WO1998024912A2 - Recombinant plague vaccine - Google Patents

Recombinant plague vaccine Download PDF

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Publication number
WO1998024912A2
WO1998024912A2 PCT/US1997/022617 US9722617W WO9824912A2 WO 1998024912 A2 WO1998024912 A2 WO 1998024912A2 US 9722617 W US9722617 W US 9722617W WO 9824912 A2 WO9824912 A2 WO 9824912A2
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recombinant
antigen
nucleic acid
molecule
group
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PCT/US1997/022617
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French (fr)
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WO1998024912A3 (en
Inventor
Elizabeth J. Haanes
Rex E. Thomas
Jorge E. Osorio
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Heska Corporation
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Priority to AU55979/98A priority Critical patent/AU5597998A/en
Publication of WO1998024912A2 publication Critical patent/WO1998024912A2/en
Publication of WO1998024912A3 publication Critical patent/WO1998024912A3/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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a recombinant vaccine to protect animals against plague. More particularly, the invention includes recombinant molecules containing isolated nucleic acid molecules that encode antigens from Yersinia, Pasteurella, or Francisella expressed in eukaryotic cells.
  • Yersinia pestis is enzootic in several wild animal reservoirs, particularly squirrels and wild mice. The disease is also frequently epizootic in prairie dogs. Plague and plague-like diseases caused by other species of Yersinia, Pasteurella, and Francisella are also enzootic in wild reservoirs. Although the enzootic reservoirs are not a severe direct threat to humans, infection of domestic cats from wild reservoirs is a very serious problem in that several pet owners and veterinarians have contracted infection from these animals; see, for example, Doll et al., 1994, Am. J. Trop. Med. Hyg., 51, 109-114.
  • plague is one of the factors responsible for the diminishing population of black-footed ferrets (Mustela nigripes) in the western United States.
  • black-footed ferrets Mustela nigripes
  • this nearly extinct species relies solely on prairie dogs as its food source; thus epizootic episodes of plague in prairie dog populations in the black-footed ferrets' habitat contribute to the depletion of this species; see, for example, Williams et al., 1994, J. Wildl. Dis., 30, 581-585.
  • methods to control plague in the wild reservoirs are also needed. It would be particularly useful to reduce plague in these wild reservoirs through the release of baited, orally delivered vaccines.
  • vaccines include a formulation comprising formalin-killed Yersinia pestis (Cutter USP, available from Greer Laboratories, Lenoire, NC) and a formulation comprising a modified live Yersinia pestis (Y. pestis EV76-6).
  • LD 50 refers to the number of bacterial cells in a dose that, when administered to a group of animals, will kill 50% of the animals in that group.
  • the LD 50 is typically about one bacterial cell.
  • FI antigen administered as an isolated subunit vaccine proved to be protective against plague challenge in mice, but less protective than the killed whole cell vaccines or avirulent live vaccines described above; see, for example, Simpson et al., 1990, Am. J. Trop. Med. Hyg., 43, 389-96.
  • animals were not well protected by intragastric administration of the vaccine formulation; see, for example, Thomas et al., 1992, Am. J. Trop. Med. Hyg., 47, 92-7.
  • the complete nucleotide sequence encoding a Y. pestis FI antigen has been disclosed in Galyov et al., 1990, FEBSLett., 277, 230-232, which is incorporated herein by reference.
  • Prokaryotic recombinant cell-based plague vaccines have also been disclosed.
  • a live recombinant Salmonella typhimurium expressing the FI antigen was protective in mice against a low-level Y. pestis challenge, i.e., less than 50 LD 50 s.
  • This vaccine formulation was hampered by instability of the construct in vivo; see, for example PCT Publication No. WO 95/18231, published July 6, 1995, by Titball et al.
  • Another example discloses vaccines comprising Salmonella minnesota, expressing FI; see, for example Russian Publication No. RU 2046145, published October 20, 1995, by Anisimov et al.
  • V antigen on the lcr plasmid of Y. pestis Another antigen being evaluated for vaccine potential is the V antigen on the lcr plasmid of Y. pestis.
  • This protein is highly antigenic and when administered with FI antigen, the two subunits are nearly as protective as the attenuated live Y. pestis vaccine; see, for example, Leary et al, 1995, Infect. Immun., 63, 2854-2858, Williamson et al., 1995, FEMS Immunol. Med. Microbiol, 12, 223-230, and PCT Publication No. WO 95/24475, published September 14, 1995, by Titball et al.
  • the V antigen however, has been shown to be associated with suppression of gamma interferon and TNF-alpha in vivo.
  • Raccoon poxvirus has been shown to be a very safe and effective vaccine vector in a variety of animal species, and particularly for cats. Protection against rabies, feline panleukopenia, and feline infectious peritonitis viruses has been demonstrated in cats vaccinated with recombinant RCN expressing antigens from the respective viruses; see, for example U.S. Patent No. 5,266,313, issued November 30, 1993, by Esposito et al., PCT Publication No. WO 93/01284, published January 21, 1993, by Scott et al, and European Publication No. EP 94306917.9, published May 10, 1995, by Wasmoen et al.
  • a particularly attractive feature of recombinant raccoon poxvirus vaccine formulations in cats is that the vaccine can be delivered orally, which is a preferred administration route both for cats and for wildlife.
  • Advantages of oral administration include a high incidence of injection site-associated sarcomas in cats, and the propensity of an orally delivered vaccine to induce mucosal immunity.
  • the inventors are not aware of any prior use of raccoon poxvirus for the expression of an antigen from a bacterial pathogen. In fact, only one citation was found for the expression of a protein from a bacterial pathogen in an animal virus vector; see, PCT Publication No. WO 90/15872, published December 27, 1990, by Fischetti et al.
  • nucleic acid immunization is apparently a safe, inexpensive method of protecting animals from disease; see, for example, Wolff et al., 1990, Science 247, 1465- 1468.
  • Nucleic acid vaccines are administered to an animal in a fashion to enable expression of protective proteins in the animal.
  • a number of delivery methods for nucleic acid vaccines are known in the art including either intramuscular or intradermal injection, intradermal scarification using skin-test applicators, and particle bombardment (e.g. "gene-gun") delivery; see, for example, Raz et al., Proc. Natl. Acad. Sci. USA, 93, 5141-5145, U.S. Patent No.
  • the present invention relates to a novel recombinant molecule in which one or more nucleic acid molecules encoding antigens from Yersinia, Pasteurella, or Francisella are operatively linked to one or more eukaryotic transcription control regions, such that the antigen(s) are expressed in eukaryotic cells.
  • Two primary embodiments of the present invention are a recombinant virus and a recombinant plasmid. Other embodiments include a recombinant animal virus genome and a recombinant eukaryotic cell.
  • Also included in the present invention are methods to produce a recombinant molecule, a recombinant plasmid, a recombinant animal virus genome, a recombinant animal virus, and a recombinant eukaryotic cell of the present invention.
  • a bacterial antigen in a live viral vector or in a eukaryotic cell
  • the idea is attractive because eukaryotic expression provides the possibility of inducing improved humoral, mucosal and cell-mediated immune responses.
  • the present invention also includes therapeutic compositions, such as vaccines, that are capable of protecting an animal from contracting plague.
  • Therapeutic compositions of the present invention include recombinant molecules that include isolated nucleic acid molecules that encode Yersinia, Pasteurella, or Francisella antigens, operatively linked to eukaryotic transcription control regions.
  • Such therapeutic compositions include a recombinant animal virus genome, a recombinant virus and a recombinant cell expressing one or more antigens derived from Yersinia, Pasteurella, or Francisella, and a recombinant plasmid that expresses one or more antigens from Yersinia, Pasteurella, or Francisella when the plasmid is delivered into a eukaryotic cell.
  • a preferred therapeutic composition of the present invention also includes an excipient, an adjuvant and/or a carrier. Also included in the present invention is a method to protect an animal from plague, which includes administering to the animal a therapeutic composition of the present invention.
  • Another embodiment of the present invention is an isolated nucleic acid molecule encoding a Yersinia pestis antigen fused, in frame, with a eukaryotic membrane anchor domain.
  • Preferred embodiments of the present invention include (a) a recombinant raccoon poxvirus genome that includes an isolated nucleic acid molecule encoding a Yersinia pestis antigen operatively linked to a poxvirus transcription control region, (b) a recombinant raccoon poxvirus including such a recombinant genome, (c) a recombinant cell including such a recombinant genome, (d) a recombinant plasmid that includes an isolated nucleic acid molecule encoding a Yersinia pestis antigen operatively linked to a eukaryotic transcription control region, and (e) a recombinant cell that includes such a recombinant plasmid.
  • a particularly preferred eukaryotic transcription control region is the human cytomegalovirus (HCMV) immediate-early promoter.
  • HCMV human cytomegalovirus
  • Particularly preferred embodiments of the present invention include, but are not limited to, an isolated nucleic acid molecule nYpFlanc 576 having nucleic acid sequence SEQ LD NO:7, and encoding protein PYpFlanc 192 , having amino acid sequence SEQ LD NO:8, and the complement of SEQ LD NO:7; recombinant molecules vRCN-pl 1- nYpFl(a)sec 544 , pCMV-nYpFl(b)sec 544 , P CMV-nYpFlanc 576 , and pCMV-nYpFlmat 474 ; recombinant virus RCN:pl l-nYpFl(a)sec 544 ; and recombinant cell BSC-1 :RCN:pl 1- n
  • the present invention discloses recombinant molecules that comprise isolated nucleic acid molecules encoding antigens from Yersinia, Pasteurella, or Francisella that are operatively linked to eukaryotic transcription control regions and, as such, are expressed under eukaryotic transcription control in eukaryotic cells. Also included in the present invention are therapeutic compositions comprising the claimed recombinant molecules, which are useful to protect animals from plague. Further included in the present invention are methods, using the claimed therapeutic compositions, to protect animals from plague.
  • Yersinia, Pasteurella, or Francisella antigens from eukaryotic transcription control regions is a novel aspect of the current invention. While not being bound by theory, the inventors believe that the current invention will have significant utility as a plague vaccine, in that the embodiments of the current invention will be efficacious in controlling plague in animal populations (including in wild animal populations); the claimed vaccines will be economical to make and use, will be safer, and will have significantly reduced side-effects relative to currently available vaccines to control plague.
  • Preferred embodiments of the present invention include recombinant live virus vaccines and genetic immunization (i.e. naked nucleic acid) vaccines.
  • Live virus vaccines and genetic immunization vaccines are advantageous because they are believed to confer more vigorous and longer-lasting immunity than subunit or killed vaccines. While not being bound by theory, it is believed that such advantages are due to the ability of the genetic information carried by the virus or the recombinant molecule to enter the cells of the treated animal, and to direct the expression of a protective compound, such as a protective protein or a protective RNA, for extended periods of time. Thus, therapeutic compositions of the present invention need not be administered frequently.
  • One particularly preferred embodiment of the present invention is an orally delivered plague vaccine for domestic cats.
  • Cats are susceptible to the pneumonic form of the disease which is much more easily transmitted to other animals, including humans. While not being bound by theory, the inventors believe that oral delivery of a therapeutic composition of the present invention will induce mucosal immunity, which will more effectively control pneumonic plague.
  • Yersinia, Pasteurella, and Francisella refer to bacterial genera, and as such, include any species belonging to any of these genera.
  • Particularly preferred bacterial species to target using embodiments of the present invention include Yersinia pestis (previously referred to as Pasteurella pestis, the name having changed about 1971) , Yersinia pseudotuberculosis, Yersinia enter ocolitica, Pasteurella multocida, and Francisella tularensis, with Y. pestis being even more preferred, as this species is believed to be the most common etiologic agent of plague.
  • a Yersinia, Pasteurella, or Francisella antigen refers to an antigen derived from any portion of a Yersinia, Pasteurella, or Francisella bacterium that is capable of being expressed from an isolated nucleic acid molecule, and that, when administered to an animal as an immunogen, will produce a humoral, mucosal, and/or cellular immune response against Yersinia, Pasteurella, or Francisella in that animal.
  • the ability of a candidate antigen to effect an immune response can be measured using techniques known to those skilled in the art, some of which are disclosed herein.
  • a or “an” entity refers to one or more of that entity; for example, an isolated nucleic acid molecule refers to one or more isolated nucleic acid molecules, or at least one isolated nucleic acid molecule.
  • an isolated nucleic acid molecule refers to one or more isolated nucleic acid molecules, or at least one isolated nucleic acid molecule.
  • the terms “a” (or “an), “one or more” and “at least one” can be used interchangeably herein.
  • the terms “comprising”, “including”, and “having” can be used interchangeably.
  • a compound “selected from the group consisting of refers to one or more of the compounds in that group, including mixtures (i.e., combinations) of two or more of the compounds.
  • An antigen of the present invention includes not only full-length antigens but also homologs of full-length Yersinia, Pasteurella, or Francisella antigens, including smaller portions of such antigens.
  • the term homolog refers to any closely related antigen or epitope capable of eliciting an immune response to the native antigen. Examples of homologs include Yersinia, Pasteurella, or Francisella proteins in which amino acids have been deleted (e.g.
  • an epitope refers to the smallest portion of an antigen that is capable of eliciting an immune response in an animal.
  • the minimal size of a protein epitope is about five amino acids. It is to be noted, however, that such an epitope might comprise a portion of the antigen other than the amino acid sequence, e.g., a carbohydrate moiety.
  • Yersinia, Pasteurella, or Francisella antigen homologs can also be the result of natural allelic or strain variation, natural mutation or laboratory-induced mutation in the genes that encode the antigens.
  • a gene includes all nucleic acid sequences related to a nucleic acid molecule that encodes a protein, such as regulatory regions that control production of the protein encoded by that nucleic acid molecule (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself.
  • allelic variant, strain variant, or variant (used interchangeably herein) of an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen refers to a nucleic acid molecule encoding an antigen at essentially the same locus (or loci) in the genome as the nucleic acid molecule in question but which, due to natural strain variations caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence.
  • Such variants typically encode proteins having similar activity to that of the protein encoded by the nucleic acid molecule to which they are being compared, but they do not necessarily have identical amino acid sequences.
  • Allelic and strain variants can also comprise alterations in the 5' or 3' untranslated regions of a gene comprising the nucleic acid molecule (e.g., in regulatory control regions). Allelic and strain variants are well known to those skilled in the art and would be expected to be found to varying extents among the surface antigen genes of a pathogenic microorganism. Also included in the definition of variants are laboratory-induced mutants, such as variants arising due to errors incorporated into a nucleic acid molecule encoding an antigen during PCR amplification.
  • Such errors can alter the nucleic acid sequence of a gene in question, and, as such, may also alter the amino acid sequence resulting in, for example, an amino acid substitution or the introduction of a stop (termination) codon, thus truncating the resultant antigen.
  • a mutant is included as a variant if the gene in question encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared, e.g., in the case of the present invention, the protein must still be capable of eliciting an immune response in an animal against a Yersinia, Pasteurella, or Francisella native antigen.
  • a bacterial antigen expressed in a recombinant eukaryotic cell might be altered in ways that will vary its presentation to an animal's immune system. While not being bound by theory, the inventors believe t hat antigens secreted from a cell, anchored to the cell's membrane, or remaining in the cytoplasm of the cell will be processed and reacted to differently by an animal's immune system. In such a way, the immune response to a particular antigen can be altered from, for example, a completely humoral response to one that includes a shift toward a cellular immune response. Methods to introduce alterations into antigens of the current invention are known to those skilled in the art, and examples are disclosed herein.
  • a nucleic acid molecule encoding a secreted protein can usually be converted into a nucleic acid molecule encoding a cytoplasmic protein by deleting the secretory signal segment from the nucleic acid molecule.
  • a nucleic acid molecule encoding a secreted protein can be engineered to be anchored to a cell's membrane by fusing a portion of a nucleic acid molecule encoding the antigen in-frame with an appropriate heterologous nucleic acid molecule encoding a membrane anchor domain.
  • Yersinia, Pasteurella, or Francisella antigens of the current invention can include any antigen, including homologs thereof, derived from these bacterial genera, that, when administered to an animal as an immunogen, using techniques known to those skilled in the art, will produce a humoral, mucosal, and/or cellular immune response against Yersinia, Pasteurella, or Francisella in that animal.
