WO1994010316A1 - Actinobacillus pleuropneumoniae outer membrane lipoprotein a and uses thereof - Google Patents

Actinobacillus pleuropneumoniae outer membrane lipoprotein a and uses thereof Download PDF

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Publication number
WO1994010316A1
WO1994010316A1 PCT/CA1993/000448 CA9300448W WO9410316A1 WO 1994010316 A1 WO1994010316 A1 WO 1994010316A1 CA 9300448 W CA9300448 W CA 9300448W WO 9410316 A1 WO9410316 A1 WO 9410316A1
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Prior art keywords
protein
pleuropneumoniae
dna
outer membrane
actinobacilluε
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WO1994010316B1 (en
Inventor
Gerald F. Gerlach
Philip J. Willson
Amalia Rossi-Campos
Andrew A. Potter
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University of Saskatchewan
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University of Saskatchewan
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Priority to EP93923430A priority Critical patent/EP0669984B1/en
Priority to DE69333365T priority patent/DE69333365T2/en
Priority to CA002148369A priority patent/CA2148369C/en
Priority to DK93923430T priority patent/DK0669984T3/en
Publication of WO1994010316A1 publication Critical patent/WO1994010316A1/en
Publication of WO1994010316B1 publication Critical patent/WO1994010316B1/en
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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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S424/00—Drug, bio-affecting and body treating compositions
    • Y10S424/825—Bacterial vaccine for porcine species, e.g. swine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/851—Haemophilus

Definitions

  • the instant invention relates generally to the prevention of disease in swine. More particularly, the present invention relates to subunit vaccines for Actinobacillus pleuropneumoniae .
  • Actinobacillus (formerly Haemophilus) pleuropneumoniae is a highly infectious porcine respiratory tract pathogen that causes porcine pleuropneumonia. Infected animals develop acute fibrinous pneumonia which leads to death or chronic lung lesions and reduced growth rates. Infection is transmitted by contact or-aerosol and the morbidity in susceptible groups can approach 100%. Persistence of the pathogen in clinically healthy pigs also poses a constant threat of transmitting disease to previously uninfected herds.
  • LppA The nucleotide sequence for this lipoprotein, termed "LppA,” has been determined
  • the active lipoprotein portion of the protein has been shown to reside in the N-terminal fatty acid containing region of the protein. Recent studie ⁇ using synthetic lipopeptides based on this protein show that even short peptides, containing two to five amino acids covalently linked to palmitate, are able to activate murine lymphocytes (Bessler, W.G., et al . , J. Immunol . (1985) 135:1900-1905).
  • A. pleuropneumoniae possesses several outer membrane proteins which are expressed only under iron limiting growth conditions (Deneer, H.G., and Potter, A.A., Infect . Immun . (1989) 57:798-804).
  • outer membrane lipoproteins from A . pleuropneumoniae have not heretofore been identified or characterized with respect to their immunogenic or protective capacity.
  • the present invention is based on the discovery of a novel subunit antigen from A. pleuropneumoniae which shows protective capability in pigs.
  • the subject invention is directed to purified, immunogenic A. pleuropneumoniae outer membrane lipoprotein A, or an immunogenic fragment thereof.
  • the instant invention is directed to an isolated nucleotide sequence encoding an immunogenic A. pleuropneumoniae outer membrane lipoprotein A, or an immunogenic fragment thereof.
  • the subject invention is directed to a DNA construct comprising the isolated nucleotide sequence described above and control sequences that are operably linked to the nucleotide sequence whereby the coding sequence can be transcribed and translated in a host cell, and at least one of the control sequences is heterologous to the coding sequence.
  • the instant invention is directed to host cells transformed with these constructs and methods of recombinantly producing the subject A. pleuropneumoniae proteins.
  • the subject invention is directed to a vaccine composition comprising a pharmaceutically acceptable vehicle and an A. pleuropneumoniae outer membrane lipoprotein A or an immunogenic fragment thereof.
  • the invention is directed to a method of treating or preventing an A. pleuropneumoniae infection in a vertebrate subject comprising administering to the subject a therapeutically effective amount of a vaccine composition as described above.
  • FIG. 1 depicts the nucleotide sequence
  • FIG. 2 depicts the nucleotide sequence
  • OxmlA are equivalent and interchangeable and define a protein from the family of proteins represented by A. pleuropneumoniae serotype 1 OmlA (depicted in Figure 1) and A . pleuropneumoniae ⁇ erotype 5 OmlA (depicted in Figure 2) .
  • the term “OmlA” also captures proteins substantially homologous and functionally equivalent to native OmlAs. Thus, the term encompasses modifications, such as deletions, additions and substitutions (generally conservative in nature) , to the native sequences, as long as immunological activity (as defined below) is not de ⁇ troyed. Such modification ⁇ of the primary amino acid sequence may result in antigens which have enhanced activity as compared to the native sequence.
  • the ⁇ e modification ⁇ may be deliberate, as through site-directed utagene ⁇ i ⁇ , or may be accidental, such as through mutations of host ⁇ which produce the lipoprotein. All of the ⁇ e modification ⁇ are included, ⁇ o long a ⁇ immunogenic activity i ⁇ retained. Accordingly, A. pleuropneumoniae ⁇ erotype 1 OmlA and A. pleuropneumoniae ⁇ erotype 5 OmlA refer not only to the amino acid ⁇ equence ⁇ depicted in
  • OxylA (or fragment ⁇ thereof) denote ⁇ a protein which occurs in neutral form or in the form of basic or acid addition salts, depending on the mode of preparation.
  • acid salt ⁇ may involve free amino groups and basic ⁇ alt ⁇ may be formed with free carboxyls.
  • Pharmaceutically acceptable basic and acid addition salts are discussed further below.
  • the protein may be modified by combination with other biological material ⁇ ⁇ uch a ⁇ lipids (either those normally as ⁇ ociated with the lipoprotein or other lipids that do not de ⁇ troy activity) and ⁇ accharide ⁇ , or by side chain modification, such as acetylation of amino groups, phosphorylation of hydroxyl ⁇ ide chain ⁇ , or oxidation of ⁇ ulfhydryl groups, as well as other modifications of the encoded primary sequence.
  • side chain modification such as acetylation of amino groups, phosphorylation of hydroxyl ⁇ ide chain ⁇ , or oxidation of ⁇ ulfhydryl groups, as well as other modifications of the encoded primary sequence.
  • OmlA included within the definition of "OmlA” herein are glycosylated and unglycosylated forms, the amino acid sequences with or without associated lipids, and amino acid sequences substantially homologous to the native sequence which retain the ability to elicit an immune response.
  • Two DNA or polypeptide sequence ⁇ are 'Substantially homologous" when at least about 65% (preferably at least about 80% to 90%, and most preferably at least about 95%) of the nucleotides or amino acid ⁇ match over a defined length of the molecule.
  • a ⁇ u ⁇ ed herein, ⁇ ub ⁇ tantially homologou ⁇ al ⁇ o refer ⁇ to ⁇ equence ⁇ ⁇ howing identity to the ⁇ pecified DNA or polypeptide ⁇ equence.
  • DNA sequence ⁇ that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular sy ⁇ tem. Defining appropriate hybridization condition ⁇ i ⁇ within the skill of the art. See, e.g., Sambrook et al . , supra ; DNA Cloning, vols I & II, supra ; Nucleic Acid Hybridization, supra .
  • the term “functionally equivalent” intends that the amino acid sequence of the subject protein i ⁇ one that will elicit an immunological re ⁇ pon ⁇ e, a ⁇ defined below, equivalent to or better than, the immunological re ⁇ pon ⁇ e elicited by a native A. pleuropneumoniae OmlA.
  • An "antigen” refers to a molecule containing one or more epitopes that will stimulate a host' ⁇ immune system to make a humoral and/or cellular antigen-specific response. The term is also used interchangeably with "immunogen.”
  • subunit antigen an antigen entity separate and discrete from a whole bacterium (live or killed) .
  • an antigen contained in a cell free extract would constitute a “subunit antigen” as would a substantially purified antigen.
  • a "hapten” is a molecule containing one or more epitopes that does not stimulate a ho ⁇ t's immune ⁇ y ⁇ tem to make a humoral or cellular response unles ⁇ linked to a carrier.
  • epitope refers to the site on an antigen or hapten to which a specific antibody molecule bind ⁇ .
  • an "immunological response" to an antigen or vaccine is the development in the host of a cellular and/ or antibody-mediated immune response to the composition or vaccine of intere ⁇ t.
  • a re ⁇ pon ⁇ e includes but is not limited to one or more of the following effects; the production of antibodies, B cells, helper T cells, ⁇ uppre ⁇ sor T cell ⁇ , and/or cytotoxic T cell ⁇ and/or y ⁇ T cell ⁇ , directed specifically to an antigen or antigens included in the composition or vaccine of intere ⁇ t.
  • immunogenic polypeptide and “immunogenic amino acid sequence” refer to a polypeptide or amino acid sequence, respectively, which elicit antibodie ⁇ that neutralize bacterial infectivity, and/or mediate antibody-complement or antibody dependent cell cytotoxicity to provide protection of an immunized ho ⁇ t.
  • immunogenic fragment i ⁇ meant a fragment of an A.
  • pleuropneumoniae OmlA which includes one or more epitopes and thus elicits antibodies that neutralize bacterial infectivity, and/or mediate antibody-complement or antibody dependent cell cytotoxicity to provide protection of an immunized host.
  • Such fragments will usually be at least about 5 amino acids in length, and preferably at least about 10 to 15 amino acids in length. There is no critical upper limit to the length of the fragment, which could comprise nearly the full length of the protein sequence, or even a fusion protein comprising fragments of two or more of the A. pleuropneumoniae subunit antigens.
  • polypeptide and protein are u ⁇ ed interchangeably and refer to any polymer of amino acid ⁇ (dipeptide or greater) linked through peptide bond ⁇ . Thu ⁇ , the term ⁇ “polypeptide” and “protein” include oligopeptide ⁇ , protein fragment ⁇ , analog ⁇ , mutein ⁇ , fu ⁇ ion proteins and the like.
  • Native proteins or polypeptides refer to protein ⁇ or polypeptide ⁇ recovered from a source occurring in nature. Thu ⁇ , the term “native outer membrane lipoprotein A” would include naturally occurring OmlA and fragment ⁇ of the ⁇ e protein ⁇ .
  • purified protein i ⁇ meant a protein ⁇ eparate and discrete from a whole organism (live or killed) with which the protein i ⁇ normally a ⁇ ociated in nature. Thu ⁇ , a protein contained in a cell free extract would con ⁇ titute a "purified protein," a ⁇ would a protein ⁇ ynthetically or recombinantly produced.
  • Recombinant polypeptide ⁇ refer to polypeptide ⁇ produced by recombinant DNA technique ⁇ ; i.e., produced from cell ⁇ tran ⁇ for ed by an exogenou ⁇ DNA • con ⁇ truct encoding the desired polypeptide.
  • Synthetic polypeptide ⁇ are those prepared by chemical ⁇ ynthe ⁇ i ⁇ .
  • a "replicon” i ⁇ any genetic element (e . g. , plasmid, chromosome, virus) that functions a ⁇ an autonomous unit of DNA replication in vivo ; i . e . , capable of replication under its own control.
  • a "vector" i ⁇ a replicon, ⁇ uch as a plasmid, phage, or co ⁇ mid, to which another DNA ⁇ egment may be at ⁇ tached ⁇ o a ⁇ to bring about the replication of the at- tached ⁇ egment.
  • double- ⁇ tranded DNA molecule refer ⁇ to the polymeric form of deoxyribonucleotide ⁇ (ba ⁇ e ⁇ adenine, guanine, thymine, or cytosine) in a double-stranded helix, both relaxed and supercoiled. This term refer ⁇ only to the primary and ⁇ econdary ⁇ tructure of the molecule, and doe ⁇ not limit it to any particular tertiary form ⁇ . Thu ⁇ , thi ⁇ term include ⁇ double- ⁇ tranded DNA found, inter alia , in linear DNA molecule ⁇ (e .g. , restriction fragments) , viruse ⁇ , pla ⁇ mids, and chromosome ⁇ .
  • linear DNA molecule ⁇ e .g. , restriction fragments
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having the ⁇ equence ho ologou ⁇ to the mRNA) .
  • a DNA "coding ⁇ equence” or a "nucleotide sequence encoding" a particular protein is a DNA sequence which is transcribed and translated into a polypeptide in vivo or in vitro when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a ⁇ tart codon at the 5' (amino) terminu ⁇ and a tran ⁇ lation stop codon at the 3' (carboxy) terminus.
  • a coding ⁇ equence can include, but i ⁇ not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic ⁇ (e .g. , mammalian) DNA, and even synthetic DNA sequences.
  • a transcription termination ⁇ equence will usually be located 3' to the coding ⁇ equence.
  • a "promoter ⁇ equence” i ⁇ a DNA regulatory region capable of binding RNA polymera ⁇ e in a cell and initiating tran ⁇ cription of a down ⁇ tream (3' direction) coding ⁇ equence.
  • RNA polymera ⁇ e RNA polymera ⁇ e.
  • Eucaryotic promoter ⁇ will often, but not alway ⁇ , contain "TATA” boxe ⁇ and "CAT” boxe ⁇ .
  • Procaryotic promoter ⁇ contain Shine-Dalgarno ⁇ equence ⁇ in addition to the -10 and -35 con ⁇ ensu ⁇ ⁇ equences.
  • control sequence ⁇ refer ⁇ collectively to promoter ⁇ equence ⁇ , ribo ⁇ ome binding ⁇ ite ⁇ , polyadenylation ⁇ ignal ⁇ , tran ⁇ cription termination ⁇ equence ⁇ , up ⁇ tream regulatory domain ⁇ , enhancer ⁇ , and the like, which collectively provide for the tran ⁇ cription and tran ⁇ lation of a coding ⁇ equence in a ho ⁇ t cell.
  • control ⁇ equences operably linked to a coding sequence are capable of effecting the expression of the coding ⁇ equence.
  • the control ⁇ equence ⁇ need not be contiguous with the coding ⁇ equence, ⁇ o long a ⁇ they function to direct the expre ⁇ ion thereof.
  • intervening untran ⁇ lated yet tran ⁇ cribed ⁇ equences can be present between a promoter sequence and the coding sequence and the promoter ⁇ equence can ⁇ till be con ⁇ idered "operably linked" to the coding ⁇ equence.
  • a control ⁇ equence "direct ⁇ the tran ⁇ cription" of a coding ⁇ equence in a cell when RNA polymera ⁇ e will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then tran ⁇ lated into the polypeptide encoded by the coding ⁇ equence.
  • a "host cell” i ⁇ a cell which ha ⁇ been tran ⁇ formed, or i ⁇ capable of tran ⁇ for ation, by an exogenou ⁇ DNA ⁇ equence.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the exogenou ⁇ DNA may be maintained on an epi ⁇ omal element, such a ⁇ a plasmid.
  • a ⁇ tably transformed cell is one in which the exogenous DNA ha ⁇ become integrated into the chromo ⁇ ome ⁇ o that it i ⁇ inherited by daughter cell ⁇ through chromo ⁇ ome replication.
  • a “clone” i ⁇ a population of cell ⁇ derived from a ⁇ ingle cell or common ance ⁇ tor by mito ⁇ is.
  • a “cell line” is a clone of a primary cell that is capable of ⁇ table growth in vitro for many generation ⁇ .
  • a "heterologou ⁇ " region of a DNA con ⁇ truct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in as ⁇ ociation with the other molecule in nature. Thu ⁇ , when the heterologou ⁇ region encode ⁇ a bacterial gene, the gene will u ⁇ ually be flanked by DNA that doe ⁇ not flank the bacterial gene in the genome of the ⁇ ource bacteria.
  • Another example of the heterologou ⁇ coding ⁇ equence i ⁇ a con ⁇ truct where the coding ⁇ equence it ⁇ elf i ⁇ not found in nature e .g. , ⁇ ynthetic ⁇ equences having codons different from the native gene
  • a composition containing A is "substantially free of" B when at least about 85% by weight of the total of A + B in the composition i ⁇ A.
  • a compri ⁇ e ⁇ at least about 90% by weight of the total of A + B in the composition, more preferably at lea ⁇ t about 95%, or even 99% by weight.
