nucleic acid hybridization assays. The following examples are provide to further illustrate the present invention.
Example I Materials and Methods
1. Bacterial Strains, Plasmid, and Growth Conditions
A. pleuropneumoniae, serotype 1 to 12 standard strains, were grown in brain-heart infusion broth (BHI, Difco Laboratories) supplemented with 0.1% NAD as described by Kamp et al . , Identification of Hemolytic and Cytotoxic Proteins of Actinobacillus pleuropneumoniae by Use of Monoclonal Antibodies, 1991, Infect. Immun. , vol. 59, pp. 3079-3085, which disclosure is hereby incorporated by reference. All E. coli strains (JM101, supE thi Δ(lac-proAB) F' [traD36 proAB÷ lad q lacZΔl5] ; TBl, ara
Δ(lacproAB) rpsL ø80dlacZΔMl5 hsdRl7 (r"m+) ; LE392, hsdR514 (r"m+) supE44 SupF58 lacYl galK2 galT22 metBl trpR55; P2392, a P2 lysogen of LE392) were cultured in Luria broth containing the appropriate antibiotics when necessary as described by Miller, J.M., Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 433, which disclosure is hereby incorporated by reference. The vector lambda-dash (λ-dash) was used as described by Chang et al. , Cloning and Characterization of a Hemolysin Gene from Actinobacillus (Haemophilus) pleuropneumoniae, ■ DNA, 1989, vol. 8, pp. 635-647, which disclosure is hereby incorporated by reference. The intact AppIIICA from λyfc32 was subcloned into pHGl65 according to the method of Stewart et al . , A pBR322 Copy Number
Derivation of pUC8 for Cloning and Expression, 1986, Plasmid, vol. 15, pp. 172-186, which disclosure is hereby incorporated by reference, as a Sail fragment to form pYFCH7.
2. Preparation of anti-A. pleuropneumoniae Serum in Pigs
The antiserum against A. pleuropneumoniae serotype 2 was prepared by intramuscular inoculation of 106 logarithmic phase live organisms into the pigs. The sera were collected before or 4-weeks after vaccination. Serum from these vaccinated pigs was shown to neutralize the leukotoxin in culture supernatants from A. pleuropneumoniae, serotype 2.
3. SDS-PAGE and Western Blotting
SDS-PAGE and Western blotting were performed as described by Chang et al. , 1987, Identification and Characterization of Pasteurella haemolytica. Infect. Immun., vol. 55, pp. 2348-2354; Chang, et al. , Cloning and Characterization of a Hemolysin Gene from Actinobacillus (Haemophilus) pleuropneumoniae, 1989, DNA, vol. 8, pp. 635-647; Chang et al . , Secretion of the Pasteurella Leukotoxin by Escherichia coli, 1989, FEMS Microbiol. Lett., vol. 60, pp. 169-174; and Chang et al . , The Actinobacillus pleuropneumoniae Hemolysin Determinant :
Unlinked AppCA and AppBD Loci Flanked by Pseudogenes, 1991,
J. Bactreiol., vol. 173, pp. 5151-5158, which disclosures are hereby incorporated by reference, using culture supernatants (5 ml) concentrated by
Cloroform/methanol/water system as described by Wessel et al . , A Method for the Quantitative Recovery of Protein in
Dilute Solution in the Presence of Detergent and Lipid,
1984, Ana. Biochem., vol. 138, pp. 141-143, which disclosure is hereby incorporated by reference, or phage lysates precipitated by 10% trichloroacetic acid (TCA) and 10% of sodium deoxycholate (DOC) at 40°C overnight as described by Bensadoun et al . , Assay of Proteins in the Presence of Interfering Materials, 1976, Ana. Biochem., vol. 70, pp. 241- 250, which disclosure is hereby incorporated by reference, and resuspended in 100 μl of sample buffer. After boiling for 2 minutes, samples (15 μl ) were subjected to SDS-PAGE. Immunoreactive proteins were visualized using porcine antitoxin and an anti-swine IgG second antibody conjugated to alkaline phosphatase as described by Chang et al . , 1987, Identification and Characterization of Pasteurella haemolytica. Infect. Immun., vol. 55, pp. 2348-2354; and Chang, et al . , Cloning and Characterization of a Hemolysin Gene from Actinobacillus (Haemophilus) pleuropneumoniae, 1989, DNA, vol. 8, pp. 635-647, which disclosures are hereby incorporated by reference.
4. Construction and Genomic Bank of A. pleuropneumoniae DNA in λ-dash
A. pleuropneumoniae chromosomal DNA was isolated and partially digested with Sau3A. The digested DNA was fractionated by sedimentation through a 10-40% sucrose gradient as described by Chang, et al . , Cloning and Characterization of a Hemolysin Gene from Actinobacillus
(Haemophilus) pleuropneumoniae, 1989, DNA, vol. 8, pp.
635-647, which disclosure is hereby incorporated by reference, and fractions containing 9- to 20-kbp fragments, as judged by agarose gel electrophoresis, were pooled and concentrated by alcohol precipitation to a final concentration of 100 μg/ml. Lambda-dash was cleaved with
BamHI and treated with alkaline phosphatase to remove terminal phosphates. After phenol extraction and concentration by ethanol precipitation, the vector DNA was mixed with size-selected A. pleuropneumoniae, serotype 2, DNA at a molar ratio of 1:4 and treated with T4 DNA ligase for 16 hours at 15°C. The ligated DNA mixture was packaged into λ particles using the commercially available in vitro packaging kit GIGAPACK plus (Stratagene Inc., La
Jolla, CA) . The phage titers were determined and amplified on P2392.