  • Preferred Yersinia, Pasteurella, and Francisella antigens include Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Pasteurella multocida, and Francisella tularensis antigens.
  • Preferred Yersinia pestis antigens of the present invention include FI antigens, V antigens, pesticin antigens, W antigens, pH 6 antigens, superoxide dismutase antigens, Yersinia outer protein (YOP) antigens, high molecular weight iron- regulated membrane protein antigens, murine toxin antigens, and/or hemin storage protein antigens. More preferred Yersinia pestis antigens of the present invention include FI antigens and V antigens. An even more preferred Yersinia pestis antigen is an FI antigen. The most preferred antigens of the present invention include
  • SEQ LD NO:2 amino acid sequences of which are presented herein as SEQ LD NO:2, SEQ LD NO:8, SEQ LD NO:10, SEQ LD NO:12, and SEQ ID NO:21, respectively, as well as homologs and smaller portions, as small as a single epitope, of such antigens.
  • SEQ LD NO:2 amino acid sequences of which are presented herein as SEQ LD NO:2, SEQ LD NO:8, SEQ LD NO:10, SEQ
  • isolated nucleic acid molecule refers to a nucleic acid molecule derived, at least partially, from Yersinia, Pasteurella, or Francisella.
  • An isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu. As such, the term “isolated” does not necessarily reflect the extent to which the nucleic acid molecule has been purified.
  • An isolated nucleic acid molecule can include DNA, RNA, or derivatives of either DNA or RNA.
  • An isolated nucleic acid molecule can be single-stranded or double-stranded.
  • nucleic acid molecules for which the nucleic acid sequence of the coding strand is disclosed in a SEQ LD NO are also recognized to include a complementary strand, the nucleic acid sequence of which can be easily determined by one skilled in the art; such complementary sequences are included as part of the present invention.
  • An isolated nucleic acid molecule can be obtained from its natural source, or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis.
  • an isolated nucleic acid molecule encodes at least one Yersinia, Pasteurella, or Francisella antigen, examples of such antigens being disclosed herein. In one embodiment, the antigen is expressed by (i.e.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a Yersinia, Pasteurella, or Francisella antigen.
  • Francisella antigen can be produced using a number of methods known to those skilled in the art; see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, and Ausubel et al.,1993, Current Protocols in Molecular Biology, Greene/Wiley Interscience. Sambrook et al, ibid., and Ausubel et al, ibid., are incorporated by reference herein in their entireties.
  • nucleic acid molecules can be produced and/or modified using a variety of techniques including, but not limited to, by classic mutagenesis and recombinant DNA techniques (e.g., site- directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification), or synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • classic mutagenesis and recombinant DNA techniques e.g., site- directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification
  • synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • Isolated nucleic acid molecules of the present invention may also (a) contain secretory signal segments (i.e., nucleic acid sequences encoding a secretory signal peptide) to enable an expressed Yersinia, Pasteurella or Francisella antigen of the present invention to be secreted from the cell that produces the antigen, (b) contain nucleic acid sequences encoding membrane anchor domains that lead to the expression of nucleic acid molecules of the present invention as antigens anchored to the cell membrane of the host cell such that the antigenic domain is outside of the cell, and/or (c) contain other fusion sequences which lead to the expression of nucleic acid molecules of the present invention as various fusion proteins.
  • secretory signal segments i.e., nucleic acid sequences encoding a secretory signal peptide
  • Nucleic acid sequences encoding signal peptides, membrane anchor domains or other protein domains are fused in- frame with isolated nucleic acid molecules encoding Yersinia, Pasteurella or Francisella antigens of the present invention by methods known to those skilled in the art.
  • the term "fused in-frame" indicates that two or more heterologous nucleic acid molecules are combined such that a single contiguous amino acid sequence is encoded. Examples of suitable signal segments and membrane anchor segments are disclosed herein.
  • Isolated nucleic acid molecules of the present invention may also include intervening and/or untranslated sequences surrounding and/or within the isolated Yersinia, Pasteurella, or Francisella nucleic acid sequences.
  • Suitable signal segments include any signal segment encoding a signal peptide capable of directing the secretion of an antigen of the present invention.
  • signal segment refers to a nucleic acid molecule encoding a secretory signal peptide
  • signal peptide refers to the peptide domain capable of directing secretion of an antigen of the present invention.
  • signal segments encode peptides of about 15 to 50 amino acids in length, and are located at the 5' end of a nucleic acid molecule encoding a secreted protein, but they can be located at other (i.e., internal) positions within a nucleic acid molecule.
  • Preferred signal segments include, but are not limited to, endogenous signal segments of the isolated Yersinia, Pasteurella, or Francisella nucleic acid molecules of the present invention, as well as tissue plasminogen activator (t-PA) , interferon, interleukin, growth hormone, histocompatibility, and viral envelope glycoprotein signal segments.
  • Suitable membrane anchor segments include any membrane anchor segment that encodes a peptide domain capable of anchoring an antigen of the present invention into a eukaryotic cell membrane.
  • the term "membrane anchor segment” refers to the nucleic acid molecule encoding a peptide domain capable of anchoring an antigen of the present invention to a eukaryotic cell membrane.
  • a membrane anchor segment encodes a protein domain comprising hydrophobic amino acids.
  • Membrane anchor segments can be located either at the 5' end or the 3 1 end of a nucleic acid molecule encoding an anchored protein.
  • a membrane anchor segment located at the 5' end usually also functions as a secretory signal segment.
  • Preferred membrane anchor segments include, but are not limited to, vesicular stomatitis virus (VSV) glycoprotein, respiratory syncytial virus G protein, herpesvirus glycoprotein, immunoglobulin, and glycosyl-phosphotidylinositol membrane anchor segments.
  • Particularly preferred membrane anchor segments include the canine herpesvirus glycoprotein G, glycoprotein E and glycoprotein I membrane anchor segments.
  • Suitable intervening and/or untranslated sequences include, but are not limited to, any sequences that enhance or regulate expression of a Yersinia, Pasteurella, or Francisella antigen of the present invention.
  • Preferred untranslated sequences include the human cytomegalovirus (HCMV) intron-A sequence and the encephalomyocarditis virus internal ribosomal entry site (EMCV-IRES).
  • Isolated nucleic acid molecules of the present invention can include any nucleic acid molecule capable of encoding a Yersinia, Pasteurella, or Francisella antigen of the current invention.
  • Preferred isolated nucleic acid molecules encode at least one Yersinia pestis antigen of the present invention, including, but not limited to, an FI antigen, a V antigen, a pesticin antigen, a W antigen, a pH 6 antigen, a superoxide dismutase antigen, a YOP antigen, a high molecular weight iron-regulated membrane protein antigen, a murine toxin antigen, and a hemin storage protein antigen.
  • More preferred isolated nucleic acid molecules encode a Yersinia pestis FI antigen and /or a V antigen.
  • An even more preferred isolated nucleic acid molecule encodes a Yersinia pestis FI antigen.
  • Particulary preferred isolated nucleic acid molecules of the present invention include nYpFl(a)sec 544 , nYpFl(b)sec 544 , nYpFlsec 510 , nY ⁇ Flanc 576 , nYpFlanc 513 , nYpFlmat 474, nYpFlmat 450 , and nYpFlmat 447 , the coding strand nucleotide sequences of which are represented herein as SEQ ID NO:l, SEQ LD NO:3, SEQ LD NO:4, SEQ ID NO:7, SEQ LD NO:9, SEQ LD NO:ll, SEQ LD NO:13, and SEQ LD NO:22, respectively
  • Isolated nucleic acid molecules nYpFl(a)sec 544 and nYpFl(b)sec 544 each encode a full-length, unprocessed FI antigen of 170 amino acids, denoted herein as PYpFlsec, 70 , having an amino acid sequence denoted herein as SEQ LD NO:2, assuming an initiation codon extending from about nucleotide 17 through about nucleotide 19 of SEQ ID NO: 1 or SEQ LD NO:4, respectively, and a stop codon extending from about nucleotide 527 through about nucleotide 529 of SEQ ID NO:l or SEQ LD NO:4, respectively.
  • PYpFlsec ⁇ o includes an N-terminal signal peptide sequence of about 21 amino acids extending from about amino acid 1 to about amino acid 21 of SEQ LD NO:2.
  • the mature (i.e., processed) form of PYpFlsec 170 is represented by PYpFlmat 149 , having the amino acid sequence SEQ LD NO:21.
  • PYpFlmat !49 is encoded by nucleic acid molecule nYpFlmat, ⁇ , having the coding strand nucleotide sequence represented by SEQ LD NO:22, assuming a first codon extending from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:22.
  • the coding region encoding PYpFlsec, 70 , not including the stop codon, is represented by nucleic acid molecule nYpFlsec 510 , having the coding strand nucleic acid sequence represented by SEQ LD NO:3.
  • Isolated nucleic acid molecule nYpFlmat 474 encodes PYpFlmat 150 , a predicted mature FI antigen of about 150 amino acids, the sequence of which is presented herein as SEQ LD NO:12, assuming an initiation codon extending from about nucleotide 7 through about nucleotide 9 of SEQ LD NO:l 1 and a stop codon extending from about nucleotide 457 through about nucleotide 459 of SEQ LD NO:l 1.
  • the coding region encoding PYpFlmat 150 is represented by nucleic acid molecule nYpFlmat 450 , having the coding strand nucleic acid sequence represented by SEQ LD NO: 13. While not being bound by theory, an expressed antigen encoded by nYpFlmat 474 would be expected to stay in the cytoplasm of the cell in which it is expressed, since it lacks a secretory signal segment.
  • Isolated nucleic acid molecule nYpFlanc 576 the coding strand of which is denoted herein as SEQ LD NO:7, comprises a coding region, not including the stop codon, encoding PYpFlanc 192 , a novel fusion protein of about 192 amino acids comprising an FI antigen of about 134 amino acids linked to the membrane anchor domain of the canine herpesvirus (CHV) glycoprotein G (i.e., about amino acid 358 through about amino acid 415 of CHV gG, disclosed as SEQ LD NO:10 in pending U.S. Patent Application Serial No. 08/602,010, by Haanes, et al., filed Feb.
  • CHV canine herpesvirus
  • nYpFlanc 576 Methods to construct nYpFlanc 576 are disclosed herein. While not being bound by theory, a protein encoded by nYpFlanc 576 would be expected to be secreted from the cytoplasm of a eukaryotic cell such that its C-terminal region is lodged in the cell's plasma membrane, and its N- terminal region is extending outside the cell.
  • the amino acid sequence of PYpFlanc 192 is presented herein as SEQ ID NO: 8, assuming an initiation codon extending from about nucleotide 1 through about nucleotide 3 of SEQ LD NO:7.
  • PYpFlanc ⁇ 92 includes an N- terminal signal peptide sequence of about 21 amino acids extending from about amino acid 1 to about amino acid 21 of SEQ LD NO:8.
  • the mature form of PYpFlanc 192 is represented by PYpFlanc 171 , having the amino acid sequence SEQ ID NO:10.
  • PYpFlanc 171 is encoded by nucleic acid molecule nYpFlanc 513 , having the coding strand nucleotide sequence represented by SEQ ID NO:9.
  • Another embodiment of the present invention is an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen that also includes at least one additional isolated nucleic acid molecule fused in-frame such that a multivalent antigen is encoded.
  • a multivalent antigen can be produced by joining two or more isolated nucleic acid molecules together in such a manner that the resulting nucleic acid molecule is expressed as a multivalent antigen containing epitopes from at least two heterologous antigens, or portions thereof.
  • Such a multivalent antigen can comprise two or more isolated nucleic acid molecules encoding Yersinia, Pasteurella or Francisella antigens, or can comprise one or more isolated nucleic acid molecules in addition to those encoding Yersinia, Pasteurella or Francisella antigens, such that the multivalent antigen is capable of protecting an animal from diseases caused by other infectious agents in addition to Yersinia, Pasteurella or Francisella.
  • multivalent antigens include, but are not limited to, Yersinia,
  • Pasteurella or Francisella antigen of the present invention attached to one or more antigens protective against one or more other infectious agents, such as, but not limited to: viruses (e.g., caliciviruses, distemper viruses, hepatitis viruses, herpesviruses, immunodeficiency viruses, infectious peritonitis viruses, leukemia viruses, panleukopenia viruses, parvoviruses, picomaviruses, rabies viruses, other cancer-causing or cancer-related viruses); bacteria (e.g., Leptospira, Rochalimaea); fungi and fungal- related microorganisms (e.g., Candida, Cryptococcus, Histoplasma); and other parasites (e.g., Babesia, Cryptosporidium, Eimeria, Encephalitozoon, Hepatozoon, Isospora, Microsporidia, Neospora, Nosema, Plasmodium, Pneumocystis, Toxoplasm
  • a recombinant molecule of the present invention includes at least one isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen, operatively linked to a eukaryotic transcription control region.
  • Such a molecule contains heterologous nucleic acid sequences, that is, nucleic acid sequences that are not naturally found adjacent to the isolated nucleic acid molecules of the present invention and that are derived from species other than Yersinia, Pasteurella, or Francisella.
  • a recombinant molecule can be either RNA or DNA, can have components from prokaryotic as well as eukaryotic sources, and must have the ability, by methods described herein, to enter eukaryotic cells and direct expression of isolated nucleic acid molecules of the present invention in those eukaryotic cells.
  • the recombinant molecule is typically a recombinant animal virus genome or a recombinant plasmid.
  • an isolated nucleic acid molecule encoding a Yersinia, Pasteurella or Francisella antigen is operatively linked to a eukaryotic transcription control region.
  • operatively linked refers to the combining of an isolated nucleic acid molecule of the present invention with a eukaryotic transcription control region in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • a eukaryotic transcription control region is a nucleic acid sequence which controls the initiation, elongation and termination of transcription in a eukaryotic cell. Particularly important transcription control regions are those which control transcription initiation, such as promoter and enhancer sequences.
  • Suitable transcription control regions include any transcription control region that can function in at least one recombinant eukaryotic cell of the present invention.
  • a variety of such transcription control regions are known to those skilled in the art.
  • Preferred transcription control regions include those which function in mammalian cells, such as, but not limited to, promoter and enhancer sequences from alphaviruses (such as Sindbis virus), vaccinia virus, raccoon poxvirus, other poxviruses, adenovirus, adeno-associated vims, cytomegaloviruses (preferably the intermediate early promoter, preferably in conjunction with intron- A), other herpesviruses, simian vims 40 (preferably the early promoter), retroviruses (such as Rous sarcoma virus), and picomavimses (particularly an internal ribosome entry site, or IRES, enhancer region).
  • alphaviruses such as Sindbis virus
  • vaccinia virus such as Sindbis virus
  • transcription control regions include those derived from mammalian genes such as actin, heat shock protein, bovine growth hormone transcription control regions, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • One type of recombinant molecule of the present invention comprises an animal vims genome.
  • Such a recombinant molecule contains an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen of the present invention, operatively linked to at least one eukaryotic transcription control region capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transformed.
  • Eukaryotic transcription control regions for recombinant vims genomes of the present invention include any that would function in the vims of choice.
  • a recombinant animal vims genome can comprise a heterologous eukaryotic transcription control region, i.e., a transcription control region that is non-native to the particular animal vims genome, being, for example, derived from another animal vims genome or from any other suitable eukaryotic gene. Suitable heterologous franscription control regions are disclosed herein.
  • a recombinant animal vims genome of the present invention can also comprise an endogenous eukaryotic transcription control region, i.e., a transcription control region that is normally found in that vims.
  • Suitable recombinant vims genomes of the present invention include a poxvims genome, a herpesvirus genome, an alphavirus genome (for example, from a Sindbis vims), a picornavirus genome (for example, from a poliovims or a mengovims), a refrovims genome, an adenovims genome, or an adeno- associated vims genome.
  • a preferred recombinant vims genome of the present invention comprises a poxvims genome, for example, an orthopoxvirus, a parapoxvirus, an entomopoxvims, or an avipoxvims (i.e., fowlpox) genome.
  • a more preferred recombinant vims genome comprises an orthopoxvirus genome, particularly a vaccinia virus or a raccoon poxvims genome.