  • treatment refers to either (i) the prevention of infection or reinfection (prophylaxi ⁇ ) , or (ii) the reduction or elimination of ⁇ ymptom ⁇ of the di ⁇ ease of interest (therapy) .
  • OmlA ⁇ Central to the pre ⁇ ent invention i ⁇ the di ⁇ covery of a family of A. pleuropneumoniae outer membrane lipoprotein ⁇ , termed OmlA ⁇ herein, which are able to elicit an immune re ⁇ pon ⁇ e in an animal to which they are ad ini ⁇ tered. All 12 of the A. pleuropneumoniae ⁇ erotype ⁇ appear to contain a gene encoding an OmlA. Thi ⁇ protein, analogs thereof and/or immunogenic fragments derived from the protein, are provided in subunit vaccine compositions and thus problems inherent in prior vaccine compositions, such as localized and ⁇ y ⁇ te ic side reaction ⁇ , a ⁇ well a ⁇ the inability to protect again ⁇ t chronic di ⁇ ea ⁇ e, are avoided.
  • the vaccine compositions can be used to treat or prevent A. pleurop.neu;noniae-induced re ⁇ piratory diseases in swine such as porcine pleuropne ⁇ monia.
  • the antigens or antibodies thereto can also be used a ⁇ diagno ⁇ tic reagents to detect the presence of an A. pleuropneumoniae infection in a ⁇ ubject.
  • the genes from the various ⁇ erotype ⁇ encoding the OmlA proteins can be cloned and u ⁇ ed to de ⁇ ign probe ⁇ for the detection of A .
  • the subunit antigens can be conveniently produced by recombinant techniques, as described herein.
  • the proteins of interest are produced in high amounts in tran ⁇ formant ⁇ , do not require extensive purification or processing, and do not cau ⁇ e le ⁇ ion ⁇ at the injection site or other ill effect ⁇ .
  • the genes encoding the A. pleuropneumoniae ⁇ erotype 1 OmlA and ⁇ erotype 5 OmlA have been i ⁇ olated and the ⁇ equence ⁇ are depicted in Figure 1 and Figure 2, re ⁇ pectively.
  • the nucleotide ⁇ equence for the serotype 1 omlA gene including the ⁇ tructural gene and flanking region ⁇ , con ⁇ i ⁇ t ⁇ of approximately 1340 ba ⁇ e pair ⁇ .
  • the open reading frame code ⁇ for a protein having approximately 365 amino acids.
  • the nucleotide sequence for the serotype 5 omlA gene, including the structural gene and flanking regions, consists of approximately 2398 base pair ⁇ .
  • the ⁇ erotype 1 and ⁇ erotype 5 OmlA protein ⁇ are approximately 65 % homologou ⁇ .
  • the invention therefore, encompasses genes encoding OmlA from all of the A. pleuropneumoniae serotype ⁇ .
  • the full-length ⁇ erotype 1 and ⁇ erotype 5 lipoprotein ⁇ both have an " apparent molecular a ⁇ of approximately 50 kDa, a ⁇ determined by di ⁇ continuou ⁇ ⁇ odium dodecyl ⁇ ulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laem li (Laem li, M.K., Nature (1970) 227:680-685).
  • the predicted molecular weight ⁇ , ba ⁇ ed on the amino acid ⁇ equences, are 39,780 and 40,213, respectively.
  • the recombinantly produced proteins are able to protect pig ⁇ from ⁇ ub ⁇ equent challenge with A. pleuropneumoniae .
  • Other OmlA protein ⁇ , from other A . pleuropneumoniae ⁇ erotypes can also be identified, purified and sequenced, using any of the various methods known to those skilled in the art.
  • the amino acid ⁇ equence ⁇ of the ⁇ ubject proteins can be determined from the purified proteins by repetitive cycles of Edman degradation, followed by amino acid analy ⁇ i ⁇ by HPLC. Other method ⁇ of amino acid sequencing are al ⁇ o known in the art.
  • Fragment ⁇ of the purified protein ⁇ can be te ⁇ ted for biological activity and active fragment ⁇ , a ⁇ de ⁇ cribed above, used in composition ⁇ in lieu of the entire protein.
  • recombinant techniques can be employed. For example a DNA library can be prepared which con ⁇ i ⁇ t ⁇ of genomic DNA from an A. pleuropneumoniae ⁇ erotype. The re ⁇ ulting clone ⁇ can be used to transform an appropriate host, such as E. coli . Individual colonies can then be screened in an immunoblot as ⁇ ay, u ⁇ ing polyclonal ⁇ erum or monoclonal antibodie ⁇ , to the desired antigen.
  • DNA fragments of a de ⁇ ired length are i ⁇ olated by, e.g., ⁇ ucro ⁇ e den ⁇ ity gradient centrifugation.
  • the ⁇ e fragment ⁇ are then ligated into any suitable expres ⁇ ion vector or replicon and thereafter the corre ⁇ ponding ho ⁇ t cell i ⁇ tran ⁇ formed with the con ⁇ tructed vector or replicon.
  • Transformed cells are plated in suitable medium.
  • a replica plate must al ⁇ o be prepared becau ⁇ e ⁇ ubsequent procedures kill these colonies.
  • the colonies are then lysed in one of a number of way ⁇ , e . g. , by exposure to chloroform vapor.
  • the ly ⁇ ed colonie ⁇ are incubated with the appropriate unlabelled antibody and developed u ⁇ ing an appropriate anti-immunoglobulin conjugate and ⁇ ubstrate. Positively reacting colonies thu ⁇ detected can be recovered from the replica plate and ⁇ ubcultured.
  • Phy ⁇ ical mapping, con ⁇ truction of deletion derivative ⁇ and nucleotide ⁇ equencing can be u ⁇ ed to characterize the encoding gene.
  • An alternative method to identify gene ⁇ encoding the proteins of the present invention once the genomic DNA library is constructed as described above, i ⁇ to prepare oligonucleotide ⁇ to probe the library and to u ⁇ e the ⁇ e probe ⁇ to i ⁇ olate the gene encoding the de ⁇ ired protein.
  • the ba ⁇ ic ⁇ trategie ⁇ for preparing oligonucleotide probe ⁇ , a ⁇ well a ⁇ ⁇ creening librarie ⁇ u ⁇ ing nucleic acid hybridization are well known to those of ordinary ⁇ kill in the art. See, e .g. , DNA Cloning: Vol. I, supra ; Nucleic Acid Hybridization, supra ; Oligonucleotide Svnthe ⁇ i ⁇ .
  • nucleotide ⁇ equence ⁇ ⁇ elected are cho ⁇ en ⁇ o as to correspond to the codons encoding a known amino acid ⁇ equence from the de ⁇ ired protein. Since the genetic code i ⁇ degenerate, it will often be nece ⁇ ary to ⁇ ynthe ⁇ ize ⁇ everal oligonucleotide ⁇ to cover all, or a rea ⁇ onable number of, the pos ⁇ ible nucleotide ⁇ equence ⁇ which encode a particular region of the protein.
  • Thu ⁇ it i ⁇ generally preferred in ⁇ electing a region upon which to ba ⁇ e the probe ⁇ , that the region not contain amino acid ⁇ who ⁇ e codon ⁇ are highly degenerate.
  • one of ⁇ kill in the art may find it de ⁇ irable to prepare probes that are fairly long, and/or encompas ⁇ region ⁇ of the amino acid ⁇ equence which would have a high degree of redundancy in corre ⁇ ponding nucleic acid ⁇ equences, particularly if this lengthy and/or redundant region i ⁇ highly characteri ⁇ tic of the protein of intere ⁇ t.
  • the selected oligonucleotide probes are labeled with a marker, such a ⁇ a radionucleotide or biotin u ⁇ ing ⁇ tandard procedure ⁇ .
  • a marker such as a ⁇ a radionucleotide or biotin u ⁇ ing ⁇ tandard procedure ⁇ .
  • the labeled set of probes i ⁇ then u ⁇ ed in the screening step, which consi ⁇ t ⁇ of allowing the ⁇ ingle- ⁇ tranded probe to hybridize to isolated ssDNA from the library, according to standard technigue ⁇ . Either stringent or permissive hybridization conditions could be appropriate, depending upon several factors, such as the length of the probe and whether the probe i ⁇ derived from the ⁇ ame ⁇ pecie ⁇ a ⁇ the library, or an evolutionarily clo ⁇ e or di ⁇ tant ⁇ pecie ⁇ .
  • DNA sequences encoding the protein ⁇ of interest can be prepared synthetically rather than cloned.
  • the DNA sequence can be designed with the appropriate codons for the particular amino acid ⁇ equence. In general, one will ⁇ elect preferred codon ⁇ for the intended ho ⁇ t if the ⁇ equence will be u ⁇ ed for expression.
  • the complete ⁇ equence is assembled from overlapping oligonucleotides prepared by ⁇ tandard methods and assembled into a complete coding sequence. See, e . g. , Edge (1981) Nature 292:756; Nambair et al . , (1984) Science 223:1299; Jay et al . , (1984) J. Biol . Chem .
  • coding sequences for the de ⁇ ired proteins can be cloned into any ⁇ uitable vector or replicon.
  • Numerou ⁇ cloning vector ⁇ are known to those of ⁇ kill in the art, and the ⁇ election of an appropriate cloning vector i ⁇ a matter of choice.
  • Example ⁇ of recombinant DNA vector ⁇ for cloning and ho ⁇ t cell ⁇ which they can tran ⁇ form include the bacteriophage ⁇ (E. coli ) , pBR322 (E. coli) , pACYC177 (E.
  • the gene can be placed under the control of a promoter, ribo ⁇ ome binding ⁇ ite (for bacterial expre ⁇ ion) and, optionally, an operator (collectively referred to herein a ⁇ "control" element ⁇ ) , ⁇ o that the DNA ⁇ equence encoding the de ⁇ ired protein i ⁇ tran ⁇ cribed into RNA in the ho ⁇ t cell tran ⁇ formed by a vector containing thi ⁇ expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence.
  • Leader sequence ⁇ can be removed by the ho ⁇ t in po ⁇ t-translational proce ⁇ ing. See , e . g. , U.S. Patent No ⁇ . 4,431,739; 4,425,437; 4,338,397.
  • regulatory ⁇ equence ⁇ which allow for regulation of the expre ⁇ ion of the protein ⁇ equence ⁇ relative to the growth of the ho ⁇ t cell.
  • Regulatory ⁇ equences are known to those of skill in the art, and examples include those which cause the expres ⁇ ion of a gene to be turned on or off in response to a chemical or phy ⁇ ical ⁇ timulu ⁇ , including the pre ⁇ ence of a regulatory compound.
  • Other type ⁇ of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • An expression vector is constructed ⁇ o that the particular coding sequence is located in the vector with the appropriate regulatory sequence ⁇ , the po ⁇ itioning and orientation of the coding sequence with re ⁇ pect to the control ⁇ equence ⁇ being ⁇ uch that the coding ⁇ equence i ⁇ tran ⁇ cribed under the "control" of the control ⁇ equence ⁇ (i.e., RNA polymera ⁇ e which bind ⁇ to the DNA molecule at the control ⁇ equence ⁇ tran ⁇ cribe ⁇ the coding ⁇ equence) .
  • Modification of the ⁇ equence ⁇ encoding the particular antigen of intere ⁇ t may be de ⁇ irable to achieve thi ⁇ end.
  • control sequences and other regulatory ⁇ equence ⁇ may be ligated to the coding ⁇ equence prior to in ⁇ ertion into a vector, ⁇ uch a ⁇ the cloning vectors described above.
  • the cod- ing sequence can be cloned directly into an expres ⁇ ion vector which already contain ⁇ the control ⁇ equence ⁇ and an appropriate re ⁇ triction ⁇ ite.
  • ⁇ equence ⁇ which cau ⁇ e the ⁇ ecretion of the polypeptide from the ho ⁇ t organi ⁇ m, with ⁇ ub ⁇ equent cleavage of the ⁇ ecretory ⁇ ignal. It may al ⁇ o be de ⁇ irable to produce mutant ⁇ or analog ⁇ of the antigen ⁇ of intere ⁇ t.
  • Mutant ⁇ or analog ⁇ may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by sub ⁇ titution of one or more nucleotide ⁇ within the sequence.
  • procaryotic expression vectors are known in the art. See, e .g. , U.S. Patent Nos. 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437;
  • the protein ⁇ of the present invention are produced by growing host cells transformed by an expres ⁇ sion vector described above under conditions whereby the protein of interest is expre ⁇ ed. The protein i ⁇ then i ⁇ olated from the ho ⁇ t cell ⁇ and purified. If the expre ⁇ ion ⁇ y ⁇ tem ⁇ ecrete ⁇ the protein into growth media, the protein can be purified directly from the media. If the protein is not secreted, it i ⁇ i ⁇ olated from cell ly ⁇ ate ⁇ . The ⁇ election of the appropriate growth condi ⁇ tion ⁇ and recovery method ⁇ are within the ⁇ kill of the art.
  • OmlA antigen ⁇ can al ⁇ o be i ⁇ olated directly from any of the A. pleuropneumoniae ⁇ erotype ⁇ .
  • Thi ⁇ i ⁇ generally accomplished by first preparing a crude extract which lacks cellular components and several extraneous proteins. The desired antigens can then be further purified, i.e., by column chromatography, HPLC, immunoadsorbent techniques or other conventional methods well known in the art.
  • the proteins of the present invention may also be produced by chemical synthesis such as solid phase peptide synthesi ⁇ , u ⁇ ing known amino acid ⁇ equence ⁇ or amino acid ⁇ equence ⁇ derived from the DNA ⁇ equence of the gene ⁇ of intere ⁇ t. Such method ⁇ are known to tho ⁇ e skilled in the art. Chemical synthe ⁇ is of peptide ⁇ may be preferable if a ⁇ mall fragment of the antigen in que ⁇ tion i ⁇ capable of rai ⁇ ing an immunological re ⁇ pon ⁇ e in the ⁇ ubject of intere ⁇ t.
  • the protein ⁇ of the pre ⁇ ent invention or their fragment ⁇ can be u ⁇ ed to produce antibodie ⁇ , both polyclonal and monoclonal.
  • polyclonal antibodie ⁇ are de ⁇ ired, a ⁇ elected mammal, (e .g. , mouse, rabbit, goat, hor ⁇ e, pig etc.) i ⁇ immunized with an antigen of the pre ⁇ ent invention, or it ⁇ fragment, or a mutated antigen. Serum from the immunized animal i ⁇ collected and treated according to known procedure ⁇ .
  • the polyclonal antibodie ⁇ can be purified by immunoaffinity chromatography, u ⁇ ing known procedure ⁇ .
  • Monoclonal antibodie ⁇ to the protein ⁇ of the pre ⁇ ent invention, and to the fragment ⁇ thereof, can al ⁇ o be readily produced by one ⁇ killed in the art.
  • the general methodology for making monoclonal antibodies by u ⁇ ing hybridoma technology i ⁇ well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other technique ⁇ ⁇ uch as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e . g. , M. Schreier et al . , Hybridoma Technigues (1980); Hammerling et al .
  • Animal ⁇ can be immunized with the compositions of the present invention by administration of the protein of intere ⁇ t, or a fragment thereof, or an analog thereof. If the fragment or analog of the protein i ⁇ u ⁇ ed, it will include the amino acid ⁇ equence of an epitope which interact ⁇ with the immune ⁇ y ⁇ tem to immunize the animal to that and ⁇ tructurally ⁇ imilar epitope ⁇ .
  • the ⁇ ubunit antigen can be a ⁇ ingle polypeptide encoding one or ⁇ everal epitope ⁇ from one or more OmlA ⁇ or two or more di ⁇ crete polypeptide ⁇ encoding different epitope ⁇ .
  • the ⁇ ubunit antigen even though carrying epitopes derived from a lipoprotein, does not require the presence of the lipid moiety. However, if the lipid is present, it need not be a lipid commonly as ⁇ ociated with the lipoprotein, ⁇ o long a ⁇ the appropriate immunologic re ⁇ ponse i ⁇ elicited.
  • the antigenic peptide may be administered linked to a carrier.