5. Southern Blotting and Library Screening A. pleuropneumoniae genomic DNA from different serotypes was prepared according to Silhavy, et al . , Experiments in Gene Fusions, 1984, p. 89-90. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., which disclosure is hereby incorporated by reference, digested with HindiII and Xbal, electrophoresed through a 0.7% agarose gel, transferred to nitrocellulose membrane and probed with 1,177 bp HindiII-Xbal fragment containing partial AppIIIA gene isolated from λyfc26. The bacteriophage library was screened by hybridization using a probe containing the lktBD genes from P. haemolytica. A DNA fragment from pYFC35 as described by Chang et al . , Secretion of the Pasteurella Leukotoxin by Escherichia coli, 1989, FEMS Microbiol. Lett., vol. 60, pp. 169-174, which disclosure is hereby incorporated by reference, containing the lktBD genes was labeled with [32P] dATP by nick-translation. Filters were hybridized in 45% formamide, 5X SSC (20X SSC containing 175.3 gm of NaCl and 88.2 gm of sodium citrate per liter, pH 7.0) , 5X Denhardt' s solution, and 100 μg/ml sheared calf thymus DNA for 12 hours at 37°C. Filters were then washed twice with 2X SSC-0.1% SDS and twice with 0.2% SSC-0.1% SDS at room temperature as described by Chang et al . , The Actinobacillus pleuropneumoniae Hemolysin Determinant : Unlinked AppCA and AppBD Loci Flanked by Pseudogenes, 1991, J. Bactreiol., vol. 173, pp. 5151-5158, which disclosure is hereby incorporated by reference. The final wash was with 0.16% SSC-0.1% SDS at 37°C. Plaques which gave positive signals were picked, rescreened, and amplified on P2392.
6. DNA Sequencing and Analysis
DNA seguencing was performed by the dideoxy chain-termination method as described by Sanger et al . , DNA Sequencing with Chain-Termination Inhibitors, 1977, Proc. Natl. Acad. Sci., vol. 74, pp. 5463-5467, which disclosure is hereby incorporated by reference. Regions from the A. pleuropneumoniae insert in bacteriophage clone λyfc28 were subcloned into Ml3mpl8 or M13mpl9 and single stranded phage DNA was prepared by standard procedures as described by Messing, J., New 113 Vectors for Cloning, 1983, Methods Enzymol., vol. 101, pp. 20-78, which disclosure is hereby incorporated by reference. The sequencing reactions utilized [35S]dATP, T7 DNA polymerase, and the commercially available SEQUENASE kit (United States Biochemicals, Cleveland) . Certain regions of the DNA insert were sequenced directly from the recombinant bacteriophage. In these cases, 1-2 μg of λyfc28 DNA was mixed with 100 ng of an oligonucleotide primer (prepared by the Analytical and Synthetic Facility, Cornell University) in a total volume of 12 μl, boiled for 5 minutes, and then cooled rapidly on ice. The sequencing reactions were performed with reagents supplied with the SEQUENASE kit (United States Biochemicals, Cleveland) according to the manufacturer's instructions. DNA sequence analysis was performed on a VAX computer using the Genetics Computer Group program package (University of Wisconsin, Madison) and programs from the PC Gene package (Intelligenetics Corp., Mountain View, CA) . The amino acid sequence alignment was carried out with the GAP and LINEUP programs (Genetics Computer Group, University of Wisconsin, Madison) and similarity was calculated according to the method of Pearson et al . , Improved Tools for Biological Sequence Comparison, 1988, Proc. Natl. Acad. Sci., vol. 5, pp. 2444-2448, which disclosure is hereby incorporated as reference.
7. Assay of Cytotoxic and Hemolytic Activity
The cytotoxic and hemolytic activity of A_j_ pleuropneumoniae cytotoxin were assayed as described by
Chang et al . , 1987, Identification and Characterization of Pasteurella haemolytica. Infect. Immun., vol. 55, pp. 2348-2354; and Chang, et al . , Cloning and Characterization of a Hemolysin Gene from Actinobacillus (Haemophilus) pleuropneumoniae, 1989, DNA, vol. 8, pp. 635-647, which disclosures are hereby incorporated by reference .
Example II Cloning the Leukotoxin Gene from A. pleuropneumoniae
To determine how many A. pleuropneumoniae serotypes could produce the 120 kDa leukotoxin, culture supernatants from A. pleuropneumoniae, 12 serotypes, were analyzed by Western blot, using antiserum against the toxin, as shown in Figure 1A. A cross-reacting polypeptide species with molecular weight 120 kDa was evident in serotypes 2, 3, 6 and 8. As a first step towards understanding the role of the leukotoxin from A^_ pleuropneumoniae in porcine pleuropneumonia, a strategy was developed to isolate the gene(s) which encode for this molecule. It was determined that the secretion determinants (BD genes) of the RTX family have extensive
DNA sequence homology. Therefore, the lktBD genes derived from pYFC35 (Chang et al . , Secretion of the Pasteurella leukotoxin by Escherichia coli, 1989, FEMS Microbiol. Lett., vol. 60, pp. 169-174, which disclosure is hereby incorporated by reference) was choosen as a probe to screen an A. pleuropneumoniae genomic library constructed in the phage vector lambda-dash. Five clones were isolated and all of these clones expressed a 120 kDa polypeptide detected by Western blotting with the anti-A. pleuropneumoniae leukotoxin antibody as shown in Figure IB. With reference to Figure 2, recombinant phage DNA were isolated from the positive recombinant clones and analyzed by restriction endonuclease mapping which overlapped with each other. Although the five clones produce the full-length leukotoxin, no cytotoxic activity could be detected in any of the phage lysates (data not shown) .