  • Preferred transcription control regions for a recombinant vims genome of the present invention include those that function in poxvimses. It will be appreciated by those skilled in the art that poxvimses undergo all aspects of viral replication, including transcription, in the cytoplasm of the infected host cell, and as such, have specialized, viral-encoded transcription-related proteins and recognition sequences. In particular, transcription of genes encoded on a poxvims genome requires a poxvims promoter. Examples of poxvims promoters include early/late promoters (i.e., working at early and late times in the infectious cycle of the vims) and late promoters.
  • poxvims promoters include the vaccinia vims p7.5 (early/late) promoter (see, for example, Cochran et al., 1985, J. Virol. 54, 30-37), the vaccinia vims pi 1 (late) promoter (see, for example, Bertholet et al., 1986, EMBO J. 5, 1951-1957), and the synthetic pSYN (late) promoter (see, for example, Davison, et al., 1990, Nucl. Acids Res. 18, 4825-4826).
  • vims genomes of the present invention are very large.
  • genes in such vimses are dispensable for growth of the vims (often referred to as non-essential genes).
  • one or more isolated nucleic acid molecules encoding a Yersinia, Pasteurella, or Francisella antigens, operatively linked to eukaryotic transcription control regions suitable for a particular vims can be located (preferably by insertion) in one or more non-essential genes.
  • an isolated nucleic acid molecule can be located between genes in the viral genome, i.e., in an intergenic region.
  • an isolated nucleic acid molecule of the present invention can be located in an essential gene. If the resulting recombinant vims is to undergo replication, this latter group of insertions requires that the essential gene be complemented in the infected host cell, either by that cell being stably transformed with the essential gene, transiently transformed with the essential gene, or having that gene supplied on a helper vims.
  • an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen can be located in any suitable non-essential gene or intergenic region.
  • Preferred non-essential genes include a thymidine kinase gene, a hemagglutination gene, an anti-inflammatory gene, and an A-type inclusion gene.
  • Preferred anti-inflammatory genes include a soluble cytokine receptor gene, a serpin gene, a complement receptor gene and an immunoglobulin receptor gene.
  • Particularly preferred non-essential genes are the thymidine kinase genes of raccoon poxvims and vaccinia vims.
  • Another recombinant molecule of the present invention comprises a recombinant plasmid which includes an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen operatively linked to a eukaryotic transcription control region.
  • a recombinant plasmid of the current invention has utility as a genetic immunization vaccine to protect an animal against plague.
  • a preferred recombinant plasmid contains an origin of replication for propagation in a bacterial host (e.g., Escherichia col ⁇ ), a mode of selection, such as an antibiotic resistance gene, and a suitable cloning site for isolated nucleic acid molecules of the present invention.
  • Any suitable eukaryotic transcription control region can be used, such as, but not limited to, those disclosed herein.
  • Particularly preferred transcription control regions include, but are not limited to, a HCMV immediate-early promoter (preferably in conjunction with intron-A), a Rous sarcoma vims long terminal repeat, and an SV40 early promoter.
  • the incorporation of polyadenylation sequences for example, bovine growth hormone or SV40 polyadenylation sequences, is also preferred.
  • a preferred recombinant plasmid can also include an enhancer region, for example, a HCMV intron-A sequence or an EMCV-IRES sequence.
  • a recombinant molecule of the present invention is a molecule that can include at least one of any isolated nucleic acid molecule that encodes an antigen from Yersinia, Pasteurella, or Francisella, operatively linked to at least one eukaryotic transcription control region capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transformed, examples of which are disclosed herein.
  • Preferred recombinant molecules include at least one of the following nucleic acid molecules: nYpFl(a)sec 544 , nYpFl(b)sec 544 , nYpFlsec 5!0 , nYpFlanc 576 , nYpFlanc 513 , nYpF 1 mat 474 ⁇ nYpF 1 mat 450 , and nYpF 1 mat ⁇ .
  • Particularly preferred recombinant molecules of the present invention include vRCN-pl l-nYpFl(a)sec 544) vRCN-pl 1- nYpF 1 anc 576 , vRCN-p 11 -nYpF 1 mat 474 , pCMV-nYpF 1 (b)sec 544 , pCMV-nYpF 1 anc 576 , and pCMV-nYpFlmat 474 .
  • vRCN refers to a recombinant raccoon poxvims genome
  • pCMV refers to a recombinant plasmid comprising the HCMV immediate-early transcription control region.
  • a recombinant vims of the present invention includes any animal vims comprising a suitable recombinant molecule, i.e. a recombinant vims genome, of the present invention.
  • Suitable recombinant vimses of the present invention include poxvimses, herpesviruses, alphavimses (for example Sindbis vims), picomaviruses (for example, poliovims or mengovims), retrovimses, adenovimses, and adeno-associated vimses.
  • a preferred recombinant vims of the present invention comprise a poxvims, for example, an orthopoxvims, a parapoxvirus, an entomopoxvims, or an avipoxvims (i.e., fowlpox).
  • a more preferred recombinant vims comprises a recombinant orthopoxvims, particularly a vaccinia vims or a raccoon poxvims.
  • An example of a more preferred embodiment of the present invention is a recombinant raccoon poxvims (RCN) comprising a nucleic acid molecule encoding an FI antigen of Yersinia pestis operatively linked to a vaccinia vims pi 1 promoter, as disclosed in Examples 1 and 2 below.
  • RCN raccoon poxvims
  • Particularly preferred recombinant vimses of the present invention comprise recombinant raccoon poxvimses RCN:p 11 -nYpF 1 (a)sec 544> RCN:p 11 -nYpF 1 anc 576, and RCN:p 11 -nYpF 1 mat 474- , with RCN:pl l-nYpFl(a)sec 544 being the more preferred.
  • One embodiment of the present invention is an attenuated recombinant vims.
  • an attenuated vims is a vims that results in less pathogenicity than its wild- type counterpart when used to infect an animal.
  • a preferred attenuated vims of the present invention causes little or no pathogenicity when used to infect an animal.
  • An attenuated recombinant vims can be produced by inactivating a viral gene that, due to that gene's inactivation, results in an attenuated vims. Methods to inactivate a gene are disclosed herein.
  • An attenuated recombinant vims can be identified by exposing animals to the vims and measuring clinical signs, such as fever, lesions, or viremia, in those animals compared to similar animals exposed to the wild-type vims. Clinical signs to measure vary with each individual vims, and are known to one skilled in the art.
  • Suitable viral genes to inactivate in order to produce an attenuated recombinant vims include any gene that when inactivated leads to an attenuated vims.
  • a preferred attenuated recombinant vims of the present invention is a vims having a recombinant genome in which a heterologous nucleic acid molecule, i.e., one encoding a Yersinia, Pasteurella, or Francisella antigen, is inserted into a viral gene, the insertion resulting in an attenuated vims.
  • a particularly preferred attenuated recombinant vims of the present invention is an attenuated recombinant vaccinia vims or raccoon poxvims.
  • a particularly preferred method of attenuation of a recombinant vaccinia vims or raccoon poxvims is by insertion of a heterologous nucleic acid molecule into the thymidine kinase (tk) locus.
  • An attenuated recombinant vims of the present invention particulary an attenuated recombinant raccoon poxvims, has utility, for example, as a therapeutic composition to protect an animal from plague. While not being bound by theory, the inventors believe that a recombinant raccoon poxvims need not be further attenuated for use as a live viral vaccine in most animals due to the low pathogenicity of wild-type RCN.
  • Another embodiment of the present invention is a recombinant cell comprising a eukaryotic host cell transformed with at least one of any recombinant molecule of the present invention. Suitable and preferred recombinant molecules with which to transform cells are disclosed herein.
  • the terms "transform” or “transformed”, as used in the present invention, refer to any way in which a recombinant molecule can be inserted into a cell.
  • Transformation techniques include, but are not limited to, transfection, viral infection with a recombinant vims, viral transduction with a recombinant vims, electroporation, microinjection, lipofection, adsorption, and protoplast fusion.
  • a recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
  • Suitable host cells to transform include any cell that can be transformed with a recombinant molecule of the present invention.
  • Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins useful in the production of a multivalent vaccines).
  • Host cells of the present invention can be any cell capable of producing at least one antigen of the present invention.
  • Preferred host cells primarily include mammalian cells.
  • Most preferred host cells include BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell line), CRFK cells (normal cat kidney cell line), BSC-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvims), COS (e.g., COS-7) cells, and Vero cells.
  • BHK cells baby hamster kidney cells
  • MDCK cells normal dog kidney cell line
  • CRFK cells normal cat kidney cell line
  • BSC-1 cells African monkey kidney cell line used, for example, to culture raccoon poxvims
  • COS e.g., COS-7 cells
  • Vero cells e.g., Vero cells.
  • Particularly preferred host cells are BHK cells, BSC-1 cells, MDCK cells, CRFK cells, CV-1 cells, COS cells, Rat-2 cells, Vero cells, and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).
  • Additional appropriate cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NLH/3T3 cells, LMTK 31 cells and/or HeLa cells.
  • fibroblast cell lines e.g., human, murine or chicken embryo fibroblast cell lines
  • myeloma cell lines e.g., Chinese hamster ovary cells
  • mouse NLH/3T3 cells e.g., mouse NLH/3T3 cells
  • LMTK 31 cells e.g., LMTK 31 cells
  • HeLa cells e.g., HeLa cells.
  • a recombinant cell of the present invention can include any eukaryotic host cell transformed with at least one of any recombinant molecule of the present invention.
  • a preferred recombinant cell includes at least one isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen operatively linked to at least one eukaryotic transcription control region capable of effectively regulating expression of the nucleic acid molecule(s) in that cell; particularly preferred nucleic acid molecules to include are nYpFl(a)sec 544 , nYpFl(b)sec 544 , nYpFlsec 510 , nYpFlanc 576 , nYpFlanc 513 , nYpFlmat 474, nYpFlmat 450 , and/or nYpFlmat ⁇ .
  • a more preferred recombinant cell includes one or more of the following recombinant molecules: vRCN-pl 1- nYpFl(a)sec 544 , vRCN-pl l-nYpFlanc 576 , vRCN-pl l-nYpFlmat 474 , pCMV- nYpF 1 (b)sec 544 , pCMV-nYpF 1 anc 576 , and pCMV-nYpF 1 mat 474 .
  • Particularly preferred recombinant cells of the present invention include BSC-1 :RCN:pll-nYpFl(a)sec 544 , BSC-1 :RCN-pl l-nYpFlanc 576 , BSC-l:RCN-pl l-nYpFlmat 474 , as well as any animal cells comprising pCMV-nYpFl(a)sec 544 , pCMV-nYpFlanc 576 , and pCMV-nYpFlmat 474 .
  • One embodiment of the present invention is a therapeutic composition that, when administered to an animal in an effective manner, is capable of protecting that animal from plague.
  • compositions of the present invention include a recombinant molecule comprising an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen operatively linked to a eukaryotic transcription control region.
  • Suitable therapeutic compositions include recombinant animal vims genomes, recombinant vimses, recombinant plasmids and recombinant cells as disclosed herein.
  • a therapeutic composition of the present invention is administered to the animal in an effective manner prior to infection in order to prevent disease, reduce disease symptoms and/or prevent transmission of the disease from asymptomatic carriers (i.e., as a preventative vaccine).
  • compositions of the present invention can be administered to any animal susceptible to such therapy, preferably to mammals, and more preferably to cats, primates, rodents, ungulates, bears, dogs, camels, and pigs.
  • Preferred animals to protect against plague include domestic cats, humans, bobcats, cougars, domestic dogs, coyotes, foxes, rock squirrels, ground squirrels, prairie dogs, black footed ferrets, domestic ferrets, pronghorn antelope, badgers, bears, wild boars, domestic pigs, camels, chipmunks, red deer, mule deer, fishers, foxes, gerbils, martens, urban mice, wild mice, polecats, rabbits, urban rats, wild rats, tree squirrels, and voles.
  • plague refers to the group of diseases most normally caused by the bacterium Yersinia pestis, but also, in some cases, similar diseases caused by other species within the genera Yersinia, Pasteurella, or Francisella.
  • plague includes, but is not limited to, diseases such as bubonic plague, septicemic plague, pneumonic plague, urban plague, rat plague, wild rodent plague, sylvatic plague, campestral plague, high plains plague, disseminated intravascular coagulopathy, la peste bubonique, The Pest, The Black Plague, and The Black Death.
  • diseases such as bubonic plague, septicemic plague, pneumonic plague, urban plague, rat plague, wild rodent plague, sylvatic plague, campestral plague, high plains plague, disseminated intravascular coagulopathy, la peste bubonique, The Pest, The Black Plague, and The Black Death.
  • Another embodiment of the present invention is a therapeutic composition to protect an animal from plague that also includes at least one additional isolated nucleic acid molecule encoding an antigen from a pathogen other than Yersinia, Pasteurella or Francisella, operatively linked to one or more eukaryotic transcription control regions, such that a multivalent therapeutic composition is produced.
  • a multivalent therapeutic composition can be produced by combining one or more additional isolated nucleic acid molecules into a recombinant molecule of the present invention, or by combining one or more recombinant molecules with a recombinant molecule of the present invention.
  • multivalent therapeutic compositions When administered to an animal, such a multivalent therapeutic composition is able to direct the expression of one or more antigens in the cells of that animal such that the animal is protected from diseases caused by other infectious agents in addition to Yersinia, Pasteurella or Francisella.
  • multivalent therapeutic compositions include, but are not limited to, a Yersinia, Pasteurella or Francisella antigen of the present invention plus one or more antigens protective against one or more other infectious agents, such as, but not limited to: vimses (e.g., calicivimses, distemper vimses, hepatitis vimses, herpesviruses, immunodeficiency vimses, infectious peritonitis vimses, leukemia vimses, panleukopenia vimses, parvovimses, picomavimses, rabies vimses, other cancer-causing or cancer-related vimses); bacteria (e.g., Leptospira, Rochalimaea);
  • compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate.
  • excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
  • Nonaqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.
  • Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran.
  • Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.
  • a therapeutic composition can include an adjuvant.
  • Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen.
  • Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of cytokines and chemokines (e.g., granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (LL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I (IGLF), transforming growth factor beta, RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), macrophage inflammatory proteins (e.g.
  • a therapeutic composition can include a carrier.
  • Carriers include compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, vimses, other cells, oils, esters, and glycols. While not being bound by theory, an advantage of a therapeutic composition comprising a recombinant vims is that a carrier is usually not required.
  • a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal.
  • a controlled release formulation comprises a composition of the present invention in a controlled release vehicle.
  • Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems.
  • Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ.
  • Preferred controlled release formulations are biodegradable (i.e., bioerodible).
  • compositions of the present invention include at least a portion of a recombinant vims genome, comprising a recombinant vims vaccine.
  • Preferred recombinant vims genomes include those based on alphavimses, poxvimses, adenovimses, adeno-associated vimses, picomavimses, herpesvimses, and retrovimses, with those based on poxvimses being particularly preferred.
  • a therapeutic composition comprising a recombinant vims of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration.
  • the recombinant molecule produces attenuated vims.
  • a number of recombinant viruses can be used, including, but not limited to, those based on alphavimses, poxvimses, adenovirases, adeno-associated vimses, picomavimses, herpesvimses, and retrovimses.
  • Preferred recombinant vims vaccines are those based on poxvimses.
  • a therapeutic composition comprising a genetic immunization (i.e., naked nucleic acid) vaccine of the present invention includes an isolated nucleic acid molecule of the present invention operatively linked to a eukaryotic transcription control region in a recombinant molecule of the present invention.
  • a genetic immunization vaccine of the present invention can comprise one or more nucleic acid molecules of the present invention in the form of, for example, a dicistronic recombinant molecule. Any suitable eukaryotic transcription control region can be used.
  • transcription control regions include the HCMV intermediate early promoter (preferably in conjunction with intron- A), Rous sarcoma vims long terminal repeat, and tissue-specific transcription control regions, as well as transcription control regions endogenous to viral vectors if viral vectors are used.
  • the incorporation of polyadenylation sequences and enhancers are also preferred.
  • a recombinant cell vaccine of the present invention includes recombinant eukaryotic cells of the present invention that express one or more Yersinia, Pasteurella, or Francisella antigens of the present invention.
  • Preferred recombinant cells for this embodiment include BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinant cells.
  • Recombinant cell vaccines of the present invention can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about 10 8 to about 10 12 cells per kilogram body weight.