  • Suitable carriers are typically large, ⁇ lowly metabolized macromolecule ⁇ ⁇ uch a ⁇ : protein ⁇ ; poly ⁇ accharides, such a ⁇ ⁇ epharo ⁇ e, agaro ⁇ e, cellulo ⁇ e, cellulo ⁇ e bead ⁇ and the like; polymeric amino acid ⁇ ⁇ uch as polyglutamic acid, polylysine, and the like; amino acid copoly er ⁇ ; and inactive viru ⁇ particle ⁇ .
  • u ⁇ eful protein ⁇ ubstrates are ⁇ erum albumin ⁇ , keyhole limpet hemocyanin, immunoglobulin molecule ⁇ , thyroglobulin, ovalbu in, and other protein ⁇ well known to tho ⁇ e skilled in the art.
  • the protein ⁇ ub ⁇ trate ⁇ may be u ⁇ ed in their na ⁇ tive form or their functional group content may be modified by, for example, ⁇ uccinylation of ly ⁇ ine re ⁇ idue ⁇ or reaction with Cy ⁇ -thiolactone.
  • a ⁇ ulfhydryl group may al ⁇ o be incorporated into the carrier (or antigen) by, for example, reaction of amino function ⁇ with 2-iminothiolane or the N-hydroxy ⁇ uccinimide e ⁇ ter of 3-(4-dithiopyridyl propionate.
  • Suitable carrier ⁇ may al ⁇ o be modified to incorporate spacer arms ( ⁇ uch a ⁇ hexamethylene diamine or other bifunctional molecule ⁇ of ⁇ imilar size) for attachment of peptide ⁇ .
  • ⁇ uitable carrier ⁇ for the protein ⁇ of the pre ⁇ ent invention include VP6 polypeptide ⁇ of rotaviru ⁇ e ⁇ , or functional fragment ⁇ thereof, a ⁇ di ⁇ clo ⁇ ed in U.S. Patent No. 5,071,651.
  • Still other ⁇ uitable carrier ⁇ include cell ⁇ , ⁇ uch as lymphocytes, since presentation in this form mim ⁇ ics the natural mode of presentation in the subject, which gives ri ⁇ e to the immunized ⁇ tate.
  • the proteins of the present invention may be coupled to erythrocytes, preferably the subject , ⁇ own erythrocytes.
  • Method ⁇ of coupling peptide ⁇ to protein ⁇ or cell ⁇ are known to tho ⁇ e of ⁇ kill in the art.
  • the novel protein ⁇ of the in ⁇ tant invention can al ⁇ o be admini ⁇ tered via a carrier viru ⁇ which expre ⁇ se ⁇ the ⁇ ame.
  • Carrier viru ⁇ e ⁇ which will find u ⁇ e with the instant invention include but are not limited to the vaccinia and other pox viruse ⁇ , adenoviru ⁇ , and herpe ⁇ viru ⁇ .
  • vaccinia virus recombinant ⁇ expressing the novel proteins can be constructed as follows.
  • This vector is then used to tran ⁇ fect cell ⁇ which are simultaneou ⁇ ly infected with vaccinia.
  • the re ⁇ ulting TK ⁇ recombinant can be ⁇ elected by culturing the cell ⁇ in the pre ⁇ ence of 5-bromodeoxy- uridine and picking viral plaque ⁇ re ⁇ istant thereto.
  • vaccine ⁇ are prepared a ⁇ injectable ⁇ , either a ⁇ liquid ⁇ olution ⁇ or ⁇ u ⁇ pen ⁇ ion ⁇ ; ⁇ olid for ⁇ ⁇ uitable for ⁇ olution in, or ⁇ u ⁇ pen ⁇ ion in, liquid vehicle ⁇ prior to injection may al ⁇ o be prepared.
  • the preparation may al ⁇ o be emul ⁇ ified or the active ingredient encap ⁇ ulated in lipo ⁇ ome vehicle ⁇ .
  • Suitable vehicle ⁇ are, for example, water, ⁇ aline, dextrose, glycerol, ethanol, or the like, and combination ⁇ thereof.
  • the vehicle may contain minor amounts of auxiliary ⁇ ubstance ⁇ ⁇ uch a ⁇ wetting or emul ⁇ ifying agent ⁇ , pH buffering agent ⁇ , or adjuvant ⁇ which enhance the effectivene ⁇ s of the vaccine.
  • Adjuvant ⁇ may include for example, muramyl dipeptides, avridine, aluminum hydroxide, oils, saponins and other substance ⁇ known in the art.
  • the vehicle compo ⁇ ition will include traditional binder ⁇ and carrier ⁇ , ⁇ uch a ⁇ , polyalkaline glycol ⁇ , or triglyceride ⁇ .
  • Such ⁇ uppo ⁇ itorie ⁇ may be formed from mixture ⁇ containing the active ingredient in the range of about 0.5% to about 10% (w/w) , preferably about 1% to about 2%.
  • Oral vehicle ⁇ include ⁇ uch normally employed excipient ⁇ a ⁇ , for example, pharmaceutical grade ⁇ of mannitol, lacto ⁇ e, ⁇ tarch, magne ⁇ ium, ⁇ tearate, ⁇ odium ⁇ accharin cellulo ⁇ e, magne ⁇ ium carbonate, and the like.
  • the ⁇ e oral vaccine compo ⁇ ition ⁇ may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, -and contain from about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.
  • Intranasal formulation ⁇ will usually include vehicles that neither cause irritation to the nasal muco ⁇ a nor ⁇ ignificantly di ⁇ turb ciliary function.
  • Diluent ⁇ ⁇ uch a ⁇ water, aqueou ⁇ ⁇ aline or other known ⁇ ub ⁇ tance ⁇ can be employed with the ⁇ ubject invention.
  • the na ⁇ al formulations may also contain preservative ⁇ ⁇ uch a ⁇ , but not limited to, chlorobutanol and benzalkonium chloride.
  • a ⁇ urfactant may be pre ⁇ ent to enhance absorption of the subject proteins by the na ⁇ al muco ⁇ a.
  • Controlled or ⁇ ustained release formulations are made by incorporating the protein into carriers or vehicle ⁇ such as liposo e ⁇ , nonre ⁇ orbable impermeable polymer ⁇ such a ⁇ ethylenevinyl acetate copolymer ⁇ and Hytrel® copolymer ⁇ , ⁇ wellable polymer ⁇ such a ⁇ hydrogel ⁇ , or re ⁇ orbable polymer ⁇ ⁇ uch a ⁇ collagen and certain polyacid ⁇ or polye ⁇ ter ⁇ such as tho ⁇ e u ⁇ ed to make re ⁇ orbable ⁇ uture ⁇ .
  • the protein ⁇ can al ⁇ o be delivered u ⁇ ing implanted mini-pump ⁇ , well known in the art.
  • the protein ⁇ (or complexe ⁇ thereof) may be formulated into vaccine compo ⁇ ition ⁇ in either neutral or ⁇ alt form ⁇ .
  • Pharmaceutically acceptable ⁇ alt ⁇ include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acid ⁇ such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed from free carboxyl groups may also be derived from inorganic base ⁇ ⁇ uch a ⁇ , for example, ⁇ odiu , pota ⁇ ium, ammonium, calcium, or ferric hydroxide ⁇ , and ⁇ uch organic ba ⁇ e ⁇ a ⁇ i ⁇ opropylamine, trimethylamine, 2-ethylamino ethanol, hi ⁇ tidine, procaine, and the like.
  • the polypeptide of intere ⁇ t, or an immunologically active fragment thereof i ⁇ ad ini ⁇ tered parenterally, u ⁇ ually by intramuscular injection in an appropriate vehicle.
  • Other modes of administration such as ⁇ ubcutaneou ⁇ , intravenou ⁇ injection and intrana ⁇ al delivery, are al ⁇ o acceptable.
  • Injectable vaccine formulation ⁇ will contain an effective amount of the active ingredient in a vehicle, the exact amount being readily determined by one ⁇ killed in the art.
  • the active ingredient may typically range from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate.
  • the quantity to be administered depends on the animal to be treated, the capacity of the animal's immune system to ⁇ ynthesize antibodie ⁇ , and the degree of protection de ⁇ ired.
  • a ⁇ little a ⁇ 0.1 to 100 ⁇ g or more, preferably 0.5 to 50 ⁇ g, more preferably 1.0 to 25 ⁇ g, of active ingredient per ml of injected ⁇ olution, should be adequate to raise an immunological re ⁇ pon ⁇ e when a do ⁇ e of 1 to 2 ml per animal i ⁇ admini ⁇ tered.
  • nucleotide ⁇ equence ⁇ (and accompanying regulatory element ⁇ ) encoding the ⁇ ubject protein ⁇ can be admini ⁇ tered directly to a subject for in vivo translation thereof.
  • gene transfer can be accomplished by transfecting the ⁇ ubject' ⁇ cell ⁇ or ti ⁇ ue ⁇ ex vivo and reintroducing the tran ⁇ formed material into the ho ⁇ t.
  • DNA can be directly introduced into the ho ⁇ t organi ⁇ m, i.e., by injection (see International Publication No. WO/90/11092; and Wolff et al . , Science (1990) 247:1465-1468).
  • Liposome-mediated gene transfer can al ⁇ o be accompli ⁇ hed u ⁇ ing known methods. See, e .
  • Targeting agent ⁇ ⁇ uch a ⁇ antibodie ⁇ directed again ⁇ t ⁇ urface antigen ⁇ expre ⁇ ed on ⁇ pecific cell types, can be covalently conjugated to the liposomal surface so that the nucleic acid can be delivered to ⁇ pecific ti ⁇ ue ⁇ and cell ⁇ susceptible to A. pleuropneumoniae .
  • the nucleic acid sequences of the ⁇ e plasmid ⁇ , a ⁇ well a ⁇ the amino ⁇ equence ⁇ of the polypeptide ⁇ encoded thereby, are controlling in the event of any conflict with the de ⁇ cription herein.
  • a licen ⁇ e may be required to make, u ⁇ e, or ⁇ ell the depo ⁇ ited material ⁇ , and no ⁇ uch licen ⁇ e i ⁇ hereby granted.
  • Enzymes were purchased from commercial sources, and used according to the manufacturers' direction ⁇ . Radionucleotides and nitrocellulo ⁇ e filters were al ⁇ o purcha ⁇ ed from commercial ⁇ ource ⁇ .
  • A. pleuropneumoniae ⁇ erotype 1 ⁇ train AP37 and A. pleuropneumoniae ⁇ erotype 5 ⁇ train AP213 were i ⁇ olated from the lung ⁇ of di ⁇ ea ⁇ ed pig ⁇ given to the We ⁇ tern College of Veterinary Medicine, Univer ⁇ ity of Saskatchewan, Saskatoon, Saskatchewan, Canada.
  • A. pleuropneumoniae serotype 7 strain AP205 was a Kansas clinical isolate obtained from M.L. Chepok, Modern Veterinary Products, Omaha, Kansas.
  • Other A. pleuropneumoniae strain ⁇ were field i ⁇ olates from herds in Saskatchewan. The E.
  • the pla ⁇ mid ⁇ pGH432 and pGH433 are expre ⁇ ion vector ⁇ containing a tac promoter, a tran ⁇ lational ⁇ tart ⁇ ite with restriction enzyme site ⁇ allowing ligation in all three reading fra e ⁇ followed by ⁇ top codon ⁇ in all reading frame ⁇ .
  • pleuropneumoniae ⁇ train ⁇ were grown on PPLO medium (Difco Laboratorie ⁇ , Detroit, MI) supplemented with 10 mg/ml S-nicotinamide adenine dinucleotide (Sigma Chemical Co., St. Loui ⁇ , MO). Plate culture ⁇ were incubated in a C0 2 -enriched (5%) atmo ⁇ phere at 37°C. Liquid culture ⁇ were grown with continuou ⁇ ⁇ haking at 37°C without C0 2 enrichment.
  • Iron re ⁇ triction wa ⁇ obtained by adding 2,2'- dipyridyl to a final concentration of 100 ⁇ mol.
  • E. coli tran ⁇ formant ⁇ were grown in Luria medium (Sambrook et al . , supra) ⁇ upplemented with ampicillin (100 mg/1) .
  • Tran ⁇ cription from the ta ⁇ -promoter wa ⁇ induced by the addition of i ⁇ opropylthioglactopyrano ⁇ ide (IPTG) to a final concentration of 1 mmol.
  • IPTG i ⁇ opropylthioglactopyrano ⁇ ide
  • Ly ⁇ i ⁇ was achieved by the addition of 5 ⁇ g of lysozyme in 250 mmol Tri ⁇ /HCl buffer pH 8 (5 min on ice) , addition of 10 ml detergent mix (5 part ⁇ 20 mmol Tri ⁇ /HCl buffer pH 8 (5 min on ice) , addition of 10 ml detergent mix (5 parts 20 mmol Tris/HCl buffer pH 7.4, 300 mmol NaCl, 2% deoxycholic acid, 2% NP-40, and 4 part ⁇ of 100 mmol Tri ⁇ /HCl buffer pH 8, 50 mmol ethylenediamine tetraacetic acid, 2% Triton X-100) , and by ⁇ onication.
  • Protein aggregate ⁇ were harve ⁇ ted by centrifugation for 30 min at 15,000 g. Aggregate protein wa ⁇ re ⁇ u ⁇ pended in H 2 0 to a concentration of 5-10 mg/ml and solubilized by the addition of an equal volume of 7 molar guanidine hydrochloride. The concentration of protein in the aggregate preparations was determined by separating serial dilutions of the protein using SDS-PAGE. The . inten ⁇ ity of the Cooma ⁇ ie blue ⁇ tained bands was compared with tho ⁇ e of a bovine ⁇ erum albumin standard (Pierce Chemical Co., Rockford, IL) .
  • A. pleuropneumoniae culture supernatant wa ⁇ obtained a ⁇ follow ⁇ .
  • A. pleuropneumoniae ⁇ erotype 1 culture supernatant was precipitated with 10% trichloroacetic (TCA; vol/vol) , emul ⁇ ified with incomplete Freund' ⁇ adjuvant, and u ⁇ ed to immunize rabbit ⁇ twice at three-week interval ⁇ .
  • TCA trichloroacetic
  • Porcine convale ⁇ cent ⁇ era were obtained from pig ⁇ experimentally infected intranasally by aerosol with A. pleuropneumoniae serotype 1 strain AP37.
  • Genomic DNA was prepared by SDS-facilitated freeze-thaw induced lysi ⁇ a ⁇ de ⁇ cribed previou ⁇ ly (Stauffer, G.V., et al . , Gene , (1981) 14:63-72). Pla ⁇ mid DNA wa ⁇ prepared from 100 ⁇ g/ml chloramphenicol-amplified culture ⁇ by alkaline ly ⁇ i ⁇ and ce ⁇ ium chloride-ethidium bromide gradient centrifugation previou ⁇ ly de ⁇ cribed (Sambrook et al . , supra) . Restriction endonuclease dige ⁇ t ⁇ were done in
  • Genomic DNA from A. pleuropneumoniae AP37 was partially digested with the re ⁇ triction endonuclea ⁇ e Sau3AI. Fragment ⁇ of 3000 Bp to 8000 Bp were isolated by ⁇ ucro ⁇ e den ⁇ ity gradient centrifugation (Sambrook et al . , supra) and ligated into the BamHI and Bglll sites of the expres ⁇ ion vector ⁇ pGH432 and pGH433, thu ⁇ allowing for fu ⁇ ion ⁇ in all three reading frame ⁇ . E. coli HB101 wa ⁇ tran ⁇ formed and plated at a den ⁇ ity of approximately 400 colonie ⁇ per plate.
  • Colonie ⁇ were replica-plated onto nitrocellulo ⁇ e di ⁇ k ⁇ , induced for 2 h with 1 mmol IPTG, and ly ⁇ ed in chloroform vapor.
  • Non ⁇ pecific binding wa ⁇ blocked with 0.5% gelatin in the wa ⁇ hing buffer and, after removal of the cellular debri ⁇ , the membrane ⁇ were incubated with rabbit ⁇ erum rai ⁇ ed again ⁇ t the A.
  • pleuropneumoniae AP37 culture ⁇ upernatant and developed u ⁇ ing goat anti-rabbit conjugate and ⁇ ub ⁇ trate a ⁇ de ⁇ cribed above.