Example III DNA Sequence of the AppIIICA Genes
A 4 kbp region from λyfc28 was subjected to DNA sequence analysis, the results of which are shown in Figure
2. As in the case of the RTX loci, there is a small open reading frame (ORF) proceeding the toxin reading frame, presumably encoding trie AppIIIC gene (Fig. 2) . The AppIIICA genes are more closely related to the lktCA from P. haemolytica than to other RTX gene family members. With reference to Table 1, there is shown summarizes of the similarities between AppIIICA and the other RTX CA genes for which sequence information is available.
Table 1
AppIIIC AppIIIA gene gene
Homology to the hemolysin locus of A. pleuropneumoniae (hlyl)
Nucleotide sequence3 68.6% 60.6% Amino acid sequence13 75.5% 71.3%
Homology to the hemolysin locus of A. pleuropneumoniae (AppII)
Nucleotide sequence 63.1% 60.4% Amino acid sequence 73.8% 65.5%
Homology to the hemolysin locus of E. coli (hly)
Nucleotide sequence 66.3% 60.5% Amino acid sequence 73.1% 70.1%
Homology to the leukotoxin locus of P. haemolytica (lkt)
Nucleotide sequence 58.1% 61.1% Amino acid sequence 72.5% 66.7%
Homology to the hemolysin locus of A. actinomvcetemcomitans (aalkt)
Nucleotide sequence 68.7% 59.2% Amino acid sequence 75.9? 65.4!
Length in amino acid residues 173 1, 049
Mr (kDa) 20.4 112.5
i 9.75 5.65
a. Percent identical residues. b. Percent identical residues assuming that the following amino acid pairs are equivalent; I and V, S and T, E and D, K and R, F and Y.
The AppIIICA sequence was examined for E. coli promoter like sequences using the homology score method described by Mulligan et al. , Analysis of the Occurrence of Promoter-Sites in DNA, 1984, Nucl. Acid Res., vol 12, pp. 789-800, which disclosure is hereby incorporated by reference. With reference to Figure 3 (Seq. ID No. 1) , there is shown a sequence, TATTAAT, similar to the consensus promoter sequence (-10 region) and one sequence, TTGTAA, similar to the consensus RNA polymerase-binding site proximal to AppIIIC. Upstream of the start codon of AppIIIC, there is a potential ribosome binding site (Fig. 3) . A ribosome binding site and a promoter sequence with consensus -10 and -35 regions lie proximal to the AppIIIA (Fig. 3) . A sequence very similar to the rho independent transcriptional terminator of E. coli downstream from AppIIIA was also observed (Fig. 3) . There are three transmembrane domains at its N-terminus and 14 glycine-rich polypeptide repeats near the C-terminus of AppIIIA as shown in Table 2 below.
Figure imgf000026_0001
Example IV
Southern Blotting Analysis
To demonstrate the distribution of the gene appZzl'A among different serotypes of A. pleuropneumoniae, the 1,177 bp Hindlll-Xbal fragment from λyfc26 was purified, nick translated with [32P]dATP, and used as a hybridization probe on genomic DNA of A. pleuropneumoniae, 12 serotypes, in Southern blots. With reference to Figure 4, the results show that the probe hybridized to a unique fragment in the DNA of A. pleuropneumoniae serotypes 2, 3 , 6 and 8. Weak signals appeared in the serotypes which have Appl and/or AppII genes (Fig. 4) .
Example V
Expression of Leukotoxic Activity in E. coli.
The Sail fragment from λyfc32 (Fig. 2) was subcloned into vector pHG165 as described by Stewart et al . , A pBR322 Copy Number Derivation of pUC8 for Cloning and Expression, 1986, Plasmid, vol. 15, pp. 172-186, which disclosure is hereby incorporated by referencem, yielding plasmid pYFC117. The AppIIICA genes of pYFC117 are likely to be expressed from an its own promoter. The plasmid was transformed into the E. coli host, TB1, and the transformant was grown to early stationary phase and examined for the expression of a 120 kDa polypeptide, and hemolytic and leukocytic activities. With reference to Figure 5, there is shown the expression of the 120 kDa leukotoxin toxin (cytotoxic protein) of the invention. The culture supernatant was toxic to BL-3 cells with 64 toxin units per ml as shown in Table 3. Pig antitoxin sera neutralized the toxic activity of culture supernatants at a dilution of 1:64. No neutralization occurred at any dilution with preimmune pig serum (data not shown) . No hemolytic activity occurred from the culture supernatant (pYFCH7) .
Table 3
Toxin leukotoxic activity
Culture supa 212 Ub pYFC117c 64 U pHG165c 0 U
a. Assay performed with a late log phase supernatant from a culture of A. pleuropneumoniae, serotype 2, grown in brain heart infusion with NAD. b. One unit of toxin activity is defined as the minimal amount of toxin, as determined by serial dilution, equired to produce the morphological change in >95% of the input BL-3 cells. c. Assay performed with early stationary phase supernatant from the E. coli host, TBl, harboring the indicated plasmid grown in LB with ampicillin (50 μg/ml) .
Results
Western blot analysis showed that neutralizing antisera to A. pleuropneumoniae could detect the 105 kDa and 120 kDa proteins secreted by this organism (Fig. 1A) . In order to understand the role of each toxin in the pathogenesis of this disease, a genomic library from A. pleuropneumoniae, ■ serotype 2, was constructed. This library was screened by a DNA probe containing the lktBD genes of Pasteurella haemolytica. A series of 5 overlapping clones (λyfc26, λyfc27, λyfc28, λyfc31 and λyfc32) which produced a 120 kDa polypeptide when expressed in E. coli were identified. Phage lysates from two of these clones, λyfc26 and λyfc27 showed two polypeptides in Western blot analysis (Fig. IB) . The reason for this is unknown, however, it is speculated that the lower band is a degraded product of the 120 kDa polypeptide since these clones have only one copy of the toxin gene (data not shown) .