  • Recombinant cell vaccines can comprise whole cells, cells stripped of cell walls or cell lysates.
  • the present invention also includes methods to protect an animal from plague using a therapeutic composition of the current invention.
  • a recombinant molecule of the present invention can be administered to an animal in a fashion to enable the recombinant molecule to enter one or more cells of the animal, such that an antigen encoded by an isolated nucleic acid molecule contained therein is expressed into a protective antigen in the animal.
  • Recombinant molecules of the present invention can be delivered to an animal by a variety of methods including, but not limited to, (a) administering a genetic immunization vaccine, e.g., naked DNA or RNA molecules, such as is taught, for example, in Wolff et al., ibid., or (b) administering a nucleic acid molecule packaged as a recombinant vims vaccine or as a recombinant cell vaccine (i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle).
  • a genetic immunization vaccine e.g., naked DNA or RNA molecules, such as is taught, for example, in Wolff et al., ibid.
  • a nucleic acid molecule packaged as a recombinant vims vaccine or as a recombinant cell vaccine i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle.
  • Genetic immunization vaccines of the present invention can be administered by a variety of methods. Suitable delivery methods include, for example, intramuscular injection, subcutaneous injection, intradermal injection, intradermal scarification, particle bombardment, oral application, and nasal application, with intramuscular injection, intradermal injection, intradermal scarification and particle bombardment being preferred.
  • a preferred single dose of a genetic immunization vaccine ranges from about 1 nanogram (ng) to about 1 milligram (mg), depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Examples of administration methods are disclosed, for example, in U.S. Patent No. 5,204,253, by Bruner, et al., issued April 20, 1993, PCT Publication No.
  • Genetic immunization vaccines of the present invention can be contained in an aqueous excipient (e.g., phosphate buffered saline) alone or with a carrier (e.g., lipid-based vehicles), or it can be bound to microparticles (e.g., gold particles).
  • aqueous excipient e.g., phosphate buffered saline
  • carrier e.g., lipid-based vehicles
  • microparticles e.g., gold particles
  • a recombinant vims vaccine of the present invention When administered to an animal, infects cells within the immunized animal and directs the production of a Yersinia, Pasteurella, or Francisella antigen that is capable of protecting the animal from plague.
  • a recombinant vims vaccine comprising an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen of the present invention is administered according to a protocol that results in the animal producing a sufficient immune response to be protected from a plague challenge.
  • a preferred single dose of a recombinant vims vaccine of the present invention is from about 1 x 10 4 to about 1 x 10 8 vims plaque forming units (pfu) per animal.
  • Administration protocols are well-known to those skilled in the art, with subcutaneous, intramuscular, intradermal, intranasal, and oral administration routes being preferred.
  • a particularly preferred method of administration, especially in cats, for a recombinant vims vaccine of the present invention is by oral delivery. Since RCN, for example, has been shown to be effective in cats when administered orally, the induction of a strong mucosal response is a possibility.
  • a preferred therapeutic composition to administer to an animal is a recombinant RCN comprising an isolated nucleic acid molecule encoding the FI antigen of Yersinia pestis.
  • a particularly preferred therapeutic composition comprises RCN:pl l-nYpFlsec 544 .
  • the efficacy of a therapeutic composition of the present invention to protect an animal from plague can be tested in a variety of ways including, but not limited to, detection of protective antibodies, detection of cellular immunity within the treated animal, or challenge of the treated animal with Yersinia, Pasteurella, or Francisella (preferably Yersinia pestis) to determine whether the treated animal is resistant to disease.
  • therapeutic compositions can be tested in a target animal and then serum from that vaccinated animal can be transferred to animal models such as mice, to test for protection by passive immunity. Such techniques are known to those skilled in the art.
  • Preferred embodiments of the present invention include (a) a recombinant raccoon poxvims genome that includes an isolated nucleic acid molecule encoding a Yersinia pestis antigen operatively linked to a poxvims transcription control region, (b) a recombinant raccoon poxvims including such a recombinant genome, (c) a recombinant cell including such a recombinant genome, (d) a recombinant plasmid that includes an isolated nucleic acid molecule encoding a Yersinia pestis antigen operatively linked to a eukaryotic transcription control region, and (e) a recombinant cell that includes such a recombinant plasmid.
  • a particularly preferred eukaryotic transcription control region is the human cytomegalovirus (HCMV) immediate-early promoter.
  • HCMV human cytomegalovirus
  • Other preferred embodiments include therapeutic compositions comprising a recombinant raccoon poxvims genome, a recombinant raccoon poxvims, a recombinant cell, and/or a recombinant plasmid as described above.
  • Example 1 The Examples include a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook, et al., ibid., Ausubel, et al., ibid., and related references.
  • Example 1
  • This example discloses the production of a recombinant raccoon poxvirus capable of expressing a secreted Y. pestis FI antigen.
  • A. Recombinant molecule pKB3poly-nYpFl(a)sec 544 , containing a nucleic acid molecule encoding the FI antigen of Yersinia pestis operatively linked to a vaccinia vims pi 1 late promoter transcription control region was produced in the following manner.
  • the pKB3poly poxvims shuttle vector was created by modifying a region of plasmid pKB3 (P,,-type) plasmid (described in U.S. Patent No. 5,348,741, by Esposito et al., issued September 20, 1994) such that the initiation codon linked to the pi 1 promoter was mutated and additional unique polylinker restriction sites were added.
  • the resulting poxvims vector referred to as pKB3poly, requires the insert DNA to provide the ATG initiation codon when inserted downstream of the pi 1 promoter.
  • the pKB3poly vector was designed such that foreign DNA cloned into the polylinker region of pKB3poly vector will recombine into the thymidine kinase (tk) gene of a wild-type orthopoxvims.
  • Plasmid YPRl (Simpson, et al., ibid., obtained from the National Institutes of Health Rocky Mountain Laboratories, Hamilton, MT) was used as a template for PCR amplification of the FI nucleic acid molecule using sense primer EJH031 5' ACG
  • PCR amplified product was digested with restriction endonucleases Sail and BamHl and gel purified, resulting in a double stranded nucleic acid molecule of about 544 base-pairs denoted herein as nYpFl(a)sec 544 the sequence of which is denoted herein as SEQ ED NO:l.
  • SEQ ED NO:l sequence of which is denoted herein as SEQ ED NO:l.
  • the PCR fragment amplified from plasmid YPRl was not derived from the same strain of Yersinia pestis as the published sequence and, as such, may comprise an allelic or strain variant of the published sequence.
  • SEQ ED NO:l contains an open reading frame of about 510 nucleotides, assuming a start codon extending from about nucleotide 17 through about nucleotide 19 of SEQ ID NO:l and a termination codon extending from about nucleotide 527 through about 529 of SEQ ED NO:l.
  • the coding region is denoted herein as nYpFlsec 510 , the coding strand of which is presented herein as SEQ ED NO:3.
  • SEQ ED NO:3 encode a full-length F 1 protein of about 170 amino acids, denoted herein as PYpFlsec 170 _ the sequence of which is presented herein as SEQ ED NO:2.
  • PYpFlsec 170 includes an N-terminal signal peptide sequence of about 21 amino acids extending from about amino acid 1 to about amino acid 21 of SEQ ED NO:2. In its native milieu, this signal peptide directs the secretion of F 1 across the inner and outer Y. pestis membranes where it is assembled into a capsule around the bacterial cell.
  • the mature form of PYpFlsec 170 is represented by PYpFlmat 149 , having the amino acid sequence SEQ ID NO:21.
  • PYpFlmat 149 is encoded by nucleic acid molecule nYpFlmat ⁇ ,, having the coding strand nucleotide sequence represented by SEQ ID NO:22, assuming a first codon extending from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:22.
  • the PCR-amplified fragment comprising nYpFl(a)sec 544 was ligated into BamHl and S /1-digested and gel-purified pKB3poly transfer vector, resulting in recombinant molecule pKB3poly-nYpFl(a)sec 544 .
  • Plasmid DNA comprising pKB3poly- nYpFl(a)sec 544 was purified using Qiagen columns (available from Qiagen, Chatsworth, CA).
  • a recombinant raccoon poxvims capable of expressing Y. pestis FI antigen was produced as follows.
  • BSC-1 African green monkey kidney cells obtained from American Type Culture Collection (ATCC), Rockville, MD
  • ATCC American Type Culture Collection
  • RCN CDC/V71- I-85A obtained from Dr.
  • the resulting recombinant vims denoted RCN:pl l-nYpFl(a)sec 544 , was plaque purified twice in RAT-2 rat embryo, thymidine kinase mutant cells (available from
  • This example demonstrates expression of cellular and secreted forms of Y. pestis F 1 antigen in RCN:p 11 -nYpF 1 (a)sec 544 -infected cells.
  • BSC-1 cells were plated into 6 well polystyrene dishes in about 2 ml of MEM medium (available from Life Technologies, Inc., Gaithersburg, MD) containing 5 % fetal bovine serum (FBS) per well. The cells were allowed to grow overnight at 37°C with 5% CO 2 . The medium was removed from the cells and replaced with about 2ml of MEM containing 1.0 % FBS.
  • MEM medium available from Life Technologies, Inc., Gaithersburg, MD
  • FBS fetal bovine serum
  • the cells were then infected with RCN:pl l-nYpFl(a)sec 544 at an MOI of approximately 0.025 pfu/cell and were further incubated for about 2 days at 37°C, 5% CO 2 until 100% cytopathic effect (CPE) was observed.
  • the culture was harvested by scraping the infected cells into the medium. The culture was centrifuged at 6000 RPM in a table-top centrifuge for 6 min. at room temperature.
  • the supernatant and cells were prepared for western blot analysis as follows.
  • the cell pellet was washed in PBS and resuspended in 50 ⁇ l of lx loading buffer (125 mM Tris, pH 6.8, 4% SDS, 0.05% Bromophenol blue, 20% glycerol, and 10% ⁇ - mercaptoethanol.).
  • the cell lysate was heated to 95°C for 5 min., and then filtered through a 0.45 ⁇ m filter unit, for example, an Ultrafree-MCTM 0.45 ⁇ m filter unit, available from Millipore Corp., Bedford, MA.
  • An about 1.9 cm 2 equivalent of the filtered sample (about 10 ⁇ l) was analyzed by western blot as described below.
  • the supernatant from the infected cells (about 2 ml) was centrifuged at 14,000 rpm for 5 min. at room temperature in a microcentrifuge and was then concentrated to 100 ⁇ l in an ultrafiltration device with a 10-kD molecular weight cutoff, for example, a Microcon- 10TM unit, available from Amicon, Inc., Beverly, MA, according to the manufacturer's instructions.
  • the supernatant was combined with an equal volume of 2X loading buffer and heated to 95°C for 5 min.
  • An about 1.9 cm 2 equivalent of the prepared concentrated supernatant (about 20 ⁇ l) was analyzed by western blot as described below.
  • Both the RCN:pl l-nYpFl(a)sec 544 infected-cell lysate and supernatant fractions revealed a eukaryotic version of FI antigen that migrated as a doublet with apparent molecular weights ranging from about 17 kD to about 22 kD.
  • the cm 2 - adjusted equivalent amounts of the cell and supernatant fractions analyzed on the western blot it appeared that the supernatant fraction contained about 4 times more FI antigen than the infected cell lysate fraction.
  • FI pestis FI
  • Native-expressed FI antigen is known to be glycosylated, which accounts for its larger observed size of 17 kD; see, for example Bennett, et al., 1974, J. Bacteriol., 117, 48-55. Since the RCN-expressed FI antigen (the lower and more abundant band of the doublet) ran at an apparent molecular weight similar, if not identical, to native FI, it was probably also post-translationally modified. While not being bound by theory, the protein migrating with a 22-kD apparent molecular weight could represent another form of post-translational modification.
  • a third, larger immunoreactive protein band migrating at a molecular weight significantly greater than 90 kD but less than 250 kD was observed in the infected-cell lysate fraction (but not in the supernatant fraction). While not being bound by theory, this band could represent a multimeric form of FI (common in 7. pestis, see, for example, Bennett, et al., ibid.) or, alternatively, could represent FI protein bound tightly to a cellular or viral protein.
  • FI common in 7. pestis
  • Vims stocks for immunization of mice were prepared as follows. BSC-1 cells were seeded into 40-225 cm 2 flasks in MEM containing 5 % FBS and incubated at 37°C for 2-3 days until a confluent monolayer was formed. The cells were infected at an MOI of 0.01 pfu/cell with RCN:pl l-nYpFl(a)sec 544. The vims stock was treated with 0.125mg/ml trypsin for 15 min.
  • the infected cells were incubated for 36-48 hrs. at 37°C until 100% CPE was obtained.
  • the infected cells were detached from the flasks with sterile glass beads and the culture was centrifuged at 5000 rpm for 15 min in a table-top centrifuge at room temperature.
  • the infected cells were resuspended in 30 ml of cold 10 mM Tris buffer, pH 9.0 and homogenized with 40 strokes in a dounce homogenizer on ice.
  • the homogenized sample was centrifuged at 300 x g for 5 min at 5-10°C in a tabletop centrifuge. The supernatant was saved on ice.
  • the pellet was resuspended in 10 ml of cold 10 mM Tris, pH 9.0, and homogenized with 20 strokes in a dounce homogenizer on ice.
  • the sample was centrifuged as before and the supernatant was removed and combined with the first supernatant.
  • the combined supernatant was sonicated on ice for 3 pulses at 6 watts of 15 seconds each with a hand held sonicator, for example, a VirSonic60TM sonicator (available from The VirTis Co., Inc., Gardiner, NY).
  • the supernatant was then layered onto three 13-ml cushions of 36% sucrose (in 10 mM Tris, pH 9.0) and centrifuged for 80 min.
  • mice of recombinant vims RCN:pl 1- nYpFl(a)sec 544 expressing the 7 pestis FI capsular antigen four groups of AJ mice, all about three weeks old, were immunized by injection into the footpad, as follows.
  • Group 1 consisting of 60 mice, received about 1 X 10 8 plaque forming units (pfu) of RCN ⁇ pll- nYpFl(a)sec 544 in about 30 ⁇ l of diluent (1 mM Tris, pH 9.0).
  • Group 2 consisting of 36 mice, received about 1 ⁇ g of FI protein purified from 7 pestis (obtained from the CDC) also in about 30 ⁇ l of diluent.
  • Group 3 consisting of 36 mice, received about 1 X 10 8 pfu of a control RCN vims, RCN-lacZ, in about 30 ⁇ l of diluent.
  • RCN-lacZ comprises the gene encoding E. coli beta-galactosidase driven by the vaccinia p7.5 promoter, which was inserted into the tk locus of RCN by a method similar to that described in Example IB above.
  • Group 4 consisting of 36 mice, received about 30 ⁇ l of diluent.
  • FI antigen The response to FI antigen by the immunized mice was measured by enzyme-linked immunosorbent assay (ELISA) assay as follows. Blood was collected from all immunized mice 5 days prior to infection and at days 10, 20, 30, 37 and 58 post-infection. Serum samples were prepared by methods well known to those skilled in the art. The serum samples were tested for anti-Fl antibodies using an ELISA for total IgM/IgG, performed as follows. Individual wells of 96-well ELISA plates were coated with purified FI antigen (about 1.0 ⁇ g in about 50 ⁇ l carbonate buffer, pH 9.6 per well), and were incubated overnight at 4°C or at 37°C for 2 hours.
  • FI antigen enzyme-linked immunosorbent assay
  • mice serum samples (1 :40 and 1:640 dilutions in 50 ⁇ l total volume, diluted in TBST) were added to duplicate wells on the plates.
  • horseradish peroxidase labeled goat anti -mouse conjugate 50 ⁇ l of a 1:2000 dilution, available from Jackson ImmunoResearch Lab., Inc., West Grove, PA
  • horseradish peroxidase labeled goat anti -mouse conjugate 50 ⁇ l of a 1:2000 dilution, available from Jackson ImmunoResearch Lab., Inc., West Grove, PA
  • 50 ⁇ l of peroxidase substrate was added to each well (using, for example, the ABTSTM peroxidase substrate available from Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD), and incubated for 15 minutes at room temperature.
  • stop solution 1% SDS
  • Eukaryotic expression vector pPVXC was produced as follows.