  • the tran ⁇ po ⁇ on TnphoA carried by a lamba phage, a ⁇ well a ⁇ the alkaline pho ⁇ phata ⁇ e-negative E. coli ⁇ train CC118, were provided by J. Beckwith, Harvard Medical School, Bo ⁇ ton, MA.
  • the utagene ⁇ i ⁇ wa ⁇ performed a ⁇ previou ⁇ ly de ⁇ cribed (Manoil, C. , and Beckwith, J. (1985) Proc. Natl . Acad. Sci . U.S .A.
  • nucleotide ⁇ equence at the in ⁇ ertion ⁇ ite wa ⁇ determined u ⁇ ing an oligonucleotide primer complementary to the fir ⁇ t 20 ba ⁇ e ⁇ of the phoA- gene in TnphoA (Chang et al . (1986) Gene 44:121-125; Manoil and Beckwith, supra) .
  • DNA ⁇ equencing wa ⁇ performed u ⁇ ing M13 vector ⁇ and the dideoxy chain termination method e ⁇ entially a ⁇ de ⁇ cribed (Sanger, F. , et al . (1977) Proc. Natl . Acad. Sci . U.S.A. 74:5463-5467).
  • Ne ⁇ ted deletion ⁇ were prepared by exonuclea ⁇ e III treatment (Henikoff, S. (1987) Methods in Enzymology 155:156-165).
  • Specific primers were synthe ⁇ ized u ⁇ ing the Pharmacia Gene A ⁇ embler (Pharmacia Canada Ltd. , Baie D'Urfe, Quebec, Canada) . Both ⁇ trand ⁇ were ⁇ equenced in their entirety.
  • the open reading frame (ORF) of the omlA gene wa ⁇ confirmed by Tnp ⁇ oA in ⁇ ertion mutagene ⁇ i ⁇ a ⁇ de ⁇ cribed above.
  • the ⁇ equence wa ⁇ analyzed u ⁇ ing the IBI/Pu ⁇ tell program and the GenBank databa ⁇ e.
  • the pellet was thawed by mixing with an equal volume of hot (70 * C) 2% SDS, 10 mM sodium acetate (pH 4.5). Then, 375 ⁇ l of hot (70 * C) H 2 0-equilibrated phenol wa ⁇ added, the tube ⁇ were vortexed, frozen at -70 C, and ⁇ pun for 10 min in an Eppendorf centrifuge. The clear ⁇ upernatant wa ⁇ removed, 2.5 volume ⁇ of ethanol wa ⁇ added, and the RNA wa ⁇ ⁇ tored at -70"C until needed. The primer exten ⁇ ion wa ⁇ done as described previously using a primer complementary to a sequence within the ORF. 7-Deaza-dGTP and AMV-reverse transcriptase were employed in order to prevent compressions. Intrin ⁇ ic Radiolabelling with r 3 H1-Palmitic Acid. Immunoprecipitation and Globomvcin Treatment
  • coli tran ⁇ formant ⁇ in Luria broth containing 1 ⁇ mol IPTG were grown with methanol, and an immunoprecipitation analy ⁇ i ⁇ wa ⁇ performed e ⁇ entially a ⁇ previou ⁇ ly de ⁇ cribed (Huang, et al . (1989) J. Bacteriol . 171:3767-3774).
  • the OmlA- ⁇ pecific ⁇ erum was obtained from immunized pigs, and protein G-Sepharose wa ⁇ u ⁇ ed to recover the OmlA-porcine antibody co plexe ⁇ .
  • the immunoprecipitated protein ⁇ were re ⁇ u ⁇ pended in SDS- ⁇ ample buffer, heated to 80°C for 5 min and ⁇ eparated by SDS-PAGE.
  • the gel ⁇ were fixed, treated with Amplify (Amer ⁇ ham Corp., Arlington Height ⁇ , IL) , dried and exposed to X-ray film.
  • Globomycin was dis ⁇ olved in 50% dimethyl ⁇ ulfoxide at a concentration of 10 mg/ml.
  • Thi ⁇ ⁇ olution wa ⁇ added to an A .
  • pleuropneumo ⁇ niae AP37 culture grown to an OD ⁇ Q of 0.6 to a final concentration of 100 ⁇ g/ml. and growth was continued for ⁇ hour.
  • the pla ⁇ id encoding thi ⁇ antigen was designated as p0M37/El (ATCC Accession No. 68954) , and the protein was designated as OmlA.
  • the nucleotide sequence of the gene encoding OmlA from pOM37/El is ⁇ hown in Figure 1.
  • the ⁇ equence wa ⁇ determined by dideoxy ⁇ equencing of overlapping deletion ⁇ generated by exonuclea ⁇ e III dige ⁇ tion.
  • the nucleotide ⁇ equence has one long open reading frame (ORF) ⁇ tarting at nucleotide position 158 and ending at position 1252.
  • ORF long open reading frame
  • the amino acid sequence of this open reading frame is al ⁇ o shown in Figure 1.
  • the predicted polypeptide has a molecular weight of 39,780, with a consensus sequence for lipid modification at amino acid residue 20.
  • Example 2 Analysis of Plasmid pOM37/E16 Colonies reacting with serum raised against the culture supernatant were ⁇ ubcultured, induced with IPTG, and examined in a We ⁇ tern blot a ⁇ de ⁇ cribed in Example 1.
  • Nucleotide sequence analysi ⁇ of pOM37/E16 revealed one ORF of 1083 Bp in length coding for a protein with a predicted molecular a ⁇ of 39,780 Da.
  • the fir ⁇ t 19 amino acid ⁇ of the polypeptide have the characteri ⁇ tic ⁇ of a lipoprotein ⁇ ignal peptide with a predicted cleavage ⁇ ite in front of the cy ⁇ teine re ⁇ idue at position 20.
  • the ORF was confirmed by two independent TnphoA-insertion ⁇ 50 bp and 530 bp down ⁇ tream from the methionine codon which, upon tran ⁇ formation of the phoA-negative E. coli ⁇ train CC118, gave ri ⁇ e to alkaline pho ⁇ phatase-po ⁇ itive tran ⁇ formant ⁇ .
  • a GenBank data ba ⁇ e homology ⁇ earch u ⁇ ing the predicted amino acid ⁇ equence of OmlA did not reveal likely ⁇ i ilaritie ⁇ (>35%) to known ORF ⁇ or polypeptide ⁇ .
  • the primer exten ⁇ ion located the beginning of the mRNA at a T-residue 76 Bp upstream of the methionine ⁇ tart codon.
  • the -10 and -30 region ⁇ are both AT-rich, and the promoter- ⁇ tructure matche ⁇ the E. coli con ⁇ en ⁇ u ⁇ characteri ⁇ tics.
  • One of the TnphoA-in ⁇ ertions was found to be located within the signal peptide.
  • the protein wa ⁇ present in whole membranes, outer membranes as prepared by ⁇ ucro ⁇ e gradient centrifugation, and membrane bleb ⁇ ; it wa ⁇ ab ⁇ ent in ⁇ arco ⁇ yl-treated outer membrane ⁇ and in high- ⁇ peed ⁇ upernatant ⁇ .
  • Styl fragment ⁇ from thi ⁇ pla ⁇ mid were treated with DNA polymera ⁇ e I Klenow fragment to fill in the 5' exten ⁇ ion ⁇ .
  • the ⁇ e fragment ⁇ were ligated into the Smal ⁇ ite of the vector, pGH432/lacI.
  • the protein wa ⁇ not expre ⁇ ed at high level ⁇ .
  • Both ⁇ trand ⁇ of the A. pleuropneumoniae ⁇ erotype 5 omlh gene were sequenced using M13 vectors as described above.
  • the nucleotide sequence and predicted amino acid ⁇ equence are ⁇ hown in Figure 2.
  • the open reading frame ⁇ hown in the figure code ⁇ for a protein ⁇ imilar to the omlA product of A. pleuropneumoniae ⁇ erotype 1, ⁇ howing approximately 65% identity at the amino acid level. Thu ⁇ , the open reading frame pre ⁇ ent in pSR213/E25 code ⁇ for the ⁇ erotype 5 equivalent of omlA.
  • A. pleuropneumoniae type ⁇ train ⁇ Genomic DNA from all 12 A. pleuropneumoniae type ⁇ train ⁇ wa ⁇ analyzed in a Southern blot u ⁇ ing the A. pleuropneumoniae AP37-derived omlA-gene a ⁇ probe. The Styl-re ⁇ tricted DNA from all A. pleuropneumoniae type ⁇ train ⁇ reacted with the probe under low ⁇ tringency condition ⁇ , and the DNA from ⁇ erotype ⁇ 1, 2, 8, 9, 11, and 12 remained hybridized to the probe under high ⁇ tringency washing conditions.
  • Example 5 The Protective Capacity of Serotype 1 OmlA Recombinant Protein The OmlA protein wa ⁇ prepared from E. coli
  • HB101/pOM37/El by IPTG-induction of a log pha ⁇ e culture followed by cell harvest and disruption, and separation of the inclusion bodies by centrifugation.
  • the inclusion bodies were ⁇ olubilized with guanidine hydrochloride and mixed with Emul ⁇ igen Plus (MVP Laboratories, Ralston, Nebra ⁇ ka) and ⁇ aline ⁇ o that the final protein concentration wa ⁇ 0.5 ⁇ g/ml, 2.5 ⁇ g/ml or 12.5 ⁇ g/ml.
  • Group ⁇ of 7 pigs were vaccinated with 2 ml of the vaccines or a placebo containing Emulsigen Plus but no protein.
  • Each group was revaccinated 21 days later and finally challenged 7 day ⁇ after the boo ⁇ t with an aero ⁇ ol of A. pleuropneumoniae (serotype 1) .
  • Clinical sign ⁇ of di ⁇ ea ⁇ e were followed for 3 day ⁇ , and 7 day ⁇ after challenge all ⁇ urvivor ⁇ were euthanized.
  • the significance of the difference in mortality rates among the different group ⁇ wa ⁇ determined u ⁇ ing a G 2 likelihood ratio te ⁇ t (Dixon, W.J., et al . , BMDP Stati ⁇ tical Software Manual. Univer ⁇ ity of California Pre ⁇ , 1988, pp. 229-273.)
  • the re ⁇ ult ⁇ are ⁇ ummarized in Table l.
  • OmlA Recombinant Protein OmlA protein wa ⁇ prepared from HB101/pSR213/E25 and formulated with E ul ⁇ igen Plu ⁇ a ⁇ de ⁇ cribed in Example 5 ⁇ o that each 2 ml do ⁇ e contained 25 ⁇ g of protein.
  • Pig ⁇ were vaccinated, boo ⁇ ted and challenged with A. pleuropneumoniae ⁇ erotype 5 ⁇ train AP213 a ⁇ de ⁇ cribed in Example 5.
  • the re ⁇ ult ⁇ ⁇ hown in Table 2 indicate that vaccination with OmlA from ⁇ erotype 5 reduced morbidity, mortality and lung damage a ⁇ ociated with Actinobacillus pleuropneumoniae infection. It i ⁇ predicted that vaccination with both ⁇ erotype l and ⁇ erotype 5 OmlA proteins would protect pigs against infection with all A. pleuropneumoniae serotype ⁇ , with the po ⁇ sible exception of serotype 11.
  • Actinobacillus pleuropneumoniae serotype 5 Actinobacillus pleuropneumoniae serotype 5.

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Abstract

Novel vaccines for use against Actinobacillus pleuropneumoniae are disclosed. The vaccines contain at least one Actinobacillus pleuropneumoniae outer membrane lipoprotein A, or an immunogenic fragment thereof. Also disclosed are DNA sequences encoding these proteins, vectors including these sequences and host cells transformed with these vectors. The vaccines can be used to treat or prevent porcine respiratory infections.

Description

ACTINOBACILLUS PLEUROPNEUMONIAE
OUTER MEMBRANE LIPOPROTEIN A AND USES THEREOF
Technical Field
The instant invention relates generally to the prevention of disease in swine. More particularly, the present invention relates to subunit vaccines for Actinobacillus pleuropneumoniae .
Background Actinobacillus (formerly Haemophilus) pleuropneumoniae is a highly infectious porcine respiratory tract pathogen that causes porcine pleuropneumonia. Infected animals develop acute fibrinous pneumonia which leads to death or chronic lung lesions and reduced growth rates. Infection is transmitted by contact or-aerosol and the morbidity in susceptible groups can approach 100%. Persistence of the pathogen in clinically healthy pigs also poses a constant threat of transmitting disease to previously uninfected herds.
The rapid onset and severity of the disease often causes losses before antibiotic therapy can become effective. Presently available vaccines are generally composed of chemically inactivated bacteria combined with oil adjuvants. However, whole cell bacterins and surface protein extracts often contain immunosuppressive components which make pigs more susceptible to infection. Furthermore, these vaccines may reduce mortality but do not reduce the number of chronic carriers in a herd. There are at least 12 recognized εerotypes of A. pleuropneumoniae with the most common in North America being serotypes 1, 5 and 7. Differences among εerotypes generally coincide with variations in the electrophoretic mobility of outer membrane proteins and enzymes, thus indicating a clonal origin of isolates from the same serotype. This antigenic variety has made the development of a successful vaccination strategy difficult. Protection after parenteral immunization with a killed bacterin or cell free extract is generally serotype specific and does not prevent chronic or latent infection. Higgins, R. , et al . , Can . Vet . J. (1985) 26:86-89; Maclnnes, J.I. and Rosendal, S . , Infect . Immun . (1987) 55:1626-1634. Thus, it would be useful to develop vaccines which protect against both death and chronicity and do not have immunosuppressive properties. One method by which this may be accomplished is to develop εubunit antigen vaccines composed of specific proteins in pure or semi-pure form. An increasing number of bacterial antigens have now been identified as lipoproteins (Anderson, B.E., et al . , J. Bacteriol . (1988) 170:4493-4500; Bricker, T.M. , et al . , Infect . Immun . (1988) 56:295-301; Hanson, M.S., and Hansen, E.J., Mol . Microbiol . (1991) 5:267-278; Hubbard, C.L., et al . , Infect . Immun . (1991) 59:1521-
1528; Nelson, M.B. , et al . , Infect . Immun . (1988) 56:128- 134; Thirkell, D., et al . , Infect . Immun . (1991) 59:781- 784) . One such lipoprotein from Haemophiluε εomnuε has been positively identified. The nucleotide sequence for this lipoprotein, termed "LppA," has been determined
(Theisen, M. , et al . , Infect . Immun . (1992) 60:826-831). These lipoproteins are generally localized in the envelope of the cell and are therefore exposed to the host's immune system. It has been shown that the murine lipoprotein from the outer membrane of Escherichia coli acts as a potent activator of murine lymphocytes, inducing both proliferation and i munoglobulin secretion (Bessler, W. , et al . , Z . Immun . (1977) 153:11-22; Melchers, F. , et al . , J. Exp. Med. (1975) 142:473-482). The active lipoprotein portion of the protein has been shown to reside in the N-terminal fatty acid containing region of the protein. Recent studieε using synthetic lipopeptides based on this protein show that even short peptides, containing two to five amino acids covalently linked to palmitate, are able to activate murine lymphocytes (Bessler, W.G., et al . , J. Immunol . (1985) 135:1900-1905).
It has been found that A. pleuropneumoniae possesses several outer membrane proteins which are expressed only under iron limiting growth conditions (Deneer, H.G., and Potter, A.A., Infect . Immun . (1989) 57:798-804). However, outer membrane lipoproteins from A . pleuropneumoniae have not heretofore been identified or characterized with respect to their immunogenic or protective capacity.
Disclosure of the Invention
The present invention is based on the discovery of a novel subunit antigen from A. pleuropneumoniae which shows protective capability in pigs.
Accordingly, in one embodiment, the subject invention is directed to purified, immunogenic A. pleuropneumoniae outer membrane lipoprotein A, or an immunogenic fragment thereof. In another embodiment, the instant invention is directed to an isolated nucleotide sequence encoding an immunogenic A. pleuropneumoniae outer membrane lipoprotein A, or an immunogenic fragment thereof. In yet another embodiment, the subject invention is directed to a DNA construct comprising the isolated nucleotide sequence described above and control sequences that are operably linked to the nucleotide sequence whereby the coding sequence can be transcribed and translated in a host cell, and at least one of the control sequences is heterologous to the coding sequence. In still further embodiments, the instant invention is directed to host cells transformed with these constructs and methods of recombinantly producing the subject A. pleuropneumoniae proteins. In another embodiment, the subject invention is directed to a vaccine composition comprising a pharmaceutically acceptable vehicle and an A. pleuropneumoniae outer membrane lipoprotein A or an immunogenic fragment thereof. In still another embodiment, the invention is directed to a method of treating or preventing an A. pleuropneumoniae infection in a vertebrate subject comprising administering to the subject a therapeutically effective amount of a vaccine composition as described above.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.