DNA sequence analysis of the 4 kb region from cloned λyfc28 indicated the presence of two reading frames which we designate AppIIIC and AppIIIA. These genes encode polypeptides of 162 and 1048 amino acids, respectively. In addition, there is a potential third open reading frame in the cloned DNA beginning at position 3752 of the sequenced region (Fig. 3) . We suspect that this represents the amino-terminal coding region of a putative ApplIIB and that a fourth gene, ApplIID, will lie distal to ApplIIB since the toxin was secreted into media by E. coli harboring pYFCH7. Western blots analysis shows that A. pleuropneumoniae, serotype 2,3,6 and 8, secreted a 120 kDa polypeptide (Fig. 1,A) . We have shown that serotype 6 secreted a 120 kDa polypeptide (Fig. 1,A, lane 6) . Southern blots analysis indicated that these four serotypes have a hybridization signal at 1278 bp when a 1.2kb
Hindlll-Xbal fragment which contains part of the AppIIIA gene was used as a probe (Fig. 5) . This result is consistent with that of the Western blots. The predicted AppIIIC and AppIIIA proteins have
74.5% and 65.5% identity with the corresponding AppIIC and AppIIA proteins from A. pleuropneumoniae, serotype 5, hemolysin determinant. The AppIIIA leukotoxin, as is the case with the other RTX toxins, does not have a classic signal sequence at its amino terminus. Instead, the amino terminus is rich in serine, threonine, and lysine (13 of the first 41 residues) and has the capability of forming a positively charged, amphiphilic alpha-helix as do the amino termini of other RTX toxins. There are three transmembrane segments at the N-terminus of AppIIIA (Fig. 3) . This structural feature has been reported to facilitate the interaction of this class of lytic toxins with target membranes.
Alignment of the predicted amino acid sequences for Hlyt, AppA, AppIIIA, and ApplA indicates that the AppIIIA proteins share little homology at their amino termini (data not shown) . Beginning at approximately residue 55, the proteins are all highly similar up to and including the region containing the glycine-rich repeats. However, the number of the glycine-rich repeats from AppIIIA is 14 as shown in Table 2. The glycine-rich repeats have been reported to be responsible for cell-binding. However, AppIIIA, lktA and aalktA, which are potent leukotoxins with no hemolytic activity, contain different glycine-rich repeats as shown in Table 2. The present invention has shown that A. pleuropneumoniae serotypes secrete three different RTX toxin. It is speculated that the AppII and ApplII toxins are required for pathogenicity or immunogenicity in pigs against A. pleuropneumoniae, serotype 2.
The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Chang, Yung-Fu
(ii) TITLE OF INVENTION: Recombinant Vaccine For Procine
Pleuropneumoniae
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Alan S. Korman
(B) STREET: 1600 Empire Tower
(C) CITY: Buffalo
(D) STATE: New York (E) COUNTRY : U.S.A.
(F) ZIP: 14202
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US UNKNOWN
(B) FILING DATE:
(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/972,229
(B) FILING DATE: 05-NOV-1992
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Korman, Alan S.
(B) REGISTRATION NUMBER: 33,932
(C) REFERENCE/DOCKET NUMBER: 19603/00001
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 716-853-8104
(B) TELEFAX: 716-853-8109
(2) INFORMATION FOR SEQ ID NO: 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3828 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Actinobacillus - pleuropneumonia
(B) STRAIN: Serotypes 2, 3, 4, 6 and 8
(C) INDIVIDUAL ISOLATE: swine (G) CELL TYPE: gram negative bacterium