  • Vector pRc/RSV available from Lnvitrogen Corp., San Diego, CA
  • Pvu restriction enzyme
  • That fragment was self-ligated to form vector pRc/RSV (Pvu), which contains a Rous Sarcoma Vims (RSV) long terminal repeat, a multiple cloning site, a bovine growth hormone polyadenylation sequence, a bacterial origin of replication, and an ampicillin resistance gene.
  • RSV Rous Sarcoma Vims
  • Expression vector pPVXC was produced by introducing a HindllUSspl fragment containing the HCMV intermediate early promoter and first intron (i.e., intron- A) into pRc/RSV(Pvu) that had been cleaved by H dIII and Nrul. This manipulation removed sequences encoding the RSV long terminal repeat from pRc/RSV( vw).
  • a recombinant plasmid, denoted herein as pCMV-nYpFl(b)sec 544 in which a nucleic acid molecule encoding a full-length FI protein is operatively linked to the ⁇ CMV immediate-early transcription control region, was produced as follows.
  • Nucleic acid molecule nYpFl(b)sec 544 which encodes PYpFlsec, 70 (i.e., SEQ ID NO:2), was produced by PCR amplification of that molecule from plasmid YPRl (described in Example 1) using forward primer JO-1, having nucleic acid sequence 5'GGCAAGCTTG AGGTAATATA TGAAAAAAAT CAG 3', represented herein as SEQ ID NO: 16 (HindlH site in bold); and reverse primer JO-2 having nucleic acid sequence 5' GGCGAATTCC TATATGGATTA TTGGTTAGAT ACGG 3', represented herein as SEQ ED NO: 17 (EcoRI site in bold).
  • the coding sequence, open reading frame and the mature processed protein encoded by S ⁇ Q LD NO:4 are all identical to those described in Example 1 for SEQ ID NO: 1. It is to be noted that the PCR fragment amplified from plasmid YPRl was not derived from the same strain of Yersinia pestis as the published sequence and, as such, may comprise an allelic or strain variant of the published sequence. Recombinant molecule pCMV-nYpFl(b)sec 544 was produced by ligating nucleic acid molecule nYpFl(b)sec 544 into pPVXC that had been cleaved by Malawi! and EcoRI and gel purified.
  • a recombinant plasmid identified herein as pCMV-nYpFlanc 576 , capable of expressing an FI antigen fused to a eukaryotic membrane anchor domain, was produced as follows.
  • Nucleic acid molecule nCgGanc 192 which encodes the membrane anchoring domain of the canine herpesvirus (CHV) glycoprotein gG gene was produced by PCR amplification from CHV viral DNA using forward primer JO-3, having a sequence 5' GGGATGACGT CGTCGGTTAT AATAATTGTA ATACCC 3', represented herein as S ⁇ Q LD NO: 18 (Tthl 111 site in bold), and reverse primer JO-4 having nucleic acid sequence 5'GGCGAATTCT TAAATATCAT AAAAATTTAA TTTCTGGGG 3', represented herein as S ⁇ Q ID NO: 19 (EcoRI site in bold).
  • CHV canine herpesvirus
  • S ⁇ Q ID NO:5 comprises nucleotides about 1072-1248 of S ⁇ Q LD NO:9 in pending U.S. Patent Application Serial No. 08/602,010, ibid.
  • SEQ ED NO:5 yields a protein of about 61 amino acids, denoted herein as PCgGanc 61 , the amino acid sequence of which is presented in SEQ ED NO:6.
  • Recombinant molecule pCMV- nYpFlanc 576 was produced by digesting pCMV-nYpFl(b)sec 544 (produced as described in Example 4B) with Tth 1111 and EcoRI, gel purifying the larger restriction fragment from this digest, and ligating this fragment with nCgGanc 192 . This manipulation resulted in the first 418 nucleotides of nYpFl(b)sec 544 being fused in-frame with nCgGanc 192 .
  • the fusion produced coding region nYpFlanc 576 the coding strand sequence of which is denoted herein as S ⁇ Q LD NO:7.
  • Translation of S ⁇ Q ⁇ D NO:7 yields a protein of about 192 amino acids, denoted PYpFlanc 192 , the amino acid sequence of which is presented in S ⁇ Q LD NO:8, assuming an initiation codon extending from about nucleotide 1 through about nucleotide 3 of S ⁇ Q LD NO:7.
  • PYpFlanc 192 includes an N-terminal signal peptide sequence of about 21 amino acids extending from about amino acid 1 to about amino acid 21 of S ⁇ Q ⁇ D NO:8.
  • PYpFlanc 192 The mature form of PYpFlanc 192 is represented by PYpFlanc 171 , having the amino acid sequence SEQ ID NO:10.
  • PYpFlanc I71 is encoded by nucleic acid molecule nYpFlanc 513 , having the coding strand sequence represented by SEQ ED NO:9.
  • a recombinant plasmid denoted herein as pCMV-nYpFlmat 474 , capable of expressing a non-secreted form of FI antigen, was produced as follows. Nucleic acid molecule nYpFlmat 474 , which encodes a non-secreted form of the 7 pestis FI antigen, was produced by PCR amplification of that molecule from plasmid YPRl (described in Example 1) using forward primer JO-5, having nucleic acid sequence 5' CCCAAGCTTA TGGACGATTT AACTGCAAGC ACC 3', represented herein as SEQ ID NO:20 (Hindl ⁇ l site in bold); and reverse primer JO-2 having nucleic acid sequence 5' GGCGAATTCC TATATGGATT ATTGGTTAGA TACGG 3', represented herein as SEQ ED NO: 17 (EcoRI site in bold).
  • nYpFlmat 474 ⁇ the coding strand nucleotide sequence of which is denoted herein as S ⁇ Q LD NO:l 1.
  • Translation of S ⁇ Q ID NO: 11 yields a protein of about 150 amino acids, denoted PYpFlmat !50 , the amino acid sequence of which is presented in S ⁇ Q LD NO:12, assuming a start codon spanning from about nucleotide 7 to about nucleotide 9 of SEQ ID NO:l 1 and a stop codon spanning from about nucleotide 457 to about nucleotide 459 of SEQ ID NO:ll.
  • the coding region of PYpFlmat 150 is referred to herein as nYpFlmat 450 , the coding strand sequence of which is represented in SEQ ED NO:13.
  • Recombinant molecule pCMV-nYpFlmat 474 was produced by ligating nucleic acid molecule nYpFlmat 474 into pPVXC that has been cleaved by HindR and EcoRI and gel purified.
  • Example 5
  • This example demonstrates the production of a recombinant protein from a recombinant plasmid of the present invention.
  • Transient expression of PYpFlsec 170 from recombinant plasmid pCMV-nYpFl(b)sec 5 4 in baby hamster kidney cells was performed as follows. Briefly, six-well polystyrene tissue culture plates were seeded with about 3 x 10 5 BHK cells/well in 2 ml of MEM NEAA Earle's salts (available from Irvine Scientific, Santa Ana, CA), supplemented with 100 mM L-glutamine and 5% FBS (complete growth media). Cells were grown to about 80% confluence (about 48 hr).
  • OptiMEM After incubation, about 500 ⁇ l OptiMEM was added and the entire mixture was overlaid onto the BHK cells that had been rinsed with OptiMEM. Cells were incubated for 4 hours at 37°C, 5% CO 2 , 90% relative humidity. The transfection mixture was then removed and replaced with about 1 ml of OptiMEM.
  • Transfected cells were incubated at 37°C, 5% CO 2 , 90% relative humidity for about 24 hr or about 48 hr, at which times the cell supematants and cells were harvested separately. The media was removed, the cells were washed twice with about 2 ml PBS and were then scraped off the plate in about 1.5 ml PBS. The cells were then pelleted by centrifugation, the PBS was removed and the cells were frozen at -70°C. The cell- supernatants were frozen without any further manipulations.
  • Example 6 This example discloses the production of a recombinant plasmid encoding a secreted form of the 7 pestis FI antigen.
  • Eukaryotic expression vector pPVXC-tPA was produced as follows.
  • a double-stranded cassette comprising the tissue plasminogen activator (t-PA) signal peptide sequence (see, for example, Wang, RF and Mullins, JI, 1995, Gene 153 (2), 197-202) was constmcted by annealing two complementary synthetic ohgonucleotides: JO-6 having nucleic acid sequence 5'AGCTTCAATC ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC AGTCTTCGTT TCGGCCGGCC CGGGAT3' (partial Hindl ⁇ l and EcoRV sites underlined, t-PA initiation codon in bold, N el site in double underline); and JO-7 having nucleic acid sequence 5'ATCCCGGGCC GGCCGAAACG AAGACTGCTC CACACAGCAG CAGCACACAG CAGAGCCCTC TCTTCATTGC ATCCATGATT
  • ohgonucleotides were annealed by methods known by those skilled in the art to produce an about 82-base-pair cassette with a 4-nucleotide overhang on the 5' end.
  • This cassette comprises the coding strand encoding the t-PA signal peptide sequence, ntPA 69 , extending from nucleotide 11 to nucleotide 79 of JO-6.
  • Translation of ntPAr ⁇ yields a protein of about 23 amino acids, denoted herein as PtPA 23 .
  • the resulting double- stranded cassette was cloned into the pPVXC plasmid (described in Example 4A) which had been previously digested with Hindl ⁇ l and EcoRV and gel purified.
  • the resulting expression vector, pPVXC-tPA contains the t-PA signal peptide sequence followed by an N ⁇ el restriction site into which a protein coding sequence may be inserted in-frame.
  • a recombinant plasmid denoted herein as pCMV-ntPA/YpFlsec 534 , in which a nucleic acid molecule encoding the mature FI protein fused in-frame with the t-PA signal peptide sequence is operatively linked to the HCMV immediate-early transcription control region, was produced as follows.
  • the nucleic acid molecule nYpFlmat 481 which encodes the mature FI protein was produced by PCR amplification from recombinant plasmid pCMV-nYpFl(b)sec 544 (described in Example 4B) using forward primer JO-8, having nucleic acid sequence 5'GGCGCCGGCG CAGATTTAAC TGCAAGCACC3' (N ⁇ el site in bold), and reverse JO-9 having nucleic acid sequence 5' GGCCTCGAGC GGAATTCTTA GGATCCTTGG TTAGATACTG TTACGG 3' (Xhol site in bold, stop codon underlined).
  • nYpFlmat 481 The resulting PCR product was digested with restriction endonucleases N ⁇ el and Xhol and gel purified, resulting in a double-stranded nucleic acid molecule of about 481 base pairs denoted herein as nYpFlmat 481 .
  • This sequence comprises a region of SEQ JD ⁇ O:l extending from nucleotide 63 to nucleotide 512, which encodes a portion of SEQ ED NO:2 extending from amino acid 14 through amino acid 163.
  • Recombinant molecule pCMV- ntPA/YpFlmat 534 was produced by ligating nucleic acid molecule nYpFlmat 481 into pPVXC-tP A that had been digested with Nael and Xhol and gel purified. This manipulation results in nYpFlmat 481 being fused in-frame with ntPA ⁇ . The fusion produces coding region ntPA/YpFlsec 534 . Translation of ntPA/YpFlsec 534 yields a protein of about 178 amino acids, denoted herein as PtPA/YpFlsec 178 .
  • Example 7 Example 7:
  • This Example demonstrates the production of recombinant protein from recombinant plasmid pCMV-ntPA/YpFlsec 534 .
  • Cell and supernatant samples were subjected to SDS PAGE followed by western blot analysis by methods similar to those described in Example 2, but with some slight modifications.
  • the cell pellets were quantified by spectrophotometric absorbance at 600nm.
  • the cells were then centrifuged and resuspended in a volume of 2X loading buffer so that 20 ⁇ l were equivalent to 0.1 O.D. units. Gels were loaded with 5 ⁇ l of cell sample per lane (0.025 OD units).
  • Supematants were concentrated approximately 67-fold with Microcon-10TM unit, and gels were loaded with 15 ⁇ l concentrated supernatant sample per lane.
  • Rabbit anti-Fl antigen antiserum as described in Example 2, was immunoreactive with antigens of about 18 kD expressed by both the cells and supernatants harvested about 48 hours after transfection with plasmid pCMV-ntP A/YpF 1 sec 534 .
  • PtPA/YpFlsec 178 was also detected by immunofluorescence following transient transfection of BHK cells. Briefly, six- well polystyrene tissue culture plates were seeded with about 3xl0 5 BHK cells per well in 2 ml of MEM NEAA Earle's salts (available from Irvine Scientific, Santa Ana, CA), supplemented with 100 nM L-glutamine and 5% FBS (complete growth media). Recombinant molecule pCMV- nfPA/YpFlsec S34 was purified using Qiagen® Maxiprep columns, according to the manufacturer's instructions.
  • the purified recombinant plasmid (1.0 ⁇ g) was mixed with 100 ⁇ l of OptiMEM® media and incubated for 10 min at room temperature.
  • the recombinant plasmid mixture was then added to the Lipofectamine mixture and incubated at room temperature for 30 min. After incubation, 800 ⁇ l of OptiMEM was added, and the entire mixture was overlaid onto BHK cells that had been rinsed with OptiMEM. Cells were incubated for 5 hours at 37°C, 5% CO 2 , and 90% relative humidity.
  • transfection mixture was then removed and replaced with 2 ml of DMEM containing 10% FBS and incubated for about 24 to 48 hours.
  • transfected cells were rinsed three times with IX PBS and then fixed in a Methanol/ Acetone (50/50) solution for 5 min on ice. Fixed cells were rinsed three times with PBS. Rabbit anti-Fl antigen antiserum (1 :50 dilution in PBS) was added, and the cells were incubated for 1 hr at 37°C.
  • This example describes the immunization of mice with recombinant molecules of the present invention and the generation of antibodies in the immunized animals.
  • Plasmid DNA for immunizations was produced as follows. Plasmid DNAs described below were purified using Qiagen® megacolumns, per manufacturer's instructions. The endotoxin level of each preparation was measured by QCL-1000 kit (available from Biowhittaker, Walkersville, MD) prior to animal immunization, and was found to be 0.043 Endotoxin Units/ug of DNA, an acceptable level for animal immunizations.
  • QCL-1000 kit available from Biowhittaker, Walkersville, MD
  • Mice were immunized with plasmid DNA using the following method. Four groups of BALB/c mice of about three weeks of age were immunized by intramuscular injection as follows.
  • Group 1 consisting of 5 mice, received about 100 ⁇ g of pPVXC plasmid DNA (produced as described in Example 4) in about 30 ⁇ l of diluent (TE).
  • Group 2 consisting of 5 mice, received about 100 ⁇ g of pCMV-nYpFl(b)sec 544 plasmid DNA (produced as described in Example 4) in about 30 ⁇ l of diluent (TE).
  • Group 3 consisting of 5 mice, received about 100 ⁇ g of pCMV-ntPA/YpFlsec 534 plasmid DNA (produced as described in Example 6) in about 30 ⁇ l of diluent (TE).
  • mice received about 1 ⁇ g of FI protein purified from 7 pestis (as described in Example 2) also in about 30 ⁇ l of diluent.
  • C The immune response to FI antigen in the immunized mice was measured by enzyme-linked immunosorbent assay (ELISA) as follows. Blood was collected from all immunized mice 5 days prior to infection and at days 10, 20, 30, 40 post-vaccination. Serum samples were prepared by methods well known to those skilled in the art. The semm samples were tested for anti-Fl antibodies using an ELISA for total IgM/IgG, performed as described in Example 3C. The antibody titers, geometric means, and standard deviations of the ELISA for serum samples taken on days -5 (i.e., 5 days prior to infection), 10, 20, 30, and 40 are summarized in Table 1. Table 1
  • Group 1 1 CMV ⁇ 40 ⁇ 40 ⁇ 40 ⁇ 40 ⁇ 40 ⁇ 40
  • Group 2 1 CMV/F1 ⁇ 40 ⁇ 40 ⁇ 40 ⁇ 40 ⁇ 40
  • Group 3 1 CMV/tPA-F1 ⁇ 40 ⁇ 40 640 2,560
  • mice vaccinated with the pCMV-ntPA/YpFlsec 534 plasmid were equivalent to those detected in animals vaccinated with the FI protein.
  • This example discloses the production of a recombinant raccoon poxvims containing the EMCV LRES and a fused 7 pestis tPA-Fl antigen.
  • Eukaryotic expression vector pCITE-tPA was constmcted as follows.