Brief Description of the Figures
Figure 1 depicts the nucleotide sequence
(SEQ ID NO:l) of the gene coding for A. pleuropneumoniae εerotype 1 outer membrane lipoprotein
A as well as the nucleotide sequence for the flanking regions from the HB101/pOM37/E16 clone. The predicted amino acid sequence is also shown.
Figure 2 depicts the nucleotide sequence
(SEQ ID NO:2) of the gene coding for A. pleuropneumoniae serotype 5 outer membrane lipoprotein A as well as the nucleotide sequence for the flanking regions from HB101/pSR213/E25. The predicted amino acid sequence is also shown. Detailed Description
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e .g. , Sa brook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual. Second Edition (1989); DNA Cloning. Vols. I and II (D.N. Glover, ed. , 1985); Oligonucleotide Synthesis
(M.J. Gait, ed., 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higginε, eds., 1984); Animal Cell Culture (R.K. Freεhney, ed. , 1986); Immobilized Cellε and Enzymes (IRL presε, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984) ; the εeries, Methods In
Enzv ology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology. Vols. I-IV (D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell Scientific Publications) .
A. Definitions
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below. The terms "outer membrane lipoprotein A" and
"OmlA" are equivalent and interchangeable and define a protein from the family of proteins represented by A. pleuropneumoniae serotype 1 OmlA (depicted in Figure 1) and A . pleuropneumoniae εerotype 5 OmlA (depicted in Figure 2) . The term "OmlA" also captures proteins substantially homologous and functionally equivalent to native OmlAs. Thus, the term encompasses modifications, such as deletions, additions and substitutions (generally conservative in nature) , to the native sequences, as long as immunological activity (as defined below) is not deεtroyed. Such modificationε of the primary amino acid sequence may result in antigens which have enhanced activity as compared to the native sequence. Theεe modificationε may be deliberate, as through site-directed utageneεiε, or may be accidental, such as through mutations of hostε which produce the lipoprotein. All of theεe modificationε are included, εo long aε immunogenic activity iε retained. Accordingly, A. pleuropneumoniae εerotype 1 OmlA and A. pleuropneumoniae εerotype 5 OmlA refer not only to the amino acid εequenceε depicted in
Figures 1 and 2, repectively, but to amino acid εequenceε homologouε thereto which retain the defined immunological activity.
Additionally, the term "OmlA" (or fragmentε thereof) denoteε a protein which occurs in neutral form or in the form of basic or acid addition salts, depending on the mode of preparation. Such acid saltε may involve free amino groups and basic εaltε may be formed with free carboxyls. Pharmaceutically acceptable basic and acid addition salts are discussed further below. In addition, the protein may be modified by combination with other biological materialε εuch aε lipids (either those normally asεociated with the lipoprotein or other lipids that do not deεtroy activity) and εaccharideε, or by side chain modification, such as acetylation of amino groups, phosphorylation of hydroxyl εide chainε, or oxidation of εulfhydryl groups, as well as other modifications of the encoded primary sequence. Thus, included within the definition of "OmlA" herein are glycosylated and unglycosylated forms, the amino acid sequences with or without associated lipids, and amino acid sequences substantially homologous to the native sequence which retain the ability to elicit an immune response. Two DNA or polypeptide sequenceε are 'Substantially homologous" when at least about 65% (preferably at least about 80% to 90%, and most preferably at least about 95%) of the nucleotides or amino acidε match over a defined length of the molecule. Aε uεed herein, εubεtantially homologouε alεo referε to εequenceε εhowing identity to the εpecified DNA or polypeptide εequence. DNA sequenceε that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular syεtem. Defining appropriate hybridization conditionε iε within the skill of the art. See, e.g., Sambrook et al . , supra ; DNA Cloning, vols I & II, supra ; Nucleic Acid Hybridization, supra .
The term "functionally equivalent" intends that the amino acid sequence of the subject protein iε one that will elicit an immunological reεponεe, aε defined below, equivalent to or better than, the immunological reεponεe elicited by a native A. pleuropneumoniae OmlA. An "antigen" refers to a molecule containing one or more epitopes that will stimulate a host'ε immune system to make a humoral and/or cellular antigen-specific response. The term is also used interchangeably with "immunogen."
By "subunit antigen" is meant an antigen entity separate and discrete from a whole bacterium (live or killed) . Thus, an antigen contained in a cell free extract would constitute a "subunit antigen" as would a substantially purified antigen.
A "hapten" is a molecule containing one or more epitopes that does not stimulate a hoεt's immune εyεtem to make a humoral or cellular response unlesε linked to a carrier.
The term "epitope" refers to the site on an antigen or hapten to which a specific antibody molecule bindε. The term iε alεo uεed interchangeably with "antigenic determinant" or "antigenic determinant εite."
An "immunological response" to an antigen or vaccine is the development in the host of a cellular and/ or antibody-mediated immune response to the composition or vaccine of intereεt. Uεually, εuch a reεponεe includes but is not limited to one or more of the following effects; the production of antibodies, B cells, helper T cells, εuppreεsor T cellε, and/or cytotoxic T cellε and/or yδ T cellε, directed specifically to an antigen or antigens included in the composition or vaccine of intereεt.
The termε "immunogenic polypeptide" and "immunogenic amino acid sequence" refer to a polypeptide or amino acid sequence, respectively, which elicit antibodieε that neutralize bacterial infectivity, and/or mediate antibody-complement or antibody dependent cell cytotoxicity to provide protection of an immunized hoεt. An "immunogenic polypeptide" aε uεed herein, includeε the full length (or near full length) εequence of an A. pleuropneumoniae OmlA, or"an immunogenic fragment thereof. By "immunogenic fragment" iε meant a fragment of an A. pleuropneumoniae OmlA which includes one or more epitopes and thus elicits antibodies that neutralize bacterial infectivity, and/or mediate antibody-complement or antibody dependent cell cytotoxicity to provide protection of an immunized host. Such fragments will usually be at least about 5 amino acids in length, and preferably at least about 10 to 15 amino acids in length. There is no critical upper limit to the length of the fragment, which could comprise nearly the full length of the protein sequence, or even a fusion protein comprising fragments of two or more of the A. pleuropneumoniae subunit antigens. The termε "polypeptide" and "protein" are uεed interchangeably and refer to any polymer of amino acidε (dipeptide or greater) linked through peptide bondε. Thuε, the termε "polypeptide" and "protein" include oligopeptideε, protein fragmentε, analogε, muteinε, fuεion proteins and the like.
"Native" proteins or polypeptides refer to proteinε or polypeptideε recovered from a source occurring in nature. Thuε, the term "native outer membrane lipoprotein A" would include naturally occurring OmlA and fragmentε of theεe proteinε.
By "purified protein" iε meant a protein εeparate and discrete from a whole organism (live or killed) with which the protein iε normally aεεociated in nature. Thuε, a protein contained in a cell free extract would conεtitute a "purified protein," aε would a protein εynthetically or recombinantly produced.
"Recombinant" polypeptideε refer to polypeptideε produced by recombinant DNA techniqueε; i.e., produced from cellε tranεfor ed by an exogenouε DNA • conεtruct encoding the desired polypeptide. "Synthetic" polypeptideε are those prepared by chemical εyntheεiε.
A "replicon" iε any genetic element (e . g. , plasmid, chromosome, virus) that functions aε an autonomous unit of DNA replication in vivo ; i . e . , capable of replication under its own control.
A "vector" iε a replicon, εuch as a plasmid, phage, or coεmid, to which another DNA εegment may be at¬ tached εo aε to bring about the replication of the at- tached εegment.
A "double-εtranded DNA molecule" referε to the polymeric form of deoxyribonucleotideε (baεeε adenine, guanine, thymine, or cytosine) in a double-stranded helix, both relaxed and supercoiled. This term referε only to the primary and εecondary εtructure of the molecule, and doeε not limit it to any particular tertiary formε. Thuε, thiε term includeε double-εtranded DNA found, inter alia , in linear DNA moleculeε (e .g. , restriction fragments) , viruseε, plaεmids, and chromosomeε. In diεcuεεing the structure of particular double-stranded DNA moleculeε, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having the εequence ho ologouε to the mRNA) .
A DNA "coding εequence" or a "nucleotide sequence encoding" a particular protein, is a DNA sequence which is transcribed and translated into a polypeptide in vivo or in vitro when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a εtart codon at the 5' (amino) terminuε and a tranεlation stop codon at the 3' (carboxy) terminus. A coding εequence can include, but iε not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic^ (e .g. , mammalian) DNA, and even synthetic DNA sequences. A transcription termination εequence will usually be located 3' to the coding εequence. A "promoter εequence" iε a DNA regulatory region capable of binding RNA polymeraεe in a cell and initiating tranεcription of a downεtream (3' direction) coding εequence. For purpoεeε of defining the present invention, the promoter sequence iε bound at the 3' terminuε by the tranεlation εtart codon (ATG) of a coding εequence and extendε upstream (5' direction) to include the minimum number of bases or elements necesεary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease SI) , aε well aε protein binding domainε (conεenεuε εequenceε) reεponεible for the binding of RNA polymeraεe. Eucaryotic promoterε will often, but not alwayε, contain "TATA" boxeε and "CAT" boxeε. Procaryotic promoterε contain Shine-Dalgarno εequenceε in addition to the -10 and -35 conεensuε εequences.
DNA "control sequenceε" referε collectively to promoter εequenceε, riboεome binding εiteε, polyadenylation εignalε, tranεcription termination εequenceε, upεtream regulatory domainε, enhancerε, and the like, which collectively provide for the tranεcription and tranεlation of a coding εequence in a hoεt cell.
"Operably linked" referε to an arrangement of elementε wherein the componentε εo deεcribed are configured εo aε to perform their usual function. Thuε, control εequences operably linked to a coding sequence are capable of effecting the expression of the coding εequence. The control εequenceε need not be contiguous with the coding εequence, εo long aε they function to direct the expreεεion thereof. Thuε, for example, intervening untranεlated yet tranεcribed εequences can be present between a promoter sequence and the coding sequence and the promoter εequence can εtill be conεidered "operably linked" to the coding εequence.
A control εequence "directε the tranεcription" of a coding εequence in a cell when RNA polymeraεe will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then tranεlated into the polypeptide encoded by the coding εequence.
A "host cell" iε a cell which haε been tranεformed, or iε capable of tranεfor ation, by an exogenouε DNA εequence.
A cell haε been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In procaryotes and yeaεtε, for example, the exogenouε DNA may be maintained on an epiεomal element, such aε a plasmid. With respect to eucaryotic cells, a εtably transformed cell is one in which the exogenous DNA haε become integrated into the chromoεome εo that it iε inherited by daughter cellε through chromoεome replication. Thiε εtability iε de onεtrated by the ability of the eucaryotic cell to eεtabliεh cell lineε or cloneε compriεed of a population of daughter cell containing the exogenouε DNA.
A "clone" iε a population of cellε derived from a εingle cell or common anceεtor by mitoεis. A "cell line" is a clone of a primary cell that is capable of εtable growth in vitro for many generationε.
A "heterologouε" region of a DNA conεtruct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in asεociation with the other molecule in nature. Thuε, when the heterologouε region encodeε a bacterial gene, the gene will uεually be flanked by DNA that doeε not flank the bacterial gene in the genome of the εource bacteria. Another example of the heterologouε coding εequence iε a conεtruct where the coding εequence itεelf iε not found in nature (e .g. , εynthetic εequences having codons different from the native gene) . Allelic variation or naturally occurring mutational eventε do not give riεe to a heterologous region of DNA, as used herein. A composition containing A is "substantially free of" B when at least about 85% by weight of the total of A + B in the composition iε A. Preferably, A compriεeε at least about 90% by weight of the total of A + B in the composition, more preferably at leaεt about 95%, or even 99% by weight. The term "treatment" aε used herein refers to either (i) the prevention of infection or reinfection (prophylaxiε) , or (ii) the reduction or elimination of εymptomε of the diεease of interest (therapy) .
B. General Methods
Central to the preεent invention iε the diεcovery of a family of A. pleuropneumoniae outer membrane lipoproteinε, termed OmlAε herein, which are able to elicit an immune reεponεe in an animal to which they are ad iniεtered. All 12 of the A. pleuropneumoniae εerotypeε appear to contain a gene encoding an OmlA. Thiε protein, analogs thereof and/or immunogenic fragments derived from the protein, are provided in subunit vaccine compositions and thus problems inherent in prior vaccine compositions, such as localized and εyεte ic side reactionε, aε well aε the inability to protect againεt chronic diεeaεe, are avoided. The vaccine compositions can be used to treat or prevent A. pleurop.neu;noniae-induced reεpiratory diseases in swine such as porcine pleuropneύmonia. The antigens or antibodies thereto can also be used aε diagnoεtic reagents to detect the presence of an A. pleuropneumoniae infection in a εubject. Similarly, the genes from the various εerotypeε encoding the OmlA proteins can be cloned and uεed to deεign probeε for the detection of A . pleuropneumoniae in tiεεue εampleε aε well aε for the detection of homologouε genes in other bacterial εtrains. The subunit antigens can be conveniently produced by recombinant techniques, as described herein. The proteins of interest are produced in high amounts in tranεformantε, do not require extensive purification or processing, and do not cauεe leεionε at the injection site or other ill effectε. The genes encoding the A. pleuropneumoniae εerotype 1 OmlA and εerotype 5 OmlA have been iεolated and the εequenceε are depicted in Figure 1 and Figure 2, reεpectively. The nucleotide εequence for the serotype 1 omlA gene, including the εtructural gene and flanking regionε, conεiεtε of approximately 1340 baεe pairε. The open reading frame codeε for a protein having approximately 365 amino acids. The nucleotide sequence for the serotype 5 omlA gene, including the structural gene and flanking regions, consists of approximately 2398 base pairε. The εtructural gene codeε for a protein of approximately 367 amino acidε. The εerotype 1 and εerotype 5 OmlA proteinε are approximately 65 % homologouε. The omlA gene from A. pleuropneumoniae εerotype
1 hybridizes with genomic DNA from all other known A . pleuropneumoniae εerotypes. The invention, therefore, encompasses genes encoding OmlA from all of the A. pleuropneumoniae serotypeε. The full-length εerotype 1 and εerotype 5 lipoproteinε both have an"apparent molecular aεε of approximately 50 kDa, aε determined by diεcontinuouε εodium dodecylεulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laem li (Laem li, M.K., Nature (1970) 227:680-685). The predicted molecular weightε, baεed on the amino acid εequences, are 39,780 and 40,213, respectively. The recombinantly produced proteins are able to protect pigε from εubεequent challenge with A. pleuropneumoniae . Other OmlA proteinε, from other A . pleuropneumoniae εerotypes, can also be identified, purified and sequenced, using any of the various methods known to those skilled in the art. For example, the amino acid εequenceε of the εubject proteins can be determined from the purified proteins by repetitive cycles of Edman degradation, followed by amino acid analyεiε by HPLC. Other methodε of amino acid sequencing are alεo known in the art. Fragmentε of the purified proteinε can be teεted for biological activity and active fragmentε, aε deεcribed above, used in compositionε in lieu of the entire protein.
In order to identify geneε encoding the subject proteins, recombinant techniques can be employed. For example a DNA library can be prepared which conεiεtε of genomic DNA from an A. pleuropneumoniae εerotype. The reεulting cloneε can be used to transform an appropriate host, such as E. coli . Individual colonies can then be screened in an immunoblot asεay, uεing polyclonal εerum or monoclonal antibodieε, to the desired antigen.