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: A. pleuropneumoniae DNA in Lambda - dash
(B) CLONE: (Lambda) yfc 26-28 and (Lambda) yfc 31-32
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :
ACGGTTCTTA AAGTGGATAA ATAATAAAAT TATGAGTTAT AAAAATGTTA AAAATTTAAC 60
AGATGATTTT ACAACTTTAG GGCATATCGC TTGGTTGTGG GCTAATTCTC
CGTTACATAA
120
GGAGTGGTCT ATCTCTTTGT TTACTAAGAA TATTTTGCCA GCCATTCAAC ATGATCAATA 180
TATTTTACTT ATGCGAGATG AGTTCCCTGT AGCGTTTTGT AGTTGGGCAA ATTTAACGTT 240
AACTAATGAA GTGAAGTATG TACGTGATGT GACGTCATTG ACTTTTGAAG ATTGGAATTC 300
AGGAGAACGA AAATGGTTGA TCGATTGGAT TGCGCCATTT GGGGATAACA
ATACGCTTTA
360 TAGATATATG CGTAAAAAAT TTCCTAATGA AGTATTCCGG GCCATTCGAG TATATCCTGG 420
TTCTACAGAA GCGAAAATCA TTCATGTTCA AGGAGGACAA ATTAATAAAT TTACAGCTAA 480
AAAATTAATA CAACAATATC AGGAAGAACT TATTCAAGTT CTTAACAATC ACAAAAAAAT 540
TGTAAGAGGA TAAAATATGA GTACTTGGTC AAGCATGTTA GCCGACTTAA AAAAAAGGGC 600
TGAAGAAGCC AAAAGACAAG TTAAAAAAGG CTACGATGTA ACTAAAAATG GTTTGCAATA 660
TGGGGTGAGT CAAGCAAAAT TACAAGCATT AGCAGCTGGT AAAGCCGTTC AAAAGTACGG 720
TAATAAATTA GTTTTAGTTA TTCCAAAAGA GTATGACGGA AGTGTTGGTA ACGGTTTCTT 780
TGATTTAGTA AAAGCAGCTG AGGAATTAGG CATTCAAGTT AAATATGTTA ACCGTAATGA 840
ATTGGAAGTT GCCCATAAAA GTTTAGGTAC CGCAGACCAA TTCTTGGGTT
TAACAGAACG
900 TGGACTTACT TTATTTGCAC CGCAACTAGA TCAGTTCTTA CAAAAACATT
CAAAAATTTC
960
TAACGTAGTG GGCAGTTCTA CTGGTGATGC AGTAAGTAAA CTTGCTAAGA GTCAAACTAT 1020
TATTTCAGGA ATTCAATCTG TATTAGGTAC TGTATTAGCA GGTATTAATC TTAATGAAGC 1080
TATTATTAGT GGCGGTTCAG AGCTCGAATT AGCTGAAGCT GGTGTTTCTT TAGCCTCTGA 1140
GCTCGTTAGC AATATTGCTA AAGGTACAAC AACAATAGAT GCTTTCACTA CACAAATCCA 1200
GAACTTTGGG AAATTAGCGG AAAATGCTAA AGGGTTAGGT GGTGTTGGCC
GCCAATTACA
1260
GAATATTTCA GGTTCTGCAT TAAGCAAAAC TGGATTAGGT TTGGATATTA TCTCAAGCTT 1320
ACTTTCAGGA GTAACTCGAA GTTTTGCTTT ACGGAATAAG AATGCTTCAA CAAGCACTAA 1380 AGTTGCTGCT GGCTTTGAAC TCTCAAATCA AGTAATTGGT GGTATTACGA
AAGCAGTATC
1440
AAGCTATATT CTTGCACAGC GTTTACGTGC TGGTTTATCA ACGACAGGTC CTGCTGCAGC 1500
ACTAATTGCG TCTAGTATTT CTTTAGCAAT CAGTCCATTG GCGTTTTTAC GTGTAGCTGA 1560
TAATTTTAAT CGTTCTAAAG AAATTGGCGA ATTTGCTGAA CGTTTCAAAA AATTGGGCTA 1620
TGACGGCGAT AAACTACTTT CAGAGTTTTA TCACGAAGCT GGTACTATTG ATGCCTCAAT 1680
TACTACAATT AGTACAGCAC TTTCTGCTAT CGCAGCTGGA ACGGCCGCCG
CGAGTGCAGG
1740
TGCATTAGTT GGCGCACCAA TTACTTTGTT GGTTACTGGT ATCACAGGAT TAATTTCTGG 1800
TATTTTAGAG TTCTCTAAAC AACCAATGTT AGATCATGTT GCATCGAAAA TTGGTAACAA 1860
AATTGACGAA TGGGAGAAAA AATACGGTAA AAATTACTTC GAGAATGGCT ATGATGCTCG 1920
TCATAAAGCT TTCTTAGAAG ATTCATTCTC ATTATTGTCT AGTTTTAATA
AACAATATGA
1980
AACTGAAAGA GCTGTTTTAA TTACACAACA ACGTTGGGAT GAATATATTG
GCGAACTTGC
2040
GGGTATTACT GGTAAAGGTG ACAAACTCTC TAGTGGTAAG GCGTATGTAG
ATTACTTTCA
2100
AGAAGGTAAA TTATTAGAGA AAAAACCTGA TGACTTTAGC AAAGTAGTTT TCGATCCAAC 2160
TAAGGGCGAA ATTGATATTT CAAATAGCCA AACGTCAACG TTGTTAAAAT TTGTTACGCC 2220
ATTATTAACA CCAGGTACAG AGTCACGTGA AAGAACTCAA ACAGGTAAAT ATGAATATAT 2280
CACGAAGTTA GTTGTAAAAG GTAAAGATAA ATGGGTTGTT AATGGCGTTA
AAGATAAAGG
2340
TGCCGTTTAT GATTATACTA ATTTAATTCA ACATGCTCAT ATTAGTTCAT
CAGTAGCACG
2400 TGGTGAAGAA TACCGTGAAG TTCGTTTGGT ATCTCATCTA GGCAATGGTA
ATGACAAAGT
2460
GTTCTTAGCT GCGGGTTCCG CAGAAATTCA CGCTGGTGAA GGTCATGATG TGGTTTATTA 2520
TGATAAAACC GATACAGGTC TTTTAGTAAT TGATGGAACC AAAGCGACTG AACAAGGGCG 2580
TTATTCTGTT ACGCGCGAAT TGAGTGGTGC TACAAAAATC CTGAGAGAAG TAATAAAAAA 2640
TCAAAAATAT GCTGTTGGTA AACGTGAAGA AACCTTGGAA TATCGTGATT ATGAATTAAC 2700
GCAATCAGGT AATAGTAACC TAAAAGCACA TGATGAATTA CATTCAGTAG
AAGAAATTGG
2760
AAGTAATCAG AGAGACGAAT TTAAAGGTAG TAAATTCAGA GATATTTTCC ATGGTGCCGA 2820
TGGTGATGAT CTATTAAATG GTAATGATGG GGATGATATT CTATACGGTG ATAAAGGTAA 2880 CGATGAGTTA AGAGGTGATA ACGGTAACGA CCAACTTTAT GGTGGTGAAG
GTGATGACAA
2940
ACTATTAGGA GGTAATGGCA ATAATTACCT CAGTGGTGGT GATGGCAATG ATGAGCTTCA 3000