  • a double-stranded cassette comprising the tissue plasminogen activator (t-PA) signal peptide sequence (Wang et al, ibid.) was constmcted by annealing two complementary synthetic ohgonucleotides: JO- 10, having nucleotide sequence 5'ATGCAATGAA GAGAGGGCTC TGCTGTGTGC TGCTGCTGTG TGGAGCAGTC TTCGTTTCTG CCGGCCCGGG TATACG3': and JO-11 having nucleotide sequence 5' GATCCGTATA CCCGGGCCGG CAGAAACGAA GACTGCTCCA CACAGCAGCA GCACACAGCA GAGCCCTCTC TTCATTGCAT3'.
  • t-PA tissue plasminogen activator
  • the annealed sequence contains three blunt cutting restriction sites: Nael (double underline); Smal (bold); and Bstl 1071 (single underline); located at the 3' end of the annealed signal sequence.
  • the annealed cassette lacks the t-PA initiation codon at the 5' end, and is designed to blunt ligate to the scl site in pCITE-4a (available from Novagen), thus utilizing the EMCV ERES preferential ATG. This changes the first amino acid from a lysine to a glutamic acid, which is a conservative change and the protein retains its original hydrophobicity.
  • the annealed cassette was ligated into the pCITE 4a+ plasmid that had been previously digested with Mscl and BamHl and gel purified.
  • the resulting nucleic acid molecule was designated pCITE-tPA.
  • nLRES-tP A DNA fragment containing IRES-tP A, denoted herein as nLRES-tP A, was PCR amplified from pCITE-tPA using forward primer JO- 12 having nucleotide sequence 5'AGGCGCGCCG 7XO4CGTTAT TTTCCACCAT ATTGCCG3'( scI site in bold, Sail site in italics), and reverse primer JO- 13 having nucleotide sequence 5'CGAATTCGG rCCGTATACC3' (EcoRI site in bold, BamHl site in italics, and Z?stl l071 site underlined).
  • Recombinant molecule pCMV-LR ⁇ S-tPA was produced by ligating nucleic acid molecule nLR ⁇ S-tPA that had been digested with Ascl and EcoRI into a modified pCMV-intA vector.
  • This modified pCMV-intA vector was created by annealing, using techniques known to those skilled in the art, the following two complementary synthetic ohgonucleotides: JO-14 having nucleotide sequence 5 'AGCTTGGCGC GCCG3 ' (Ht ⁇ dlll site in italics, Ascl site in bold, first base of BamHl site underlined), and JO- 15 having nucleotide sequence 5'GATCCGGCGC GCCA3' (BamH site underlined, Ascl site in bold, first base of HwdLTI site underlined).
  • C Recombinant molecule pCMV-IRES-ntPA YpFlsec 525 , containing a nucleic acid molecule encoding the mature FI antigen of 7 pestis fused in- frame with the t-PA signal peptide sequence and operatively linked to a CMV promoter and EMCV IRES, was produced as follows.
  • Nucleic acid molecule nYpFlmat 468 was PCR amplified from pCMV-nYpFl(b)sec 544 (described in Example 4B) using forward primer JO-8 and reverse primer JO- 16, having nucleic acid sequence 5'CGGAATTCTT AGG ⁇ rCCTTG GTTAGATACG GTTACGG3' (EcoRI site in bold, stop codon underlined, BamHl site in italics). The resulting PCR product was digested with restriction endonucleases NgoMI and EcoRI and gel purified, resulting in a double-stranded molecule of 468 base pairs denoted herein as nYpFlmat 468 .
  • Recombinant molecule pCMV- ⁇ R ⁇ S- ntPA/YpFlsec 525 was produced by ligating nucleic acid molecule nYpFlmat 468 into the pCMV-IR ⁇ S-tPA vector that had been digested with NgoMl and EcoRI and gel purified.
  • D. A recombinant raccoon poxvims (RC ⁇ V) capable of expressing 7 pestis FI antigen was produced as follows.
  • Recombinant cell Vero:RC ⁇ :IR ⁇ S-ntPA/ ⁇ pFlsec 525 containing nucleic acid molecule ntPA/YpFlsec 525 operatively linked to a vaccinia vims pi 1 late promoter transcription control region and EMCV ERES was produced in the following manner.
  • Recombinant molecule pCMV-IRES-ntPA/YpFlsec 525 was digested with Sail and EcoRI to generate a fragment containing nucleic acid molecule ntPA/YpFlsec 525 operatively linked to a CMV promoter and ⁇ MCV IRES.
  • This example demonstrates enhanced expression of 7 pestis FI antigen in cells infected with a recombinant raccoon pox vims of the present invention.
  • Vero cells were plated at 7 xlO cells/well in six well dishes with MEM+5%FBS one day prior to infection. Cells were infected in duplicate at an MOI of approximately 0.5 with vimses that had pre-treated with trypsin (1 mg/ml) for 15 min at 37°C. Upon infection, the media was changed to MEM without FBS, and the infected cells were incubated for about 24 to 48 hr.
  • the infected cells were harvested by washing cells into media, and recovering the cells by centrifugation at 10,000 rpm for 5 min at room temperature. The supernatants and cells were prepared for western blot analysis as described in Example 2. About 10 ⁇ l of each cell fraction and 30 ⁇ l of each concentrated media sample were loaded on a 4-20% SDS-PAGE gel and run for 1 hr at 200 V.
  • the separated proteins were transferred to nitrocellulose using a Bio-Rad transfer apparatus at 100 V for 1 hr.
  • the filter was subjected to western blot analysis using polyclonal rabbit anti-Fl antigen antisemm (described in Example 2). Filters were scanned and analyzed for density with a NIH image program. Analysis of the results, in comparison with a known quantity of FI antigen, indicated that the presence of the EMCV ERES motif led to an about two-fold increase in FI protein production; that is, cells infected with RCN:ERES-ntPA/YpFlsec 525 produced about twice as much protein as did cells infected with RCN:p 11 -nYpF 1 (a)sec 544 .
  • Example 11 Example 11 :
  • This example discloses the production of recombinant mengoviruses containing several forms of the 7 pestis FI antigen.
  • Recombinant molecule pMV-nYpFlmat 450 encoding the mature FI antigen in frame with the polyprotein coding region of mengovims, was prepared as follows.
  • the mature FI coding region was amplified from recombinant molecule pCMV-LRES- ntPA/YpFlsec 525 (produced as described in Example 9C) using forward primer JO-17, having the nucleotide sequence 5'GGGGCTAGCA GATTTAACTG CAAGCACCAC and reverse primer JO- 18 having the nucleotide sequence 5'GGGGCTAGCT GGTTAGATAC GGTTACGGTT ACAGCAGC (Nhel sites shown in bold).
  • the amplified fragment was purified using QIAquickTM PCR purification kit (available from Qiagen Inc., Valencia, CA) as recommended by manufacturer.
  • the PCR-amplified fragment was re-circularized by ligation, digested with restriction endonuclease Nhel, and gel purified, resulting in a double-stranded nucleic acid molecule of about 450 base pairs denoted herein as nYpFlmat 450 .
  • Recombinant molecule pMV-nYpFlmat 450 was produced by ligating nucleic acid molecule nYpFlmat 450 into menogvims plasmid pCoCe (available from the University of Wisconsin, Madison, WI; see also U.S.
  • Patent No. 5,229,111 by Duke et al, issued July 20, 1993) that had been digested with Nhel and gel purified. This manipulation resulted in nYpFlmat 450 being fused in-frame with the sequence encoding the mengovims polyprotein.
  • Recombinant molecule pMV-ntPA/YpFlsec sl9 encoding the mature FI antigen fused with the t-PA signal peptide sequence, and in frame with the polyprotein coding region of mengovims, was prepared as follows.
  • the mature FI coding region fused to the t-PA signal peptide sequence was amplified from recombinant molecule pCMV-LRES-ntPA/YpFlsec 525 (produced as described in Example 9C) using forward primer JO- 14, having the nucleotide sequence 5 ' GGGGCTAGCC GATGCAATGA AGAGAGGGCT CT 3' and reverse primer JO-13 (Nhel site shown in bold).
  • the amplified fragment was purified and digested with restriction endonuclease Nhel as described in Example 11 A, resulting in a double-stranded nucleic acid molecule of about 519 base pairs denoted herein as ntPA/YpF 1 sec 519 .
  • Recombinant molecule pMV- ntPA/YpFlsec 519 was produced by ligating nucleic acid molecule ntPA-YpFlsec 519 into the mengovims pCoCe plasmid described in Example 11 A that had been digested with Nhel and gel purified. This manipulation resulted in ntPA/YpFlsec 519 being fused in- frame with the sequence encoding the mengovims polyprotein.
  • Recombinant molecule pMV-ntPA/YpFlanc 705 encoding the mature FI antigen fused with the t-PA signal peptide sequence and the CHV gG membrane anchor sequence (described in Example 4C), and in frame with the polyprotein coding region of mengovims, was prepared as follows.
  • the mature FI coding region fused to the t-PA signal peptide sequence was amplified from recombinant molecule pCMV-IRES- ntPA/YpFlsec 525 as in Example 1 IB using forward primer JO-14, and reverse primer JO-15 having nucleotide sequence 5'CGGAATTCTT AGGATCCTTG
  • nucleic acid molecule ntPA/YpFlanc 705 was produced by ligating nucleic acid molecule ntPA/YpFlan ⁇ os into the mengovims pCoCe plasmid described in Example 11 A that had been digested with Nhel and gel purified. This manipulation resulted in ntPA/YpFlanc 705 being fused in- frame with the sequence encoding the mengovims polyprotein.
  • Example 12
  • This example describes the production of recombinant mengoviruses from the recombinant molecules described in Example 11.
  • RNA was produced from recombinant molecules pMV-nYpFlmat 45 o, pMV-ntPA/YpFlsec 519 , andpMV-ntPA/YpFlanc 705 using in vitro transcription, as follows. Two ⁇ g of each recombinant molecule was linearized with BamHl (pMV-nYpFlmat 450 and pMV-ntPA/YpFlsec 519 ) or H dlLI (pMV- ntPA/YpF 1 anc 705 ) in a 50 ⁇ l reaction mix. Each DNA was extracted once with phenol/C ⁇ Cl 3 and precipitated.
  • RNA from each recombinant molecule was then synthesized by in vitro transcription using MEGAscriptTM kit (available from Ambion Inc., USA) in 20 ⁇ l of reaction volume containing 1 ⁇ g of each template DNA essentially as described by manufacturer, producing mengovims RNA molecules rMV- nYpFlmat 450 , rMV-ntPA/YpFlsec 519 , and rMV-ntPA/YpFlanc 705 .
  • the yield of each mengovims RNA was evaluated by running an aliquot of RNA on 1% non-denaturing agarose gel.
  • HeLa cells were grown in a T225 cm 2 tissue culture flask to -80% confluency in D-MEM- 10%, which is D-MEM media supplemented with 10% FBS, 2 mM glutamine, 100 ⁇ g/ml streptomycin, 100 units/ml penicillin, IX MEM vitamins mixture (all reagents available from LTI). The cells were trypsinized using a standard protocol and resuspended in D-MEM- 10%.
  • RNA samples were washed three times in ice-cold OPTI-MEM I media (available from LTI) and resuspended in 500 ⁇ l aliquots of 5x10 6 cells for each RNA sample. Approximately 2 ⁇ g of each mengovims RNA, produced as described in Example 12 A, was added to an aliquot of cells and the mixtures were immediately subjected to two pulses of electrical discharge in BTX-500 electroporation device (Electro cell manipulator 600, BTX Inc., Santa Clara, CA) with the following settings: 400 V, 800 ⁇ F, 13 ohms.
  • BTX-500 electroporation device Electro cell manipulator 600, BTX Inc., Santa Clara, CA
  • the transfected recombinant cells denoted herein as HeLa:MV- nYpFlmat 450 , HeLa:MV-ntPA/YpFlsec 519 , and HeLa:MV-ntPA/YpFlanc 705 were resuspended in 10 ml of OPTI-MEM supplemented with 1% of FBS and were transferred into tissue culture flasks.
  • Infectious vimses denoted herein as MV- nYpFlmat 450 , MV-ntPA/YpFlsec 5 ⁇ 9 , and MV-ntPA YpFlanc 705 were collected after complete CPE was observed, at approximately 2 days post electroporation.
  • the infected cells were lysed by two freeze-thaw cycles followed by clarification of cell lysates by centrifugation for 15 min at 3,000 rpm in GP8R refrigerated centrifuge (Forma Scientific Inc.).
  • GCA GGT AAA TAC ACT GAT GCT GTA ACC GTA ACC GTA TCT AAC CAA TAA 529 Ala Gly Lys Tyr Thr Asp Ala Val Thr Val Thr Val Ser Asn Gin 160 165 170 TCCATATAGG GATCC 544
  • MOLECULE TYPE Genomic DNA
  • SEQUENCE DESCRIPTION SEQ ID NO : 3 :
  • MOLECULE TYPE protein
  • AATAACCACC AATTCACTAC AAAAGTGATT GGCAAGGATT CTAGAGATTT TGATATCTCT 300
  • MOLECULE TYPE primer
  • SEQUENCE DESCRIPTION SEQ ID NO: 18: GGGATGACGT CGTCGGTTAT AATAATTGTA ATACCC 36
  • MOLECULE TYPE primer
  • MOLECULE TYPE primer
  • SEQUENCE DESCRIPTION SEQ ID NO: 20:
  • TTCTTTGTTC GCTCAATTGG TTCCAAAGGC GGTAAACTTG CAGCAGGTAA ATACACTGAT 420

Abstract

The present invention relates to a recombinant vaccine to protect animals against plague. More particularly, the invention includes recombinant molecules containing isolated nucleic acid molecules that encode proteins from Yersinia, Pasteurella, or Francisella bacteria expressed in eukaryotic cells. In one embodiment, the recombinant molecule is an animal virus genome; in another embodiment the recombinant molecule is a recombinant plasmid. The present invention also includes recombinant viruses comprising a recombinant animal virus genome and recombinant cells comprising either a recombinant virus or a recombinant plasmid. The present invention further includes therapeutic compositions comprising such recombinant molecules, viruses and cells, as well as methods to protect animals from plague.

Description

RECOMBINA T PLAGUE VACCLNE Field of the Invention The present invention relates to a recombinant vaccine to protect animals against plague. More particularly, the invention includes recombinant molecules containing isolated nucleic acid molecules that encode antigens from Yersinia, Pasteurella, or Francisella expressed in eukaryotic cells.
Background of the Invention Plague remains a significant problem in the western United States. Yersinia pestis is enzootic in several wild animal reservoirs, particularly squirrels and wild mice. The disease is also frequently epizootic in prairie dogs. Plague and plague-like diseases caused by other species of Yersinia, Pasteurella, and Francisella are also enzootic in wild reservoirs. Although the enzootic reservoirs are not a severe direct threat to humans, infection of domestic cats from wild reservoirs is a very serious problem in that several pet owners and veterinarians have contracted infection from these animals; see, for example, Doll et al., 1994, Am. J. Trop. Med. Hyg., 51, 109-114. For this reason, an effective vaccine to protect cats (and thus cat owners and veterinarians) against plague is needed. Additionally, plague is one of the factors responsible for the diminishing population of black-footed ferrets (Mustela nigripes) in the western United States. In addition to contracting plague themselves, this nearly extinct species relies solely on prairie dogs as its food source; thus epizootic episodes of plague in prairie dog populations in the black-footed ferrets' habitat contribute to the depletion of this species; see, for example, Williams et al., 1994, J. Wildl. Dis., 30, 581-585. Thus, methods to control plague in the wild reservoirs are also needed. It would be particularly useful to reduce plague in these wild reservoirs through the release of baited, orally delivered vaccines.
Current plague vaccine formulations suffer from significant problems. Commercially available vaccines include a formulation comprising formalin-killed Yersinia pestis (Cutter USP, available from Greer Laboratories, Lenoire, NC) and a formulation comprising a modified live Yersinia pestis (Y. pestis EV76-6). Although both of these vaccine formulations protect mice from lethal plague challenge (i.e., up to 5 x 103 50% lethal doses (LD50s) for the killed vaccine and up to 5 x 106 LD50s for the modified live vaccine), these formulations result in severe side effects, and thus have not come under wide-spread use in either humans or other animals; see, for example, Russell et al., 1995, Vaccine, 13, 1551-1556. As used herein, the term "LD50" refers to the number of bacterial cells in a dose that, when administered to a group of animals, will kill 50% of the animals in that group. When a strain of Y. pestis is administered to a group of mice, the LD50 is typically about one bacterial cell.