More specifically, after preparation of a DNA library, DNA fragments of a deεired length are iεolated by, e.g., εucroεe denεity gradient centrifugation. Theεe fragmentε are then ligated into any suitable expresεion vector or replicon and thereafter the correεponding hoεt cell iε tranεformed with the conεtructed vector or replicon. Transformed cells are plated in suitable medium. A replica plate must alεo be prepared becauεe εubsequent procedures kill these colonies. The colonies are then lysed in one of a number of wayε, e . g. , by exposure to chloroform vapor. Thiε releaεeε the antigen from the poεitive colonieε. The lyεed colonieε are incubated with the appropriate unlabelled antibody and developed uεing an appropriate anti-immunoglobulin conjugate and εubstrate. Positively reacting colonies thuε detected can be recovered from the replica plate and εubcultured. Phyεical mapping, conεtruction of deletion derivativeε and nucleotide εequencing can be uεed to characterize the encoding gene.
An alternative method to identify geneε encoding the proteins of the present invention, once the genomic DNA library is constructed as described above, iε to prepare oligonucleotideε to probe the library and to uεe theεe probeε to iεolate the gene encoding the deεired protein. The baεic εtrategieε for preparing oligonucleotide probeε, aε well aε εcreening librarieε uεing nucleic acid hybridization, are well known to those of ordinary εkill in the art. See, e .g. , DNA Cloning: Vol. I, supra ; Nucleic Acid Hybridization, supra ; Oligonucleotide Svntheεiε. supra ; Sambrook et al . , supra . The particular nucleotide εequenceε εelected are choεen εo as to correspond to the codons encoding a known amino acid εequence from the deεired protein. Since the genetic code iε degenerate, it will often be neceεεary to εyntheεize εeveral oligonucleotideε to cover all, or a reaεonable number of, the posεible nucleotide εequenceε which encode a particular region of the protein. Thuε, it iε generally preferred in εelecting a region upon which to baεe the probeε, that the region not contain amino acidε whoεe codonε are highly degenerate. In certain circumεtanceε, one of εkill in the art may find it deεirable to prepare probes that are fairly long, and/or encompasε regionε of the amino acid εequence which would have a high degree of redundancy in correεponding nucleic acid εequences, particularly if this lengthy and/or redundant region iε highly characteriεtic of the protein of intereεt. It may alεo be deεirable to uεe two probeε (or εetε of probeε) , each to different regions of the gene, in a single hybridization experiment. Automated oligonucleotide syntheεiε haε made the prepara¬ tion of large familieε of probes relatively εtraight- forward. While the exact length of the probe employed iε not critical, generally it iε recognized in the art that probes from about 14 to about 20 base pairs are usually effective. Longer probes of about 25 to about 60 base pairs are alεo uεed. The selected oligonucleotide probes are labeled with a marker, such aε a radionucleotide or biotin uεing εtandard procedureε. The labeled set of probes iε then uεed in the screening step, which consiεtε of allowing the εingle-εtranded probe to hybridize to isolated ssDNA from the library, according to standard technigueε. Either stringent or permissive hybridization conditions could be appropriate, depending upon several factors, such as the length of the probe and whether the probe iε derived from the εame εpecieε aε the library, or an evolutionarily cloεe or diεtant εpecieε. The εelection of the appropriate conditionε iε within the εkill of the art. See, generally, Nucleic Acid hybridization, supra . The baεic requirement iε that hybridization conditionε be of εufficient εtringency εo that εelective hybridization occurs; i.e., hybridization is due to a εufficient degree of nucleic acid homology (e . g. , at leaεt about 75%), aε oppoεed to nonεpecific binding. Once a clone from the εcreened library has been identified by poεitive hybridization, it can be confirmed by reεtriction enzyme analysis and DNA sequencing that the particular library insert contains a gene for the desired protein.
Alternatively, DNA sequences encoding the proteinε of interest can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the particular amino acid εequence. In general, one will εelect preferred codonε for the intended hoεt if the εequence will be uεed for expression. The complete εequence is assembled from overlapping oligonucleotides prepared by εtandard methods and assembled into a complete coding sequence. See, e . g. , Edge (1981) Nature 292:756; Nambair et al . , (1984) Science 223:1299; Jay et al . , (1984) J. Biol . Chem . 259:6311. Once coding sequences for the deεired proteins have been prepared or iεolated, they can be cloned into any εuitable vector or replicon. Numerouε cloning vectorε are known to those of εkill in the art, and the εelection of an appropriate cloning vector iε a matter of choice. Exampleε of recombinant DNA vectorε for cloning and hoεt cellε which they can tranεform include the bacteriophage λ (E. coli ) , pBR322 (E. coli) , pACYC177 (E. coli) , pKT230 (gram-negative bacteria) , pGVH06 (gram-negative bacteria) , pLAFRl (gram-negative bacteria) , pME290 (non-.E. coli gram-negative bacteria) , pHVl4 (E. coli and Bacillus subtiliε) , pBD9 (Bacillus) , pIJ6l (Streptomyceε) , pUC6 (Streptomyces) , YIp5 (Saccharomyceε) , YCpl9 (Saccharomyceε) and bovine papilloma viruε (mammalian cellε) . See, generally , DNA Cloning; Volε. I & II, supra ; Sambrook et al . , supra; B. Perbal, supra.
The gene can be placed under the control of a promoter, riboεome binding εite (for bacterial expreεεion) and, optionally, an operator (collectively referred to herein aε "control" elementε) , εo that the DNA εequence encoding the deεired protein iε tranεcribed into RNA in the hoεt cell tranεformed by a vector containing thiε expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. Leader sequenceε can be removed by the hoεt in poεt-translational proceεεing. See , e . g. , U.S. Patent Noε. 4,431,739; 4,425,437; 4,338,397.
In addition to control sequences, it may be desirable to add regulatory εequenceε which allow for regulation of the expreεεion of the protein εequenceε relative to the growth of the hoεt cell. Regulatory εequences are known to those of skill in the art, and examples include those which cause the expresεion of a gene to be turned on or off in response to a chemical or phyεical εtimuluε, including the preεence of a regulatory compound. Other typeε of regulatory elements may also be present in the vector, for example, enhancer sequences.
An expression vector is constructed εo that the particular coding sequence is located in the vector with the appropriate regulatory sequenceε, the poεitioning and orientation of the coding sequence with reεpect to the control εequenceε being εuch that the coding εequence iε tranεcribed under the "control" of the control εequenceε (i.e., RNA polymeraεe which bindε to the DNA molecule at the control εequenceε tranεcribeε the coding εequence) . Modification of the εequenceε encoding the particular antigen of intereεt may be deεirable to achieve thiε end. For example, in some caseε it may be neceεεary to modify the εequence εo that it may be attached to the control εequenceε with the appropriate orientation; i.e., to maintain the reading frame. The control sequences and other regulatory εequenceε may be ligated to the coding εequence prior to inεertion into a vector, εuch aε the cloning vectors described above. Alternatively, the cod- ing sequence can be cloned directly into an expresεion vector which already containε the control εequenceε and an appropriate reεtriction εite.
In εome caεeε, it may be desirable to add εequenceε which cauεe the εecretion of the polypeptide from the hoεt organiεm, with εubεequent cleavage of the εecretory εignal. It may alεo be deεirable to produce mutantε or analogε of the antigenε of intereεt. Mutantε or analogε may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by subεtitution of one or more nucleotideε within the sequence. Technique™ for modifying nucleotide sequenceε, such as site-directed utagenesis, are well known to those εkilled in the art. See, e .g. , Sa brook et al . , supra ; DNA Cloning. Vols. I and II, supra; Nucleic Acid Hybridization, supra .
A number of procaryotic expression vectors are known in the art. See, e .g. , U.S. Patent Nos. 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437;
4,418,149; 4,411,994; 4,366,246; 4,342,832; see also U.K. Patent Applications GB 2,121,054; GB 2,008,123; GB 2,007,675; and European Patent Application 103,395. Yeast expresεion vectorε are alεo known in the art. See, e .g. , U.S. Patent Noε. 4,446,235; 4,443,539; 4,430,428; see alεo European Patent Applicationε 103,409; 100,561; 96,491.
Depending on the expreεεion εyεtem and hoεt . selected, the proteinε of the present invention are produced by growing host cells transformed by an expres¬ sion vector described above under conditions whereby the protein of interest is expreεεed. The protein iε then iεolated from the hoεt cellε and purified. If the expreεεion εyεtem εecreteε the protein into growth media, the protein can be purified directly from the media. If the protein is not secreted, it iε iεolated from cell lyεateε. The εelection of the appropriate growth condi¬ tionε and recovery methodε are within the εkill of the art. OmlA antigenε can alεo be iεolated directly from any of the A. pleuropneumoniae εerotypeε. Thiε iε generally accomplished by first preparing a crude extract which lacks cellular components and several extraneous proteins. The desired antigens can then be further purified, i.e., by column chromatography, HPLC, immunoadsorbent techniques or other conventional methods well known in the art.
The proteins of the present invention may also be produced by chemical synthesis such as solid phase peptide synthesiε, uεing known amino acid εequenceε or amino acid εequenceε derived from the DNA εequence of the geneε of intereεt. Such methodε are known to thoεe skilled in the art. Chemical syntheεis of peptideε may be preferable if a εmall fragment of the antigen in queεtion iε capable of raiεing an immunological reεponεe in the εubject of intereεt.
The proteinε of the preεent invention or their fragmentε can be uεed to produce antibodieε, both polyclonal and monoclonal. If polyclonal antibodieε are deεired, a εelected mammal, (e .g. , mouse, rabbit, goat, horεe, pig etc.) iε immunized with an antigen of the preεent invention, or itε fragment, or a mutated antigen. Serum from the immunized animal iε collected and treated according to known procedureε. If εeru containing polyclonal antibodieε iε uεed, the polyclonal antibodieε can be purified by immunoaffinity chromatography, uεing known procedureε.
Monoclonal antibodieε to the proteinε of the preεent invention, and to the fragmentε thereof, can alεo be readily produced by one εkilled in the art. The general methodology for making monoclonal antibodies by uεing hybridoma technology iε well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniqueε εuch as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e . g. , M. Schreier et al . , Hybridoma Technigues (1980); Hammerling et al . , Monoclonal Antibodies and T-cell Hvbridomaε (1981); Kennett et al . , Monoclonal Antibodies (1980); see alεo U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890. Panels of monoclonal antibodieε produced againεt the antigen of interest, or fragment thereof, can be screened for various properties; i.e., for iεotype, epitope, affinity, etc. Monoclonal antibodies are uεeful in purification, uεing immunoaffinity techniqueε, of the individual antigenε which they are directed againεt.
Animalε can be immunized with the compositions of the present invention by administration of the protein of intereεt, or a fragment thereof, or an analog thereof. If the fragment or analog of the protein iε uεed, it will include the amino acid εequence of an epitope which interactε with the immune εyεtem to immunize the animal to that and εtructurally εimilar epitopeε.
If εynthetic or recombinant proteinε are employed, the εubunit antigen can be a εingle polypeptide encoding one or εeveral epitopeε from one or more OmlAε or two or more diεcrete polypeptideε encoding different epitopeε. The εubunit antigen, even though carrying epitopes derived from a lipoprotein, does not require the presence of the lipid moiety. However, if the lipid is present, it need not be a lipid commonly asεociated with the lipoprotein, εo long aε the appropriate immunologic reεponse iε elicited.
Prior to immunization, it may be desirable to increase the immunogenicity of the particular protein, or an analog of the protein, or particularly fragments of the protein. This can be accomplished in any one of εeveral wayε known to those of skill in the art. For example, the antigenic peptide may be administered linked to a carrier. Suitable carriers are typically large, εlowly metabolized macromoleculeε εuch aε: proteinε; polyεaccharides, such aε εepharoεe, agaroεe, celluloεe, celluloεe beadε and the like; polymeric amino acidε εuch as polyglutamic acid, polylysine, and the like; amino acid copoly erε; and inactive viruε particleε. Especially uεeful protein εubstrates are εerum albuminε, keyhole limpet hemocyanin, immunoglobulin moleculeε, thyroglobulin, ovalbu in, and other proteinε well known to thoεe skilled in the art.
The protein εubεtrateε may be uεed in their na¬ tive form or their functional group content may be modified by, for example, εuccinylation of lyεine reεidueε or reaction with Cyε-thiolactone. A εulfhydryl group may alεo be incorporated into the carrier (or antigen) by, for example, reaction of amino functionε with 2-iminothiolane or the N-hydroxyεuccinimide eεter of 3-(4-dithiopyridyl propionate. Suitable carrierε may alεo be modified to incorporate spacer arms (εuch aε hexamethylene diamine or other bifunctional moleculeε of εimilar size) for attachment of peptideε.
Other εuitable carrierε for the proteinε of the preεent invention include VP6 polypeptideε of rotaviruεeε, or functional fragmentε thereof, aε diεcloεed in U.S. Patent No. 5,071,651. Alεo uεeful iε a fuεion product of a viral protein and the εubject immunogens made by methods discloεed in U.S. Patent No. 4,722,840. Still other εuitable carrierε include cellε, εuch as lymphocytes, since presentation in this form mim¬ ics the natural mode of presentation in the subject, which gives riεe to the immunized εtate. Alternatively, the proteins of the present invention may be coupled to erythrocytes, preferably the subject,ε own erythrocytes. Methodε of coupling peptideε to proteinε or cellε are known to thoεe of εkill in the art.
The novel proteinε of the inεtant invention can alεo be adminiεtered via a carrier viruε which expreεseε the εame. Carrier viruεeε which will find uεe with the instant invention include but are not limited to the vaccinia and other pox viruseε, adenoviruε, and herpeε viruε. By way of example, vaccinia virus recombinantε expressing the novel proteins can be constructed as follows. The DNA encoding the particular protein iε firεt inεerted into an appropriate vector εo that it iε adjacent to a vaccinia promoter and flanking vaccinia DNA sequenceε, such aε the sequence encoding thymidine kinase (TK) . This vector is then used to tranεfect cellε which are simultaneouεly infected with vaccinia. Homologouε recombination εerveε to inεert the vaccinia promoter pluε the gene encoding the inεtant protein into the viral genome. The reεulting TK~ recombinant can be εelected by culturing the cellε in the preεence of 5-bromodeoxy- uridine and picking viral plaqueε reεistant thereto.
It iε alεo poεεible to immunize a εubject with a protein of the preεent invention, or a protective fragment thereof, or an analog thereof, which iε adminiεtered alone, or mixed with a pharmaceutically acceptable vehicle or excipient. Typically, vaccineε are prepared aε injectableε, either aε liquid εolutionε or εuεpenεionε; εolid for ε εuitable for εolution in, or εuεpenεion in, liquid vehicleε prior to injection may alεo be prepared. The preparation may alεo be emulεified or the active ingredient encapεulated in lipoεome vehicleε. The active immunogenic ingredient iε often mixed with vehicles containing excipients which are pharmaceutically acceptable and compatible with the ac¬ tive ingredient. Suitable vehicleε are, for example, water, εaline, dextrose, glycerol, ethanol, or the like, and combinationε thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary εubstanceε εuch aε wetting or emulεifying agentε, pH buffering agentε, or adjuvantε which enhance the effectiveneεs of the vaccine. Adjuvantε may include for example, muramyl dipeptides, avridine, aluminum hydroxide, oils, saponins and other substanceε known in the art. Actual methodε of preparing εuch dosage forms are known, or will be appar¬ ent, to those skilled in the art. See, e.g.. Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pennεylvania, 15th edition, 1975. The compoεition or formulation to be adminiεtered will, in any event, contain a quantity of the protein adequate to achieve the deεired immunized εtate in the individual being treated. Additional vaccine formulationε which are εuit¬ able for other modeε of adminiεtration include εup- poεitorieε and, in εome caεeε, aeroεol, intranaεal, oral formulationε, and εuεtained releaεe formulationε. For εuppositories, the vehicle compoεition will include traditional binderε and carrierε, εuch aε, polyalkaline glycolε, or triglycerideε. Such εuppoεitorieε may be formed from mixtureε containing the active ingredient in the range of about 0.5% to about 10% (w/w) , preferably about 1% to about 2%. Oral vehicleε include εuch normally employed excipientε aε, for example, pharmaceutical gradeε of mannitol, lactoεe, εtarch, magneεium, εtearate, εodium εaccharin celluloεe, magneεium carbonate, and the like. Theεe oral vaccine compoεitionε may be taken in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, -and contain from about 10% to about 95% of the active ingredient, preferably about 25% to about 70%.