AGTATTAGGC AATGGTTTTA ATGTGCTTCG TGGCGGTAAA GGCGATGATA AACTTTATGG 3060
TAGCTCAGGT TCTGATTTAC TTGATGGTGG AGAAGGTAAT GATTATCTAG AAGGAGGCGA 3120
TGGTAGCGAT TTTTATGTTT ATCGTTCCAC TTCAGGTAAT CATACTATTT ATGATCAAGG 3180
TAAAGCTAGC GATTCAGATA AGCTATATTT GTCAGATCTT TCTTTTGATA
ATATTTTAGT
3240
TAAAAGGGTT AACGATAACC TTGAGTTTAG AAGCAATAAT AACAGTAATA GTGGTGTGCT 3300
TACGATCAAG GACTGGTTCA AAGGCGGCAA TAGTTACAAT CATAAAATTG AACAAATTGT 3360 TGATAAAAAT GGTAGAAAAT TGACAGCTGG GAATTTAGGA AATAACTTCC
ATGATACTCA
3420
ACAAGCTAGT AGTTTACTTA AAAATGTTAC ACAAGAACAA AATGAAAGCA ATTTATCTTC 3480
ACTTAAAACT GAATTAGGTA AAATTATTAC TAATGCAGGT AATTTTGGTG TGGCAAAACA 3540
AGGTAATACT GGAATCAATA CAGCTGCCTT GAACAATGAA GTGAATAAAA TCATTTCTTC 3600
TGCTAATACC TTTGCTACTT CACAATTGGG TGGCTCAGGG ATGGGAACAT TACCATCAAC 3660
GAATGTAAAT TCAATGATGC TAGGTAACCT AGCTAGAGCA GCTTAATCAT
CTGCAATAAT
3720
CAATAGCAAT CCTATGGTTA TTCTAGGATT GCTATTTTAT TTATGGAGTC ACAAATGCCT 3780
TTTAACGAAA AAATAGATTA CGGATTACAT GCATTGGTAA TTCTCGCG
3828 (2) INFORMATION FOR SEQ ID NO:2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1244 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Actinobacillus pleuropneumoniae
(B) STRAIN: Serotypes 2, 3, 4, 6 and 8 (C) INDIVIDUAL ISOLATE: Swine
(G) CELL TYPE: Gram negative bacterium
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :
Met Ser Tyr Lys Asn Val Lys Asn Leu Thr Asp Asp Phe Thr Thr Leu
1 5 10
15
Gly His lie Ala Trp Leu Trp Ala Asn Ser Pro Leu His Lys Glu Trp
20 25 30 Ser lie Ser Leu Phe Thr Lys Asn lie Leu Pro Ala lie
Gin His Asp
35 40 45
Gin Tyr lie Leu Leu Met Arg Asp Glu Phe Pro Val Ala Phe Cys Ser
50 55 60
Trp Ala Asn Leu Thr Leu Thr Asn Glu Val Lys Tyr Val Arg Asp Val
65 70 75
80
Thr Ser Leu Thr Phe Glu Asp Trp Asn Ser Gly Glu Arg Lys Trp Leu
85 90
95
lie Asp Trp lie Ala Pro Phe Gly Asp Asn Asn Thr Leu Tyr Arg Tyr
100 105
110
Met Arg Lys Lys Phe Pro Asn Glu Val Phe Arg Ala lie Arg Val Tyr
115 120 125
Pro Gly Ser Thr Glu Ala Lys lie lie His Val Gin Gly Gly Gin He
130 135 140 Asn Lys Phe Thr Ala Lys Lys Leu He Gin Gin Tyr Gin
Glu Glu Leu
145 150 155
160
He Gin Val Leu Asn Asn His Lys Lys He Val Arg Gly Met Ser Thr
165 170
175
Trp Ser Ser Met Leu Ala Asp Leu Lys Lys Arg Ala Glu Glu Ala Lys
180 185
190
Arg Gin Val Lys Lys Gly Tyr Asp Val Thr Lys Asn Gly Leu Gin Tyr
195 200 205
Gly Val Ser Gin Ala Lys Leu Gin Ala Leu Ala Ala Gly Lys Ala Val
210 215 220
Gin Lys Tyr Gly Asn Lys Leu Val Leu Val He Pro Lys Glu Tyr Asp
225 230 235
240
Gly Ser Val Gly Asn Gly Phe Phe Asp Leu Val Lys Ala Ala Glu Glu
245 250
255 Leu Gly He Gin Val Lys Tyr Val Asn Arg Asn Glu Leu
Glu Val Ala
260 265
270
His Lys Ser Leu Gly Thr Ala Asp Gin Phe Leu Gly Leu
Thr Glu Arg
275 280 285
Gly Leu Thr Leu Phe Ala Pro Gin Leu Asp Gin Phe Leu Gin Lys His
290 295 300
Ser Lys He Ser Asn Val Val Gly Ser Ser Thr Gly Asp Ala Val Ser
305 310 315
320
Lys Leu Ala Lys Ser Gin Thr He He Ser Gly He Gin Ser Val Leu
325 330
335
Gly Thr Val Leu Ala Gly He Asn Leu Asn Glu Ala He He Ser Gly
340 345
350
Gly Ser Glu Leu Glu Leu Ala Glu Ala Gly Val Ser Leu Ala Ser Glu
355 360 365 Leu Val Ser Asn He Ala Lys Gly Thr Thr Thr He Asp
Ala Phe Thr
370 375 380
Thr Gin He Gin Asn Phe Gly Lys Leu Ala Glu Asn Ala Lys Gly Leu
385 390 395
400
Gly Gly Val Gly Arg Gin Leu Gin Asn He Ser Gly Ser Ala Leu Ser
405 410
415
Lys Thr Gly Leu Gly Leu Asp He He Ser Ser Leu Leu Ser Gly Val
420 425
430
Thr Arg Ser Phe Ala Leu Arg Asn Lys Asn Ala Ser Thr Ser Thr Lys
435 440 445
Val Ala Ala Gly Phe Glu Leu Ser Asn Gin Val He Gly Gly He Thr
450 455 460
Lys Ala Val Ser Ser Tyr He Leu Ala Gin Arg Leu Arg Ala Gly Leu
465 470 475
480 Ser Thr Thr Gly Pro Ala Ala Ala Leu He Ala Ser Ser
He Ser Leu
485 490
495
Ala He Ser Pro Leu Ala Phe Leu Arg Val Ala Asp Asn
Phe Asn Arg
500 505