In several animal models, protection from challenge with virulent Yersinia pestis has been correlated with increased antibody titer toward the Fraction 1 (FI) capsular antigen. In previous studies, FI antigen administered as an isolated subunit vaccine proved to be protective against plague challenge in mice, but less protective than the killed whole cell vaccines or avirulent live vaccines described above; see, for example, Simpson et al., 1990, Am. J. Trop. Med. Hyg., 43, 389-96. Furthermore, animals were not well protected by intragastric administration of the vaccine formulation; see, for example, Thomas et al., 1992, Am. J. Trop. Med. Hyg., 47, 92-7. The complete nucleotide sequence encoding a Y. pestis FI antigen has been disclosed in Galyov et al., 1990, FEBSLett., 277, 230-232, which is incorporated herein by reference.
Prokaryotic recombinant cell-based plague vaccines have also been disclosed. For example, a live recombinant Salmonella typhimurium expressing the FI antigen was protective in mice against a low-level Y. pestis challenge, i.e., less than 50 LD50s. This vaccine formulation was hampered by instability of the construct in vivo; see, for example PCT Publication No. WO 95/18231, published July 6, 1995, by Titball et al. Another example discloses vaccines comprising Salmonella minnesota, expressing FI; see, for example Russian Publication No. RU 2046145, published October 20, 1995, by Anisimov et al. However, the safety of live attenuated Salmonella vaccines is questionable, and different attenuated Salmonella species would be required to protect different animals due to the species specificity of Salmonella infection (e.g., S. typhimurium primarily infects mice and S. typhi primarily infects humans).
Another antigen being evaluated for vaccine potential is the V antigen on the lcr plasmid of Y. pestis. This protein is highly antigenic and when administered with FI antigen, the two subunits are nearly as protective as the attenuated live Y. pestis vaccine; see, for example, Leary et al, 1995, Infect. Immun., 63, 2854-2858, Williamson et al., 1995, FEMS Immunol. Med. Microbiol, 12, 223-230, and PCT Publication No. WO 95/24475, published September 14, 1995, by Titball et al. The V antigen, however, has been shown to be associated with suppression of gamma interferon and TNF-alpha in vivo. Raccoon poxvirus (RCN) has been shown to be a very safe and effective vaccine vector in a variety of animal species, and particularly for cats. Protection against rabies, feline panleukopenia, and feline infectious peritonitis viruses has been demonstrated in cats vaccinated with recombinant RCN expressing antigens from the respective viruses; see, for example U.S. Patent No. 5,266,313, issued November 30, 1993, by Esposito et al., PCT Publication No. WO 93/01284, published January 21, 1993, by Scott et al, and European Publication No. EP 94306917.9, published May 10, 1995, by Wasmoen et al. A particularly attractive feature of recombinant raccoon poxvirus vaccine formulations in cats is that the vaccine can be delivered orally, which is a preferred administration route both for cats and for wildlife. Advantages of oral administration include a high incidence of injection site-associated sarcomas in cats, and the propensity of an orally delivered vaccine to induce mucosal immunity. The inventors are not aware of any prior use of raccoon poxvirus for the expression of an antigen from a bacterial pathogen. In fact, only one citation was found for the expression of a protein from a bacterial pathogen in an animal virus vector; see, PCT Publication No. WO 90/15872, published December 27, 1990, by Fischetti et al.
Similarly, nucleic acid immunization is apparently a safe, inexpensive method of protecting animals from disease; see, for example, Wolff et al., 1990, Science 247, 1465- 1468. Nucleic acid vaccines are administered to an animal in a fashion to enable expression of protective proteins in the animal. A number of delivery methods for nucleic acid vaccines are known in the art including either intramuscular or intradermal injection, intradermal scarification using skin-test applicators, and particle bombardment (e.g. "gene-gun") delivery; see, for example, Raz et al., Proc. Natl. Acad. Sci. USA, 93, 5141-5145, U.S. Patent No. 5,204,253, issued April 20, 1993, by Bruner et al., and PCT Publication No. WO 95/19799, published July 27, 1995, by McCabe. Summary of the Invention The present invention relates to a novel recombinant molecule in which one or more nucleic acid molecules encoding antigens from Yersinia, Pasteurella, or Francisella are operatively linked to one or more eukaryotic transcription control regions, such that the antigen(s) are expressed in eukaryotic cells. Two primary embodiments of the present invention are a recombinant virus and a recombinant plasmid. Other embodiments include a recombinant animal virus genome and a recombinant eukaryotic cell. Also included in the present invention are methods to produce a recombinant molecule, a recombinant plasmid, a recombinant animal virus genome, a recombinant animal virus, and a recombinant eukaryotic cell of the present invention. Although it is uncommon to express a bacterial antigen in a live viral vector or in a eukaryotic cell, the idea is attractive because eukaryotic expression provides the possibility of inducing improved humoral, mucosal and cell-mediated immune responses. The present invention also includes therapeutic compositions, such as vaccines, that are capable of protecting an animal from contracting plague. Therapeutic compositions of the present invention include recombinant molecules that include isolated nucleic acid molecules that encode Yersinia, Pasteurella, or Francisella antigens, operatively linked to eukaryotic transcription control regions. Such therapeutic compositions include a recombinant animal virus genome, a recombinant virus and a recombinant cell expressing one or more antigens derived from Yersinia, Pasteurella, or Francisella, and a recombinant plasmid that expresses one or more antigens from Yersinia, Pasteurella, or Francisella when the plasmid is delivered into a eukaryotic cell. A preferred therapeutic composition of the present invention also includes an excipient, an adjuvant and/or a carrier. Also included in the present invention is a method to protect an animal from plague, which includes administering to the animal a therapeutic composition of the present invention.
Another embodiment of the present invention is an isolated nucleic acid molecule encoding a Yersinia pestis antigen fused, in frame, with a eukaryotic membrane anchor domain. Preferred embodiments of the present invention include (a) a recombinant raccoon poxvirus genome that includes an isolated nucleic acid molecule encoding a Yersinia pestis antigen operatively linked to a poxvirus transcription control region, (b) a recombinant raccoon poxvirus including such a recombinant genome, (c) a recombinant cell including such a recombinant genome, (d) a recombinant plasmid that includes an isolated nucleic acid molecule encoding a Yersinia pestis antigen operatively linked to a eukaryotic transcription control region, and (e) a recombinant cell that includes such a recombinant plasmid. A particularly preferred eukaryotic transcription control region is the human cytomegalovirus (HCMV) immediate-early promoter. Particularly preferred embodiments of the present invention include, but are not limited to, an isolated nucleic acid molecule nYpFlanc576 having nucleic acid sequence SEQ LD NO:7, and encoding protein PYpFlanc192, having amino acid sequence SEQ LD NO:8, and the complement of SEQ LD NO:7; recombinant molecules vRCN-pl 1- nYpFl(a)sec544, pCMV-nYpFl(b)sec544, PCMV-nYpFlanc576, and pCMV-nYpFlmat474; recombinant virus RCN:pl l-nYpFl(a)sec544; and recombinant cell BSC-1 :RCN:pl 1- nYpFl(a)sec544.
DETAILED DESCRIPTION OF THE INVENTION The present invention discloses recombinant molecules that comprise isolated nucleic acid molecules encoding antigens from Yersinia, Pasteurella, or Francisella that are operatively linked to eukaryotic transcription control regions and, as such, are expressed under eukaryotic transcription control in eukaryotic cells. Also included in the present invention are therapeutic compositions comprising the claimed recombinant molecules, which are useful to protect animals from plague. Further included in the present invention are methods, using the claimed therapeutic compositions, to protect animals from plague.
Expression of Yersinia, Pasteurella, or Francisella antigens from eukaryotic transcription control regions is a novel aspect of the current invention. While not being bound by theory, the inventors believe that the current invention will have significant utility as a plague vaccine, in that the embodiments of the current invention will be efficacious in controlling plague in animal populations (including in wild animal populations); the claimed vaccines will be economical to make and use, will be safer, and will have significantly reduced side-effects relative to currently available vaccines to control plague.
Preferred embodiments of the present invention include recombinant live virus vaccines and genetic immunization (i.e. naked nucleic acid) vaccines. Live virus vaccines and genetic immunization vaccines are advantageous because they are believed to confer more vigorous and longer-lasting immunity than subunit or killed vaccines. While not being bound by theory, it is believed that such advantages are due to the ability of the genetic information carried by the virus or the recombinant molecule to enter the cells of the treated animal, and to direct the expression of a protective compound, such as a protective protein or a protective RNA, for extended periods of time. Thus, therapeutic compositions of the present invention need not be administered frequently.
One particularly preferred embodiment of the present invention is an orally delivered plague vaccine for domestic cats. Cats are susceptible to the pneumonic form of the disease which is much more easily transmitted to other animals, including humans. While not being bound by theory, the inventors believe that oral delivery of a therapeutic composition of the present invention will induce mucosal immunity, which will more effectively control pneumonic plague.
As used herein, the terms Yersinia, Pasteurella, and Francisella refer to bacterial genera, and as such, include any species belonging to any of these genera. Particularly preferred bacterial species to target using embodiments of the present invention include Yersinia pestis (previously referred to as Pasteurella pestis, the name having changed about 1971) , Yersinia pseudotuberculosis, Yersinia enter ocolitica, Pasteurella multocida, and Francisella tularensis, with Y. pestis being even more preferred, as this species is believed to be the most common etiologic agent of plague.
A Yersinia, Pasteurella, or Francisella antigen refers to an antigen derived from any portion of a Yersinia, Pasteurella, or Francisella bacterium that is capable of being expressed from an isolated nucleic acid molecule, and that, when administered to an animal as an immunogen, will produce a humoral, mucosal, and/or cellular immune response against Yersinia, Pasteurella, or Francisella in that animal. The ability of a candidate antigen to effect an immune response can be measured using techniques known to those skilled in the art, some of which are disclosed herein. It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, an isolated nucleic acid molecule refers to one or more isolated nucleic acid molecules, or at least one isolated nucleic acid molecule. As such, the terms "a" (or "an), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. Furthermore, a compound "selected from the group consisting of refers to one or more of the compounds in that group, including mixtures (i.e., combinations) of two or more of the compounds. An antigen of the present invention includes not only full-length antigens but also homologs of full-length Yersinia, Pasteurella, or Francisella antigens, including smaller portions of such antigens. As used herein, the term homolog refers to any closely related antigen or epitope capable of eliciting an immune response to the native antigen. Examples of homologs include Yersinia, Pasteurella, or Francisella proteins in which amino acids have been deleted (e.g. a truncated version of the protein, such as a peptide), inserted, inverted, substituted, and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristylation, prenylation, palmitoylation, amidation and or addition of glycerophosphatidyl inositol) such that the homolog includes at least one epitope capable of eliciting an immune response against Yersinia, Pasteurella, or Francisella. As used herein, an epitope refers to the smallest portion of an antigen that is capable of eliciting an immune response in an animal. The minimal size of a protein epitope, as defined herein, is about five amino acids. It is to be noted, however, that such an epitope might comprise a portion of the antigen other than the amino acid sequence, e.g., a carbohydrate moiety. Yersinia, Pasteurella, or Francisella antigen homologs can also be the result of natural allelic or strain variation, natural mutation or laboratory-induced mutation in the genes that encode the antigens. As used herein, a gene includes all nucleic acid sequences related to a nucleic acid molecule that encodes a protein, such as regulatory regions that control production of the protein encoded by that nucleic acid molecule (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself. An allelic variant, strain variant, or variant (used interchangeably herein) of an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen refers to a nucleic acid molecule encoding an antigen at essentially the same locus (or loci) in the genome as the nucleic acid molecule in question but which, due to natural strain variations caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. Such variants typically encode proteins having similar activity to that of the protein encoded by the nucleic acid molecule to which they are being compared, but they do not necessarily have identical amino acid sequences. Allelic and strain variants can also comprise alterations in the 5' or 3' untranslated regions of a gene comprising the nucleic acid molecule (e.g., in regulatory control regions). Allelic and strain variants are well known to those skilled in the art and would be expected to be found to varying extents among the surface antigen genes of a pathogenic microorganism. Also included in the definition of variants are laboratory-induced mutants, such as variants arising due to errors incorporated into a nucleic acid molecule encoding an antigen during PCR amplification. Such errors can alter the nucleic acid sequence of a gene in question, and, as such, may also alter the amino acid sequence resulting in, for example, an amino acid substitution or the introduction of a stop (termination) codon, thus truncating the resultant antigen. Such a mutant is included as a variant if the gene in question encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared, e.g., in the case of the present invention, the protein must still be capable of eliciting an immune response in an animal against a Yersinia, Pasteurella, or Francisella native antigen.
It will be appreciated by those skilled in the art that a bacterial antigen expressed in a recombinant eukaryotic cell might be altered in ways that will vary its presentation to an animal's immune system. While not being bound by theory, the inventors believe that antigens secreted from a cell, anchored to the cell's membrane, or remaining in the cytoplasm of the cell will be processed and reacted to differently by an animal's immune system. In such a way, the immune response to a particular antigen can be altered from, for example, a completely humoral response to one that includes a shift toward a cellular immune response. Methods to introduce alterations into antigens of the current invention are known to those skilled in the art, and examples are disclosed herein. For example, a nucleic acid molecule encoding a secreted protein can usually be converted into a nucleic acid molecule encoding a cytoplasmic protein by deleting the secretory signal segment from the nucleic acid molecule. Also, a nucleic acid molecule encoding a secreted protein can be engineered to be anchored to a cell's membrane by fusing a portion of a nucleic acid molecule encoding the antigen in-frame with an appropriate heterologous nucleic acid molecule encoding a membrane anchor domain.
Yersinia, Pasteurella, or Francisella antigens of the current invention can include any antigen, including homologs thereof, derived from these bacterial genera, that, when administered to an animal as an immunogen, using techniques known to those skilled in the art, will produce a humoral, mucosal, and/or cellular immune response against Yersinia, Pasteurella, or Francisella in that animal. Preferred Yersinia, Pasteurella, and Francisella antigens include Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Pasteurella multocida, and Francisella tularensis antigens. Preferred Yersinia pestis antigens of the present invention include FI antigens, V antigens, pesticin antigens, W antigens, pH 6 antigens, superoxide dismutase antigens, Yersinia outer protein (YOP) antigens, high molecular weight iron- regulated membrane protein antigens, murine toxin antigens, and/or hemin storage protein antigens. More preferred Yersinia pestis antigens of the present invention include FI antigens and V antigens. An even more preferred Yersinia pestis antigen is an FI antigen. The most preferred antigens of the present invention include
PYpFlsec170, PYpFlanc192, PYpFlanc!7 PYpFlmat15fΛandPYpFlmat,49 the amino acid sequences of which are presented herein as SEQ LD NO:2, SEQ LD NO:8, SEQ LD NO:10, SEQ LD NO:12, and SEQ ID NO:21, respectively, as well as homologs and smaller portions, as small as a single epitope, of such antigens. It is to be understood that other Yersinia, Pasteurella, or Francisella species, share at least some of the above preferred antigens with Y. pestis (see, for example, Carter et al., 1980, Infect. Immun., 28, 638-40), but also comprise other antigens useful in the present invention.