Intranasal formulationε will usually include vehicles that neither cause irritation to the nasal mucoεa nor εignificantly diεturb ciliary function. Diluentε εuch aε water, aqueouε εaline or other known εubεtanceε can be employed with the εubject invention. The naεal formulations may also contain preservativeε εuch aε, but not limited to, chlorobutanol and benzalkonium chloride. A εurfactant may be preεent to enhance absorption of the subject proteins by the naεal mucoεa.
Controlled or εustained release formulations are made by incorporating the protein into carriers or vehicleε such as liposo eε, nonreεorbable impermeable polymerε such aε ethylenevinyl acetate copolymerε and Hytrel® copolymerε, εwellable polymerε such aε hydrogelε, or reεorbable polymerε εuch aε collagen and certain polyacidε or polyeεterε such as thoεe uεed to make reεorbable εutureε. The proteinε can alεo be delivered uεing implanted mini-pumpε, well known in the art. Furthermore, the proteinε (or complexeε thereof) may be formulated into vaccine compoεitionε in either neutral or εalt formε. Pharmaceutically acceptable εaltε include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acidε such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic baseε εuch aε, for example, εodiu , potaεεium, ammonium, calcium, or ferric hydroxideε, and εuch organic baεeε aε iεopropylamine, trimethylamine, 2-ethylamino ethanol, hiεtidine, procaine, and the like.
To immunize a εubject, the polypeptide of intereεt, or an immunologically active fragment thereof, iε ad iniεtered parenterally, uεually by intramuscular injection in an appropriate vehicle. Other modes of administration, however, such as εubcutaneouε, intravenouε injection and intranaεal delivery, are alεo acceptable. Injectable vaccine formulationε will contain an effective amount of the active ingredient in a vehicle, the exact amount being readily determined by one εkilled in the art. The active ingredient may typically range from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate. The quantity to be administered depends on the animal to be treated, the capacity of the animal's immune system to εynthesize antibodieε, and the degree of protection deεired. With the preεent vaccine formulationε, aε little aε 0.1 to 100 μg or more, preferably 0.5 to 50 μg, more preferably 1.0 to 25 μg, of active ingredient per ml of injected εolution, should be adequate to raise an immunological reεponεe when a doεe of 1 to 2 ml per animal iε adminiεtered. Other effective doεageε can be readily eεtabliεhed by one of ordinary εkill in the art through routine trialε eεtabliεhing doεe reεponεe curveε. The εubject iε immunized by administration of the particular antigen or fragment thereof, or analog thereof, in at least one dose, and preferably two doseε. Moreover, the animal may be adminiεtered aε many doεeε as is required to maintain a state of immunity to pneumonia. An alternative route of administration involves gene therapy or nucleic acid immunization. Thus, nucleotide εequenceε (and accompanying regulatory elementε) encoding the εubject proteinε can be adminiεtered directly to a subject for in vivo translation thereof. Alternatively, gene transfer can be accomplished by transfecting the εubject'ε cellε or tiεεueε ex vivo and reintroducing the tranεformed material into the hoεt. DNA can be directly introduced into the hoεt organiεm, i.e., by injection (see International Publication No. WO/90/11092; and Wolff et al . , Science (1990) 247:1465-1468). Liposome-mediated gene transfer can alεo be accompliεhed uεing known methods. See, e . g. , Hazinski et al . , Am . J. Reεpir. Cell Mol . Biol . (1991) 4:206-209; Brigha et al . , Am . J. Wed. Sci . (1989) 298:278-281; Canonico et al . , Clin . Reε . (1991) 39:219A; and Nabel et al., Science (1990) 249:1285-1288. Targeting agentε, εuch aε antibodieε directed againεt εurface antigenε expreεεed on εpecific cell types, can be covalently conjugated to the liposomal surface so that the nucleic acid can be delivered to εpecific tiεεueε and cellε susceptible to A. pleuropneumoniae .
Below are examples of specific embodiments for carrying out the present invention. The exampleε are of- fered for illustrative purposeε only, and are not intended to limit the εcope of the preεent invention in any way.
Depoεits of Strains Useful in Practicing the Invention A deposit of biologically pure cultures of the following εtrainε waε made with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, under the proviεionε of the Budapeεt Treaty. The acceεεion number indicated waε aεεigned after εucceεεful viability teεting, and the requiεite feeε were paid. Theεe depoεitε are provided merely aε a convenience to thoεe of εkill in the art, and are not an admiεεion that a deposit is required. The nucleic acid sequences of theεe plasmidε, aε well aε the amino εequenceε of the polypeptideε encoded thereby, are controlling in the event of any conflict with the deεcription herein. A licenεe may be required to make, uεe, or εell the depoεited materialε, and no εuch licenεe iε hereby granted.
Strain Depoεit Date ATCC No.
HB101/pOM37/El (in E. coli) 4/7/92 68954
HB101/pSR213/E25 (in E. coli) 10/8/92 69083
C. Experimental
Materials and Methodε Enzymes were purchased from commercial sources, and used according to the manufacturers' directionε. Radionucleotides and nitrocelluloεe filters were alεo purchaεed from commercial εourceε.
In the cloning of DNA fragmentε, except where noted, all DNA manipulationε were done according to εtandard procedureε. See Sambrook et al . , supra . Reεtriction enzymeε, T4 DNA ligaεe, E. coli , DNA polymeraεe I, Klenow fragment, and other biological reagentε were purchaεed from commercial supplierε and uεed according to the manufacturerε' directionε. Double εtranded DNA fragmentε were εeparated on agaroεe gelε.
Bacterial Strainε. Plaε idε and Media
A. pleuropneumoniae εerotype 1 εtrain AP37 and A. pleuropneumoniae εerotype 5 εtrain AP213 were iεolated from the lungε of diεeaεed pigε given to the Weεtern College of Veterinary Medicine, Univerεity of Saskatchewan, Saskatoon, Saskatchewan, Canada. A. pleuropneumoniae serotype 7 strain AP205 was a Nebraska clinical isolate obtained from M.L. Chepok, Modern Veterinary Products, Omaha, Nebraska. Other A. pleuropneumoniae strainε were field iεolates from herds in Saskatchewan. The E. coli εtrain HB101 (hsdM, hsdR, recA) waε used in all transformationε uεing plasmid DNA. E. coli εtrainε NM538 (supF , hsdR) and NM539 (supF, hsdR, P2cox) served as hostε for the bacteriophage λ library. The plaεmidε pGH432 and pGH433 are expreεεion vectorε containing a tac promoter, a tranεlational εtart εite with restriction enzyme siteε allowing ligation in all three reading fra eε followed by εtop codonε in all reading frameε. A. pleuropneumoniae εtrainε were grown on PPLO medium (Difco Laboratorieε, Detroit, MI) supplemented with 10 mg/ml S-nicotinamide adenine dinucleotide (Sigma Chemical Co., St. Louiε, MO). Plate cultureε were incubated in a C02-enriched (5%) atmoεphere at 37°C. Liquid cultureε were grown with continuouε εhaking at 37°C without C02 enrichment.
Iron reεtriction waε obtained by adding 2,2'- dipyridyl to a final concentration of 100 μmol. E. coli tranεformantε were grown in Luria medium (Sambrook et al . , supra) εupplemented with ampicillin (100 mg/1) . Tranεcription from the taσ-promoter waε induced by the addition of iεopropylthioglactopyranoεide (IPTG) to a final concentration of 1 mmol.
Preparation and Analysis of Culture Supernatantε, Outer Membranes and Protein Aggregates.
Culture supernatantε, outer membraneε, and aggregated protein were prepared aε previouεly deεcribed (Gerlach et al . , Infect . Immun . (1992) 60:892-898;
Deneer, H.G., and Potter, -A.A. , Infect . Immun . (1989) 57:798-804). Culture εupernatantε were mixed with two volu eε of absolute ethanol and kept at -20°C for 1 h. Precipitateε were recovered by centrifugation and reεuεpended in water. Outer membraneε were prepared by εarkoεyl εolubilization aε previouεly deεcribed (Deneer and Potter, supra) . For the preparation of protein aggregateε, broth cultures (50 ml) in mid log phase (OD^ of 0.6) were induced by the addition of 1 mmol isopropyl- thiogalactoεide (IPTG; final concentration) . After 2 hourε of vigorouε εhaking at 37°C, cellε were harveεted by centrifugation, reεuεpended in 2 ml of 25% εucroεe, 50 mmol Triε/HCl buffer pH 8, and frozen at -70°C. Lyεiε was achieved by the addition of 5 μg of lysozyme in 250 mmol Triε/HCl buffer pH 8 (5 min on ice) , addition of 10 ml detergent mix (5 partε 20 mmol Triε/HCl buffer pH 8 (5 min on ice) , addition of 10 ml detergent mix (5 parts 20 mmol Tris/HCl buffer pH 7.4, 300 mmol NaCl, 2% deoxycholic acid, 2% NP-40, and 4 partε of 100 mmol Triε/HCl buffer pH 8, 50 mmol ethylenediamine tetraacetic acid, 2% Triton X-100) , and by εonication. Protein aggregateε were harveεted by centrifugation for 30 min at 15,000 g. Aggregate protein waε reεuεpended in H20 to a concentration of 5-10 mg/ml and solubilized by the addition of an equal volume of 7 molar guanidine hydrochloride. The concentration of protein in the aggregate preparations was determined by separating serial dilutions of the protein using SDS-PAGE. The . intenεity of the Coomaεεie blue εtained bands was compared with thoεe of a bovine εerum albumin standard (Pierce Chemical Co., Rockford, IL) .
Western Blotting
Whole cell lysateε of A . pleuropneumoniae grown in broth under iron-reεtricted conditionε were εeparated by SDS-PAGE and electroblotted onto nitrocelluloεe membraneε eεεentially aε deεcribed by Towbin et al. (Towbin et al . , Proc . Natl . Acad . Sci . U. S .A . (1979) 76:4350-4354). Nonεpecific binding waε blocked by incubation in 0.5% gelatine in waεhing buffer (150 mmol εaline, 30 mmol Triε-HCl, 0.05% Triton-XlOO) . Antibody and alkaline phosphatase conjugate (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) were added in waεhing buffer, and each incubated for 1 h at room temperature. Blots were developed with a subεtrate containing 5-bromo-4-chloro-3-indolyl phoεphate (BCIP) and nitro blue tetrazolium (NBT) (ImmunoSelect, BRL, Gaithersburg, MD) in 100 mmol Tris/HCl buffer pH 9.5, 50 mmol NaCl, 5 mmol MgCl2. Preparation of Antiεera
Serum againεt an A. pleuropneumoniae culture supernatant waε obtained aε followε. A. pleuropneumoniae εerotype 1 culture supernatant was precipitated with 10% trichloroacetic (TCA; vol/vol) , emulεified with incomplete Freund'ε adjuvant, and uεed to immunize rabbitε twice at three-week intervalε. Porcine convaleεcent εera were obtained from pigε experimentally infected intranasally by aerosol with A. pleuropneumoniae serotype 1 strain AP37.
Preparation of DNA and Southern Blotting
Genomic DNA was prepared by SDS-facilitated freeze-thaw induced lysiε aε deεcribed previouεly (Stauffer, G.V., et al . , Gene , (1981) 14:63-72). Plaεmid DNA waε prepared from 100 μg/ml chloramphenicol-amplified cultureε by alkaline lyεiε and ceεium chloride-ethidium bromide gradient centrifugation previouεly deεcribed (Sambrook et al . , supra) . Restriction endonuclease digeεtε were done in
T4 DNA polymeraεe buffer (Sambrook et al . , supra) εupplemented with 1 mmol dithiothreitol and 3 mmol εpermidine. Digeεted DNA waε εeparated on 0.7% agaroεe gels and transferred onto nitro cellulose by capillary blotting. [32P]-labelled probes were prepared by random priming (Feinberg, A.P., and Vogelstein, B. (1983) Anal. Bioαheτn. 132:6-13), and unincorporated nucleotideε were removed by passage through a Sephadex G-50 column. Filters were prehybridized in 5x Denhardt's εolution-6x SSC (lx SSC is 0.15 mol NaCl, 0.015 mol sodium citrate
(pH 8))-0.5% SDS at 65°C. Filterε were hybridized in the εame εolution at 55°C and waεhed at 55°C in 3x SSC-0.5% (low εtringency) , or at 65°C in O.lx SSC-0.5% SDS (high εtringency) . Preparation and Screening of the A. pleuropneumoniae Serotype 1 Expreεεion Library
Genomic DNA from A. pleuropneumoniae AP37 was partially digested with the reεtriction endonucleaεe Sau3AI. Fragmentε of 3000 Bp to 8000 Bp were isolated by εucroεe denεity gradient centrifugation (Sambrook et al . , supra) and ligated into the BamHI and Bglll sites of the expresεion vectorε pGH432 and pGH433, thuε allowing for fuεionε in all three reading frameε. E. coli HB101 waε tranεformed and plated at a denεity of approximately 400 colonieε per plate. Colonieε were replica-plated onto nitrocelluloεe diεkε, induced for 2 h with 1 mmol IPTG, and lyεed in chloroform vapor. Nonεpecific binding waε blocked with 0.5% gelatin in the waεhing buffer and, after removal of the cellular debriε, the membraneε were incubated with rabbit εerum raiεed againεt the A. pleuropneumoniae AP37 culture εupernatant and developed uεing goat anti-rabbit conjugate and εubεtrate aε deεcribed above.
Transposon Mutagenesiε
The tranεpoεon TnphoA, carried by a lamba phage, aε well aε the alkaline phoεphataεe-negative E. coli εtrain CC118, were provided by J. Beckwith, Harvard Medical School, Boεton, MA. The utageneεiε waε performed aε previouεly deεcribed (Manoil, C. , and Beckwith, J. (1985) Proc. Natl . Acad. Sci . U.S .A. 82:8129-8133) and the nucleotide εequence at the inεertion εite waε determined uεing an oligonucleotide primer complementary to the firεt 20 baεeε of the phoA- gene in TnphoA (Chang et al . (1986) Gene 44:121-125; Manoil and Beckwith, supra) . Nucleotide Seguence Analvεiε
DNA εequencing waε performed uεing M13 vectorε and the dideoxy chain termination method eεεentially aε deεcribed (Sanger, F. , et al . (1977) Proc. Natl . Acad. Sci . U.S.A. 74:5463-5467). Neεted deletionε were prepared by exonucleaεe III treatment (Henikoff, S. (1987) Methods in Enzymology 155:156-165). Specific primers were syntheεized uεing the Pharmacia Gene Aεεembler (Pharmacia Canada Ltd. , Baie D'Urfe, Quebec, Canada) . Both εtrandε were εequenced in their entirety. The open reading frame (ORF) of the omlA gene waε confirmed by TnpλoA inεertion mutageneεiε aε deεcribed above. The εequence waε analyzed uεing the IBI/Puεtell program and the GenBank databaεe.