510
Ser Lys Glu He Gly Glu Phe Ala Glu Arg Phe Lys Lys
Leu Gly Tyr
515 520 525
Asp Gly Asp Lys Leu Leu Ser Glu Phe Tyr His Glu Ala Gly Thr He
530 535 540
Asp Ala Ser He Thr Thr He Ser Thr Ala Leu Ser Ala He Ala Ala
545 550 555
560
Gly Thr Ala Ala Ala Ser Ala Gly Ala Leu Val Gly Ala Pro He Thr
565 570
575
Leu Leu Val Thr Gly He Thr Gly Leu He Ser Gly He Leu Glu Phe
580 585
590 Ser Lys Gin Pro Met Leu Asp His Val Ala Ser Lys He
Gly Asn Lys
595 600 605
He Asp Glu Trp Glu Lys Lys Tyr Gly Lys Asn Tyr Phe Glu Asn Gly
610 615 620
Tyr Asp Ala Arg His Lys Ala Phe Leu Glu Asp Ser Phe Ser Leu Leu
625 630 635
640
Ser Ser Phe Asn Lys Gin Tyr Glu Thr Glu Arg Ala Val Leu He Thr
645 650
655
Gin Gin Arg Trp Asp Glu Tyr He Gly Glu Leu Ala Gly He Thr Gly
660 665
670
Lys Gly Asp Lys Leu Ser Ser Gly Lys Ala Tyr Val Asp Tyr Phe Gin
675 680 685
Glu Gly Lys Leu Leu Glu Lys Lys Pro Asp Asp Phe Ser Lys Val Val
690 695 700 Phe Asp Pro Thr Lys Gly Glu He Asp He Ser Asn Ser
Gin Thr Ser
705 710 715
720
Thr Leu Leu Lys Phe Val Thr Pro Leu Leu Thr Pro Gly
Thr Glu Ser
725 730
735
Arg Glu Arg Thr Gin Thr Gly Lys Tyr Glu Tyr He Thr
Lys Leu Val
740 745
750
Val Lys Gly Lys Asp Lys Trp Val Val Asn Gly Val Lys
Asp Lys Gly
755 760 765
Ala Val Tyr Asp Tyr Thr Asn Leu He Gin His Ala His He Ser Ser
770 775 780
Ser Val Ala Arg Gly Glu Glu Tyr Arg Glu Val Arg Leu Val Ser His
785 790 795
800
Leu Gly Asn Gly Asn Asp Lys Val Phe Leu Ala Ala Gly Ser Ala Glu
805 810
815 He His Ala Gly Glu Gly His Asp Val Val Tyr Tyr Asp
Lys Thr Asp
820 825
830
Thr Gly Leu Leu Val He Asp Gly Thr Lys Ala Thr Glu Gin Gly Arg
835 840 845
Tyr Ser Val Thr Arg Glu Leu Ser Gly Ala Thr Lys He Leu Arg Glu
850 855 860
Val He Lys Asn Gin Lys Tyr Ala Val Gly Lys Arg Glu Glu Thr Leu
865 870 875
880
Glu Tyr Arg Asp Tyr Glu Leu Thr Gin Ser Gly Asn Ser Asn Leu Lys
885 890
895
Ala His Asp Glu Leu His Ser Val Glu Glu He Gly Ser Asn Gin Arg
900 905
910
Asp Glu Phe Lys Gly Ser Lys Phe Arg Asp He Phe His Gly Ala Asp
915 920 925 Gly Asp Asp Leu Leu Asn Gly Asn Asp Gly Asp Asp He
Leu Tyr Gly
930 935 940
Asp Lys Gly Asn Asp Glu Leu Arg Gly Asp Asn Gly Asn Asp Gin Leu
945 950 955
960
Tyr Gly Gly Glu Gly Asp Asp Lys Leu Leu Gly Gly Asn Gly Asn Asn
965 970
975
Tyr Leu Ser Gly Gly Asp Gly Asn Asp Glu Leu Gin Val Leu Gly Asn
980 985
990
Gly Phe Asn Val Leu Arg Gly Gly Lys Gly Asp Asp Lys Leu Tyr Gly
995 1000 1005
Ser Ser Gly Ser Asp Leu Leu Asp Gly Gly Glu Gly Asn Asp Tyr Leu
1010 1015 1020
Glu Gly Gly Asp Gly Ser Asp Phe Tyr Val Tyr Arg Ser Thr Ser Gly
1025 1030 1035
1040 Asn His Thr He Tyr Asp Gin Gly Lys Ala Ser Asp Ser
Asp Lys Leu
1045 1050
1055
Tyr Leu Ser Asp Leu Ser Phe Asp Asn He Leu Val Lys Arg Val Asn
1060 1065
1070
Asp Asn Leu Glu Phe Arg Ser Asn Asn Asn Ser Asn Ser Gly Val Leu
1075 1080 1085
Thr He Lys Asp Trp Phe Lys Gly Gly Asn Ser Tyr Asn His Lys He
1090 1095 1100
Glu Gin He Val Asp Lys Asn Gly Arg Lys Leu Thr Ala Gly Asn Leu
1105 1110 1115
1120
Gly Asn Asn Phe His Asp Thr Gin Gin Ala Ser Ser Leu Leu Lys Asn
1125 1130
1135
Val Thr Gin Glu Gin Asn Glu Ser Asn Leu Ser Ser Leu Lys Thr Glu
1140 1145
1150 Leu Gly Lys He He Thr Asn Ala Gly Asn Phe Gly Val
Ala Lys Gin
1155 1160 1165
Gly Asn Thr Gly He Asn Thr Ala Ala Leu Asn Asn Glu Val Asn Lys
1170 1175 1180
He He Ser Ser Ala Asn Thr Phe Ala Thr Ser Gin Leu Gly Gly Ser
1185 1190 1195
1200
Gly Met Gly Thr Leu Pro Ser Thr Asn Val Asn Ser Met Met Leu Gly
1205 1210
1215
Asn Leu Ala Arg Ala Ala Met Glu Ser Gin Met Pro Phe Asn Glu Lys
1220 1225
1230
He Asp Tyr Gly Leu His Ala Leu Val He Leu Ala 1235 1240

Claims

We claim :
1. A DNA molecule encoding a leukotoxin secreted from Actinobacillus pleuropneumonia, serotype 2, said leukotoxin having an approximate molecular size of 120 kilodaltons .
2. A DNA molecule according to claim 1, wherein said DNA molecule encodes for the AppIIIA amino acid sequence, or a mutant thereof, as shown in Figure 3.
3. A DNA molecule according to claim 1, wherein said DNA molecule encodes for the AppIIIC amino acid sequence, or a mutant thereof, as shown in Figure 3.
4. A DNA molecule according to claim 1, wherein said DNA molecule encodes for an amino acid sequence as shown in Figure 3.
5. A DNA molecule according to claim 1, wherein said DNA molecule has the AppIIIA nucleotide sequence shown in Figure 3, or a mutant DNA sequence thereof.
6. A DNA molecule according to claim 1, wherein said DNA molecule has the AppIIIC nucleotide sequence shown in Figure 3 , or a mutant DNA sequence thereof.
7. A DNA molecule according to claim 1, wherein said DNA molecule has the nucleotide sequence shown in Figure 3 , or a mutant DNA sequence thereof .
8. An isolated recombinant DNA vector containing a DNA sequence or a mutant DNA sequence thereof, encoding aleukotoxin secreted from Actinobacillus pleuropneumonia, serotype 2, said leukotoxin having an approximate molecular size of 120 kilodaltons.
9. A DNA vector according to claim 8, wherein said DNA sequence is inserted into said vector in proper orientation and correct reading frame.
10. A DNA vector according to claim 8, wherein said DNA sequence is operatively linked to a promotor sequence.
11. A DNA vector according to claim 8, wherein said
DNA sequence comprises the DNA according to claims 1, 2, 3, 4, 5, 6 and 7.
12. A DNA vector according to claim 11, wherein said vector is a plasmid.
13. A DNA vector according to claim 12, wherein said plasmid is pYFC117.
14. A cell transformed with the DNA vector of claim 8, 9, 10, 11, 12 and 13.
15. A cell according to claim 14, wherein said cell is a bacteria cell.
16. A cell according to claim 15, wherein said bactereia cell is E. coli.
17. A cell according to claim 16, wherein said E^ coli isolate is E. coli TBl, mutants, recombinants and genetically engieered derivitives thereof.
18. A vaccine formulation comprising a biologically active amount of the leukotoxin produced according to claims 1 , 8 and 14 .
19. A method of protecting a pig against porcine pleuropneumonia comprising administering to the pig an immunologically effective amount of the vaccine formulation of claim 18.
20. An expression system for leukotoxin secreted from A. pleuropneumonia, which comprises the recombinant DNA vector of claim 8, wherein the DNA is operably linked to a control sequence.
21. A monoclonal antibody which recognizes an antigenic determinant on the leukotoxin produced according to claims 1, 8, 14, 18, and 20.
22. A hybridoma which produces the monoclonal antibody of claim 21.
23. A method of producing the 120 kDa leukotoxin secreted from A. pleuropneumonia, serotypes, 2, 3, 4, 6 and
8, comprising: providing a recombinant cell according to claim
14; culturing the cells as a culture mixture under conditions which allow the cell to produce the leukotoxin; and isolating the leukotoxin from the culture mixture.
PCT/US1993/010500 1992-11-05 1993-11-04 Recombinant vaccine for porcine pleuropneumoniae WO1994009821A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US97222992A 1992-11-05 1992-11-05
US07/972,229 1992-11-05
US7228593A 1993-06-03 1993-06-03
US08/072,285 1993-06-03

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CN113980101A (en) * 2021-09-11 2022-01-28 江苏南农高科技股份有限公司 Actinobacillus pleuropneumoniae subunit vaccine

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Publication number Priority date Publication date Assignee Title
US5925354A (en) * 1995-11-30 1999-07-20 Michigan State University Riboflavin mutants as vaccines against Actinobacillus pleuropneumoniae
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CN113980101A (en) * 2021-09-11 2022-01-28 江苏南农高科技股份有限公司 Actinobacillus pleuropneumoniae subunit vaccine
CN113980101B (en) * 2021-09-11 2023-06-30 江苏南农高科技股份有限公司 Actinobacillus pleuropneumoniae subunit vaccine

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