Primer Extension Mapping
RNA was prepared from A. pleuropneumoniae AP37 essentially as described by Emory and Belasco (Emory, S.A., and Belaεco, J.G. (1990) J. Bacteriol . 172:4472- 4481). Briefly, 25 ml of bacterial culture (OD^ = 0.4) waε cooled on cruεhed ice'-and centrifuged. The bacterial pellet waε resuspended in 250 μl of 10% sucroεe, 10 mM sodium acetate (pH 4.5), and frozen at -70'C. The pellet was thawed by mixing with an equal volume of hot (70*C) 2% SDS, 10 mM sodium acetate (pH 4.5). Then, 375 μl of hot (70*C) H20-equilibrated phenol waε added, the tubeε were vortexed, frozen at -70 C, and εpun for 10 min in an Eppendorf centrifuge. The clear εupernatant waε removed, 2.5 volumeε of ethanol waε added, and the RNA waε εtored at -70"C until needed. The primer extenεion waε done as described previously using a primer complementary to a sequence within the ORF. 7-Deaza-dGTP and AMV-reverse transcriptase were employed in order to prevent compressions. Intrinεic Radiolabelling with r3H1-Palmitic Acid. Immunoprecipitation and Globomvcin Treatment
Labelling waε done eεεentially aε deεcribed previouεly (Ichihara, S. et al . (1981) J. Biol . Chem . 256:3125-3129). Briefly, [9,10-3H] palmitic acid with a specific radioactivity of 55 Ci/mmol in toluene (Amersham Corp., Arlington Heights, IL) waε lyophilized and diεεolved in iεopropanol to a concentration of 5 mCi/ml. A. pleuropneumoniae AP37 (in PPLO-broth) and E. coli tranεformantε (in Luria broth containing 1 μmol IPTG were grown with methanol, and an immunoprecipitation analyεiε waε performed eεεentially aε previouεly deεcribed (Huang, et al . (1989) J. Bacteriol . 171:3767-3774). The OmlA- εpecific εerum was obtained from immunized pigs, and protein G-Sepharose waε uεed to recover the OmlA-porcine antibody co plexeε. The immunoprecipitated proteinε were reεuεpended in SDS-εample buffer, heated to 80°C for 5 min and εeparated by SDS-PAGE. The gelε were fixed, treated with Amplify (Amerεham Corp., Arlington Heightε, IL) , dried and exposed to X-ray film. Globomycin was disεolved in 50% dimethylεulfoxide at a concentration of 10 mg/ml. Thiε εolution waε added to an A . pleuropneumo¬ niae AP37 culture grown to an OD^Q of 0.6 to a final concentration of 100 μg/ml. and growth was continued for ι hour. Cells were pelleted, resuspended in sample buffer and analyzed by SDS-PAGE and electroblotting onto nitrocelluloεe, aε deεcribed above, uεing the O lA- εpecific εerum. EXAMPLES Example 1 Cloning and Expresεion of the A. pleuropneumoniae Serotype 1 omlA Gene An expreεεion library of A. pleuropneumoniae εtrain AP37 εerotype 1 in the vector pGH432 lad waε εcreened with rabbit polyclonal antiεerum generated againεt a concentrated culture εupernatant of A. pleuropneumoniae by a colony im unoblot aεεay aε deεcribed above. Colonieε reacting with εerum raiεed againεt the culture εupernatant were εubcultured, induced with IPTG, and examined in a Western blot using porcine convalescent serum. From among thoεe cloneε which reacted in the colony immunoblot aεεay, one clone which alεo reacted with convaleεcent εerum waε εelected for further εtudy. The E. coli tranεformant produced a protein which co-migrated with an i munoreactive protein from A. pleuropneumoniae AP37, and had an electrophoretic mobility of 50k Da. Upon IPTG induction, thiε transformant produced the immunoreactive protein in
•aggregated form. The plaέ id encoding thiε antigen was designated as p0M37/El (ATCC Accession No. 68954) , and the protein was designated as OmlA.
Physical mapping showed that the plasmid contained a 5,000 Bp insert. Several deletion derivatives were constructed, and it was observed that transformants containing the deletion derivative POM37/E17 produced a truncated protein, thuε indicating that the encoding gene overlaps the Kpnl restriction enzyme site.
The nucleotide sequence of the gene encoding OmlA from pOM37/El is εhown in Figure 1. The εequence waε determined by dideoxy εequencing of overlapping deletionε generated by exonucleaεe III digeεtion. The nucleotide εequence has one long open reading frame (ORF) εtarting at nucleotide position 158 and ending at position 1252. The amino acid sequence of this open reading frame is alεo shown in Figure 1. The predicted polypeptide has a molecular weight of 39,780, with a consensus sequence for lipid modification at amino acid residue 20. In order to confirm thiε, cellε were labelled with [3H]-paImitate and immunoprecipitated with rabbit antiεera generated againεt the recombinant protein aε deεcribed above. Following polyacrylamide gel electrophoresis and autoradiography, one band with an apparent molecular weight of 50,000 was observed, indicating that lipid modification of the polypeptide had occurred. Further, when globo ycin was added, no [3H]- palmitate-labelled material was viεible on the autoradiogram. Globomycin iε a εpecific inhibitor of εignal peptidaεe II. Thuε, the omlA gene product iε a lipoprotein. Thiε may explain why it migrateε on polyacrylamide gelε with an apparent molecular weight of 50,000 when the predicted value iε leεs than 40,000. Immunoreactive product was expresεed in
•tranεformantε even in the ^abεence of IPTG induction. Thiε εuggeεts that a promoter recognizable by E. coli waε located on the A. pleuropneumoniae-derived DNA upstream of the ORF. The εimultaneouε inducibility by IPTG, aε well aε the truncated polypeptide produced by E. coli pOM37/E17 tranεformantε/ indicated the location of the carboxy-terminal of the omlA 'gene aε well aε itε direction of tranεcription. •Example 2 Analysis of Plasmid pOM37/E16 Colonies reacting with serum raised against the culture supernatant were εubcultured, induced with IPTG, and examined in a Weεtern blot aε deεcribed in Example 1. The εmalleεt plaε id expreεsing the full-length OmlA protein was deεignated pOM37/El6. Nucleotide sequence analysiε of pOM37/E16 revealed one ORF of 1083 Bp in length coding for a protein with a predicted molecular aεε of 39,780 Da. It waε preceded by a Shine-Dalgarno conεenεuε εequence AAGGAA 8 Bp upεtream of the methionine codon. The protein encoded by the nucleotide sequence of pOM37/E16 is identical to that shown in Figure 1.
The firεt 19 amino acidε of the polypeptide have the characteriεticε of a lipoprotein εignal peptide with a predicted cleavage εite in front of the cyεteine reεidue at position 20. The ORF was confirmed by two independent TnphoA-insertionε 50 bp and 530 bp downεtream from the methionine codon which, upon tranεformation of the phoA-negative E. coli εtrain CC118, gave riεe to alkaline phoεphatase-poεitive tranεformantε. A GenBank data baεe homology εearch uεing the predicted amino acid εequence of OmlA did not reveal likely εi ilaritieε (>35%) to known ORFε or polypeptideε. The primer extenεion located the beginning of the mRNA at a T-residue 76 Bp upstream of the methionine εtart codon. The -10 and -30 regionε are both AT-rich, and the promoter-εtructure matcheε the E. coli conεenεuε characteriεtics. One of the TnphoA-inεertions was found to be located within the signal peptide. The expreεεion of a functional PhoA protein in thiε fuεion iε probably due to itε location behind the hydrophobic core of the εignal peptide. The transcriptional εtart εite aε determined by primer extension analysis is preceded by a -10 and -30 region εimilar to thoεe common in E. coli promoterε, Roεenberg, M. , and Court, D. , (1979) Annu . Rev. Genet. 13:319-353, and thiε finding is in accordance with the expresεion found in noninduced E. coli tranεformants. Downεtream of the ORF, a palindromic sequence of 26 bp in length iε preεent which might act aε a terminator sequence. Adhya, S., and Gottes an, M. , (1978) Annu. J?ev. Biochem . 47:967-996.
The predicted εignal peptide cleavage εite reεulting in an amino-terminal cyεteine reεidue of the mature protein waε confirmed by labelling of the E. coli tranεformantε with [14C]-palmitate and εubεequent immunoprecipitation uεing porcine anti-O lA εerum. In addition, it was shown that growth of A. pleuropneumoniae AP37 in the preεence of globomycin inhibited the palmitate-labelling of OmlA aε well aε the proceεεing of the OmlA precurεor protein.
The expreεεion of the OmlA protein waε independent from the level of iron in the growth medium. The protein waε present in whole membranes, outer membranes as prepared by εucroεe gradient centrifugation, and membrane blebε; it waε abεent in εarcoεyl-treated outer membraneε and in high-εpeed εupernatantε.
Example 3
Cloning, Expression and Seguencing of the A . pleuropneumoniae Serotype 5 omlA Gene Genomic DNA from A. pleuropneumoniae εerotype 5 εtrain AP213 waε digested to completion with Styl and ligated into the Wcol site of the pGH432 lacl-derivative, pAA505. HB101 recombinants were εcreened with convaleεcent εerum obtained from a pig which had been infected with A. pleuropneumoniae εerotype 5. One positive clone, HB101/pSR213/El, was selected for further analysiε. HB101/pSR213/El waε εhown to contain three Styl fragmentε. In order to iεolate the DNA coding for the immunoreactive protein, Styl fragmentε from thiε plaεmid were treated with DNA polymeraεe I Klenow fragment to fill in the 5' extenεionε. Theεe fragmentε were ligated into the Smal εite of the vector, pGH432/lacI. A εeroreactive clone, deεignated HB101/pSR213/E4, waε iεolated and εhown to produce a εeroreactive protein with an apparent molecular weight of 50 kDa. However, the protein waε not expreεεed at high levelε. To increaεe the level of expreεεion, plaεmid pSR213/Er waε digeεted with Bgill (which cutε the vector εequence upεtream of the gene) and then partially digeεted with Asel (which cutε at the beginning of the coding region of the gene). The 5' extenεionε were filled in with DNA polymeraεe I Klenow fragment, and the plaεmid recircularized by ligation. The reεulting clone, HB101/pSR213/E25 (ATCC Acceεεion No. 69083), overexpreεεed the εeroreactive protein.
Both εtrandε of the A. pleuropneumoniae εerotype 5 omlh gene were sequenced using M13 vectors as described above. The nucleotide sequence and predicted amino acid εequence are εhown in Figure 2. The open reading frame εhown in the figure codeε for a protein εimilar to the omlA product of A. pleuropneumoniae εerotype 1, εhowing approximately 65% identity at the amino acid level. Thuε, the open reading frame preεent in pSR213/E25 codeε for the εerotype 5 equivalent of omlA.
Example 4 Diεtribution of the omlA gene in the
A. pleuropneumoniae type εtrainε. Genomic DNA from all 12 A. pleuropneumoniae type εtrainε waε analyzed in a Southern blot uεing the A. pleuropneumoniae AP37-derived omlA-gene aε probe. The Styl-reεtricted DNA from all A. pleuropneumoniae type εtrainε reacted with the probe under low εtringency conditionε, and the DNA from εerotypeε 1, 2, 8, 9, 11, and 12 remained hybridized to the probe under high εtringency washing conditions.
Whole cell lysates from all A. pleuropneumoniae type strains, grown under iron-restricted conditionε, were analyzed in a Weεtern blot uεing the εerum from pigs immunized with the recombinant OmlA protein. The same εtrainε that hybridized to the DNA probe under high εtringency washing conditions bound the anti-O lA sera, and the whole cell lyεateε from the A. pleuropneumoniae type εtrainε for εerotypeε 1, 9, and 11 reacted more εtrongly than thoεe of εerotypeε 2 , 8, and 12.
Example 5 The Protective Capacity of Serotype 1 OmlA Recombinant Protein The OmlA protein waε prepared from E. coli
HB101/pOM37/El by IPTG-induction of a log phaεe culture followed by cell harvest and disruption, and separation of the inclusion bodies by centrifugation. The inclusion bodies were εolubilized with guanidine hydrochloride and mixed with Emulεigen Plus (MVP Laboratories, Ralston, Nebraεka) and εaline εo that the final protein concentration waε 0.5 μg/ml, 2.5 μg/ml or 12.5 μg/ml. Groupε of 7 pigs were vaccinated with 2 ml of the vaccines or a placebo containing Emulsigen Plus but no protein. Each group was revaccinated 21 days later and finally challenged 7 dayε after the booεt with an aeroεol of A. pleuropneumoniae (serotype 1) . Clinical signε of diεeaεe were followed for 3 dayε, and 7 dayε after challenge all εurvivorε were euthanized. The significance of the difference in mortality rates among the different groupε waε determined uεing a G2 likelihood ratio teεt (Dixon, W.J., et al . , BMDP Statiεtical Software Manual. Univerεity of California Preεε, 1988, pp. 229-273.) The reεultε are εummarized in Table l.
Table 1. Protective Capacity of OmlA Againεt Challenge with Actinobacillus pleuropneumoniae εerotype 1.
GROUP MORTALITY CLINICAL SCORE Day 1 Day 2 Day 3 Day 1 Day 2 Day 3
Figure imgf000044_0001
Within 2 dayε of challenge, all of the pigε which received the placebo were dead while only 1 of the OmlA-vaccinateε had died. Clinical εignε of diεeaεe were εignificantly lower in the vaccinates on day 1 post- challenge, the only day on which a comparison could be made due to high mortality in the placebo group. Thuε, the omlk gene product of A. pleuropneumoniae (εerotype 1) iε an effective immunogen for the prevention of porcine pleuropneumonia cauεed by A. pleuropneumoniae . Immunization of pigε with the recombinant OmlA protein induced a εtrong immune reεponεe and εignificantly lowered mortality. Theεe reεultε demonstrate that protection against A. pleuropneumoniae εerotype 1 can be achieved by immunization with a single protein antigen. Since the recombinant protein used for the vaccination trial waε produced aε an aggregate in E. coli , the lipid modification doeε not appear to be neceεsary for the induction of a protective immune responεe.
Example 6
The Protective Capacity of Serotype 5
OmlA Recombinant Protein OmlA protein waε prepared from HB101/pSR213/E25 and formulated with E ulεigen Pluε aε deεcribed in Example 5 εo that each 2 ml doεe contained 25 μg of protein. Pigε were vaccinated, booεted and challenged with A. pleuropneumoniae εerotype 5 εtrain AP213 aε deεcribed in Example 5. The reεultε εhown in Table 2 indicate that vaccination with OmlA from εerotype 5 reduced morbidity, mortality and lung damage aεεociated with Actinobacillus pleuropneumoniae infection. It iε predicted that vaccination with both εerotype l and εerotype 5 OmlA proteins would protect pigs against infection with all A. pleuropneumoniae serotypeε, with the poεsible exception of serotype 11.
Figure imgf000046_0001
Table 2.
Protective Capacity of OmlA Against Challenge with
Actinobacillus pleuropneumoniae serotype 5.
Figure imgf000046_0003
Figure imgf000046_0002
Thuε, εubunit vaccineε for uεe againεt A. pleuropneumoniae are disclosed, as are methods of making and using the same. Although preferred embodiments of the subject invention have been described in some detail, it iε underεtood that obviouε variationε can be made without departing from the εpirit and the εcope of the invention aε defined by the appended claims.

Claims

1. A purified, Actinobacilluε pleuropneumoniae outer membrane protein, wherein the protein is an immunogenic Actinobacilluε pleuropneumoniae outer membrane lipoprotein A, or an immunogenic fragment thereof.
2. The protein of claim 1 wherein said protein is serotype 1 outer membrane liporprotein A comprising an amino acid sequence subεtantially homologouε and functionally equivalent to the amino acid εequence of SEQ ID N0:1, or an immunogenic fragment thereof.
3. The protein of claim 1 wherein εaid protein iε εerotype 5 outer membrane lipoprotein A compriεing an amino acid εequence εubstantially homologous and functionally equivalent to the amino acid sequence of SEQ ID NO:2, or an immunogenic fragment thereof.
4. An isolated nucleotide sequence comprising a sequence encoding an immunogenic Actinobacilluε pleuropneumoniae outer membrane protein according to any of claims 1-3.
5. A DNA construct comprising:
(a) a nucleotide sequence according to claim 4; and
(b) control sequences that are operably linked to said nucleotide sequence whereby said nucleotide sequence can be transcribed and translated in a host cell, and wherein at least one of said control εequences iε heterologouε to εaid nucleotide εequence.
6. A host cell tranεformed by a DNA conεtruct according to claim 5.
7. A method of producing an immunogenic Actinobacilluε pleuropneumoniae outer membrane protein, εaid method compriεing:
(a) providing a population of hoεt cellε according to claim 6; and
(b) growing εaid population of cellε under conditionε whereby the protein encoded by εaid DNA conεtruct iε expressed.
8. A vaccine composition comprising a pharmaceutically acceptable vehicle and at leaεt one Actinobacilluε pleuropneumoniae outer membrane protein according to any of claimε 1-3.
9. The vaccine compoεition of claim 8 further compriεing an adjuvant.
10. A method of treating or preventing an Actinobacilluε pleuropneumoniae infection in a vertebrate εubject compriεing adminiεtering to εaid εubject a therapeutically effective amount of a vaccine composition according to claim 8.
11. A method of treating or preventing an Actinobacilluε pleuropneumoniae infection in a vertebrate subject comprising administering to said subject a therapeutically effective amount of a vaccine composition according to claim 9.
PCT/CA1993/000448 1992-11-05 1993-11-03 Actinobacillus pleuropneumoniae outer membrane lipoprotein a and uses thereof Ceased WO1994010316A1 (en)

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