As used herein, the term "isolated nucleic acid molecule" refers to a nucleic acid molecule derived, at least partially, from Yersinia, Pasteurella, or Francisella. An isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu. As such, the term "isolated" does not necessarily reflect the extent to which the nucleic acid molecule has been purified. An isolated nucleic acid molecule can include DNA, RNA, or derivatives of either DNA or RNA. An isolated nucleic acid molecule can be single-stranded or double-stranded. Certain isolated nucleic acid molecules for which the nucleic acid sequence of the coding strand is disclosed in a SEQ LD NO are also recognized to include a complementary strand, the nucleic acid sequence of which can be easily determined by one skilled in the art; such complementary sequences are included as part of the present invention. An isolated nucleic acid molecule can be obtained from its natural source, or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. According to the present invention, an isolated nucleic acid molecule encodes at least one Yersinia, Pasteurella, or Francisella antigen, examples of such antigens being disclosed herein. In one embodiment, the antigen is expressed by (i.e. under the direction of) a recombinant molecule of which the isolated nucleic acid molecule is a part, via operative linkage of that nucleic acid molecule to a eukaryotic transcription control region. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a Yersinia, Pasteurella, or Francisella antigen. An isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or
Francisella antigen can be produced using a number of methods known to those skilled in the art; see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, and Ausubel et al.,1993, Current Protocols in Molecular Biology, Greene/Wiley Interscience. Sambrook et al, ibid., and Ausubel et al, ibid., are incorporated by reference herein in their entireties. For example, nucleic acid molecules can be produced and/or modified using a variety of techniques including, but not limited to, by classic mutagenesis and recombinant DNA techniques (e.g., site- directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification), or synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Isolated nucleic acid molecules of the present invention may also (a) contain secretory signal segments (i.e., nucleic acid sequences encoding a secretory signal peptide) to enable an expressed Yersinia, Pasteurella or Francisella antigen of the present invention to be secreted from the cell that produces the antigen, (b) contain nucleic acid sequences encoding membrane anchor domains that lead to the expression of nucleic acid molecules of the present invention as antigens anchored to the cell membrane of the host cell such that the antigenic domain is outside of the cell, and/or (c) contain other fusion sequences which lead to the expression of nucleic acid molecules of the present invention as various fusion proteins. Nucleic acid sequences encoding signal peptides, membrane anchor domains or other protein domains are fused in- frame with isolated nucleic acid molecules encoding Yersinia, Pasteurella or Francisella antigens of the present invention by methods known to those skilled in the art. As used herein, the term "fused in-frame" indicates that two or more heterologous nucleic acid molecules are combined such that a single contiguous amino acid sequence is encoded. Examples of suitable signal segments and membrane anchor segments are disclosed herein.
Isolated nucleic acid molecules of the present invention may also include intervening and/or untranslated sequences surrounding and/or within the isolated Yersinia, Pasteurella, or Francisella nucleic acid sequences.
Suitable signal segments include any signal segment encoding a signal peptide capable of directing the secretion of an antigen of the present invention. As used herein, the term "signal segment" refers to a nucleic acid molecule encoding a secretory signal peptide, and the term "signal peptide" refers to the peptide domain capable of directing secretion of an antigen of the present invention. Typically, signal segments encode peptides of about 15 to 50 amino acids in length, and are located at the 5' end of a nucleic acid molecule encoding a secreted protein, but they can be located at other (i.e., internal) positions within a nucleic acid molecule. Preferred signal segments include, but are not limited to, endogenous signal segments of the isolated Yersinia, Pasteurella, or Francisella nucleic acid molecules of the present invention, as well as tissue plasminogen activator (t-PA) , interferon, interleukin, growth hormone, histocompatibility, and viral envelope glycoprotein signal segments. Suitable membrane anchor segments include any membrane anchor segment that encodes a peptide domain capable of anchoring an antigen of the present invention into a eukaryotic cell membrane. As used herein, the term "membrane anchor segment" refers to the nucleic acid molecule encoding a peptide domain capable of anchoring an antigen of the present invention to a eukaryotic cell membrane. Typically, a membrane anchor segment encodes a protein domain comprising hydrophobic amino acids. Membrane anchor segments can be located either at the 5' end or the 31 end of a nucleic acid molecule encoding an anchored protein. A membrane anchor segment located at the 5' end (e.g., as in a class II transmembrane glycoprotein gene) usually also functions as a secretory signal segment. Preferred membrane anchor segments include, but are not limited to, vesicular stomatitis virus (VSV) glycoprotein, respiratory syncytial virus G protein, herpesvirus glycoprotein, immunoglobulin, and glycosyl-phosphotidylinositol membrane anchor segments. Particularly preferred membrane anchor segments include the canine herpesvirus glycoprotein G, glycoprotein E and glycoprotein I membrane anchor segments.
Suitable intervening and/or untranslated sequences include, but are not limited to, any sequences that enhance or regulate expression of a Yersinia, Pasteurella, or Francisella antigen of the present invention. Preferred untranslated sequences include the human cytomegalovirus (HCMV) intron-A sequence and the encephalomyocarditis virus internal ribosomal entry site (EMCV-IRES).
Isolated nucleic acid molecules of the present invention can include any nucleic acid molecule capable of encoding a Yersinia, Pasteurella, or Francisella antigen of the current invention. Preferred isolated nucleic acid molecules encode at least one Yersinia pestis antigen of the present invention, including, but not limited to, an FI antigen, a V antigen, a pesticin antigen, a W antigen, a pH 6 antigen, a superoxide dismutase antigen, a YOP antigen, a high molecular weight iron-regulated membrane protein antigen, a murine toxin antigen, and a hemin storage protein antigen. More preferred isolated nucleic acid molecules encode a Yersinia pestis FI antigen and /or a V antigen. An even more preferred isolated nucleic acid molecule encodes a Yersinia pestis FI antigen. Particulary preferred isolated nucleic acid molecules of the present invention include nYpFl(a)sec544, nYpFl(b)sec544, nYpFlsec510, nYρFlanc576, nYpFlanc513, nYpFlmat474, nYpFlmat450, and nYpFlmat447, the coding strand nucleotide sequences of which are represented herein as SEQ ID NO:l, SEQ LD NO:3, SEQ LD NO:4, SEQ ID NO:7, SEQ LD NO:9, SEQ LD NO:ll, SEQ LD NO:13, and SEQ LD NO:22, respectively, as well as variants of these nucleic acid molecules and/or smaller portions of these nucleic acid molecules, capable of encoding at least one epitope (i.e., at least about 15 nucleotides). It is to be understood that isolated nucleic acid molecules encoding antigens from other Yersinia, Pasteurella, or Francisella species, as disclosed above, are also useful in the present invention.
Isolated nucleic acid molecules nYpFl(a)sec544and nYpFl(b)sec544, the nucleic acid sequence of the coding strands of which are denoted herein as SEQ LD NO:l and SEQ LD NO:4, respectively, each encode a full-length, unprocessed FI antigen of 170 amino acids, denoted herein as PYpFlsec,70, having an amino acid sequence denoted herein as SEQ LD NO:2, assuming an initiation codon extending from about nucleotide 17 through about nucleotide 19 of SEQ ID NO: 1 or SEQ LD NO:4, respectively, and a stop codon extending from about nucleotide 527 through about nucleotide 529 of SEQ ID NO:l or SEQ LD NO:4, respectively. As disclosed in Galyov, et al., ibid., PYpFlsecπo includes an N-terminal signal peptide sequence of about 21 amino acids extending from about amino acid 1 to about amino acid 21 of SEQ LD NO:2. The mature (i.e., processed) form of PYpFlsec170is represented by PYpFlmat149, having the amino acid sequence SEQ LD NO:21. PYpFlmat!49 is encoded by nucleic acid molecule nYpFlmat,^, having the coding strand nucleotide sequence represented by SEQ LD NO:22, assuming a first codon extending from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:22. The coding region encoding PYpFlsec,70, not including the stop codon, is represented by nucleic acid molecule nYpFlsec510, having the coding strand nucleic acid sequence represented by SEQ LD NO:3.
Isolated nucleic acid molecule nYpFlmat474, denoted herein as SEQ LD NO:l 1, encodes PYpFlmat150, a predicted mature FI antigen of about 150 amino acids, the sequence of which is presented herein as SEQ LD NO:12, assuming an initiation codon extending from about nucleotide 7 through about nucleotide 9 of SEQ LD NO:l 1 and a stop codon extending from about nucleotide 457 through about nucleotide 459 of SEQ LD NO:l 1. The coding region encoding PYpFlmat150, not including the stop codon, is represented by nucleic acid molecule nYpFlmat450, having the coding strand nucleic acid sequence represented by SEQ LD NO: 13. While not being bound by theory, an expressed antigen encoded by nYpFlmat474 would be expected to stay in the cytoplasm of the cell in which it is expressed, since it lacks a secretory signal segment. Isolated nucleic acid molecule nYpFlanc576, the coding strand of which is denoted herein as SEQ LD NO:7, comprises a coding region, not including the stop codon, encoding PYpFlanc192, a novel fusion protein of about 192 amino acids comprising an FI antigen of about 134 amino acids linked to the membrane anchor domain of the canine herpesvirus (CHV) glycoprotein G (i.e., about amino acid 358 through about amino acid 415 of CHV gG, disclosed as SEQ LD NO:10 in pending U.S. Patent Application Serial No. 08/602,010, by Haanes, et al., filed Feb. 15, 1996; this application is incorporated herein by reference in its entirety). Methods to construct nYpFlanc576 are disclosed herein. While not being bound by theory, a protein encoded by nYpFlanc576 would be expected to be secreted from the cytoplasm of a eukaryotic cell such that its C-terminal region is lodged in the cell's plasma membrane, and its N- terminal region is extending outside the cell. The amino acid sequence of PYpFlanc192 is presented herein as SEQ ID NO: 8, assuming an initiation codon extending from about nucleotide 1 through about nucleotide 3 of SEQ LD NO:7. PYpFlancι92 includes an N- terminal signal peptide sequence of about 21 amino acids extending from about amino acid 1 to about amino acid 21 of SEQ LD NO:8. The mature form of PYpFlanc192is represented by PYpFlanc171, having the amino acid sequence SEQ ID NO:10. PYpFlanc171 is encoded by nucleic acid molecule nYpFlanc513, having the coding strand nucleotide sequence represented by SEQ ID NO:9.
Another embodiment of the present invention is an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen that also includes at least one additional isolated nucleic acid molecule fused in-frame such that a multivalent antigen is encoded. Such a multivalent antigen can be produced by joining two or more isolated nucleic acid molecules together in such a manner that the resulting nucleic acid molecule is expressed as a multivalent antigen containing epitopes from at least two heterologous antigens, or portions thereof. Such a multivalent antigen can comprise two or more isolated nucleic acid molecules encoding Yersinia, Pasteurella or Francisella antigens, or can comprise one or more isolated nucleic acid molecules in addition to those encoding Yersinia, Pasteurella or Francisella antigens, such that the multivalent antigen is capable of protecting an animal from diseases caused by other infectious agents in addition to Yersinia, Pasteurella or Francisella. Examples of multivalent antigens include, but are not limited to, Yersinia,
Pasteurella or Francisella antigen of the present invention attached to one or more antigens protective against one or more other infectious agents, such as, but not limited to: viruses (e.g., caliciviruses, distemper viruses, hepatitis viruses, herpesviruses, immunodeficiency viruses, infectious peritonitis viruses, leukemia viruses, panleukopenia viruses, parvoviruses, picomaviruses, rabies viruses, other cancer-causing or cancer-related viruses); bacteria (e.g., Leptospira, Rochalimaea); fungi and fungal- related microorganisms (e.g., Candida, Cryptococcus, Histoplasma); and other parasites (e.g., Babesia, Cryptosporidium, Eimeria, Encephalitozoon, Hepatozoon, Isospora, Microsporidia, Neospora, Nosema, Plasmodium, Pneumocystis, Toxoplasma, as well as helminth parasites).
A recombinant molecule of the present invention includes at least one isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen, operatively linked to a eukaryotic transcription control region. Such a molecule contains heterologous nucleic acid sequences, that is, nucleic acid sequences that are not naturally found adjacent to the isolated nucleic acid molecules of the present invention and that are derived from species other than Yersinia, Pasteurella, or Francisella. A recombinant molecule can be either RNA or DNA, can have components from prokaryotic as well as eukaryotic sources, and must have the ability, by methods described herein, to enter eukaryotic cells and direct expression of isolated nucleic acid molecules of the present invention in those eukaryotic cells. In the case of the present invention, the recombinant molecule is typically a recombinant animal virus genome or a recombinant plasmid.
According to the present invention, an isolated nucleic acid molecule encoding a Yersinia, Pasteurella or Francisella antigen is operatively linked to a eukaryotic transcription control region. The phrase "operatively linked" refers to the combining of an isolated nucleic acid molecule of the present invention with a eukaryotic transcription control region in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, a eukaryotic transcription control region is a nucleic acid sequence which controls the initiation, elongation and termination of transcription in a eukaryotic cell. Particularly important transcription control regions are those which control transcription initiation, such as promoter and enhancer sequences. Suitable transcription control regions include any transcription control region that can function in at least one recombinant eukaryotic cell of the present invention. A variety of such transcription control regions are known to those skilled in the art. Preferred transcription control regions include those which function in mammalian cells, such as, but not limited to, promoter and enhancer sequences from alphaviruses (such as Sindbis virus), vaccinia virus, raccoon poxvirus, other poxviruses, adenovirus, adeno-associated vims, cytomegaloviruses (preferably the intermediate early promoter, preferably in conjunction with intron- A), other herpesviruses, simian vims 40 (preferably the early promoter), retroviruses (such as Rous sarcoma virus), and picomavimses (particularly an internal ribosome entry site, or IRES, enhancer region). Other preferred transcription control regions include those derived from mammalian genes such as actin, heat shock protein, bovine growth hormone transcription control regions, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
One type of recombinant molecule of the present invention comprises an animal vims genome. Such a recombinant molecule contains an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen of the present invention, operatively linked to at least one eukaryotic transcription control region capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transformed. Eukaryotic transcription control regions for recombinant vims genomes of the present invention include any that would function in the vims of choice. As used herein, a recombinant animal vims genome can comprise a heterologous eukaryotic transcription control region, i.e., a transcription control region that is non-native to the particular animal vims genome, being, for example, derived from another animal vims genome or from any other suitable eukaryotic gene. Suitable heterologous franscription control regions are disclosed herein. A recombinant animal vims genome of the present invention can also comprise an endogenous eukaryotic transcription control region, i.e., a transcription control region that is normally found in that vims. Such endogenous transcription control regions can be situated at their normal (i.e., natural) position in the viral genome, or at a non-natural position, i.e. inserted into another viral gene or into an intergenic region of the viral genome. Suitable recombinant vims genomes of the present invention include a poxvims genome, a herpesvirus genome, an alphavirus genome (for example, from a Sindbis vims), a picornavirus genome (for example, from a poliovims or a mengovims), a refrovims genome, an adenovims genome, or an adeno- associated vims genome. A preferred recombinant vims genome of the present invention comprises a poxvims genome, for example, an orthopoxvirus, a parapoxvirus, an entomopoxvims, or an avipoxvims (i.e., fowlpox) genome. A more preferred recombinant vims genome comprises an orthopoxvirus genome, particularly a vaccinia virus or a raccoon poxvims genome.
Preferred transcription control regions for a recombinant vims genome of the present invention include those that function in poxvimses. It will be appreciated by those skilled in the art that poxvimses undergo all aspects of viral replication, including transcription, in the cytoplasm of the infected host cell, and as such, have specialized, viral-encoded transcription-related proteins and recognition sequences. In particular, transcription of genes encoded on a poxvims genome requires a poxvims promoter. Examples of poxvims promoters include early/late promoters (i.e., working at early and late times in the infectious cycle of the vims) and late promoters. Particularly preferred poxvims promoters include the vaccinia vims p7.5 (early/late) promoter (see, for example, Cochran et al., 1985, J. Virol. 54, 30-37), the vaccinia vims pi 1 (late) promoter (see, for example, Bertholet et al., 1986, EMBO J. 5, 1951-1957), and the synthetic pSYN (late) promoter (see, for example, Davison, et al., 1990, Nucl. Acids Res. 18, 4825-4826).
Many recombinant vims genomes of the present invention, particularly poxvims genomes, are very large. One skilled in the art will know that a portion of genes in such vimses are dispensable for growth of the vims (often referred to as non-essential genes). As such, one or more isolated nucleic acid molecules encoding a Yersinia, Pasteurella, or Francisella antigens, operatively linked to eukaryotic transcription control regions suitable for a particular vims, can be located (preferably by insertion) in one or more non-essential genes. Alternatively, an isolated nucleic acid molecule can be located between genes in the viral genome, i.e., in an intergenic region. In some recombinant vims genomes, an isolated nucleic acid molecule of the present invention can be located in an essential gene. If the resulting recombinant vims is to undergo replication, this latter group of insertions requires that the essential gene be complemented in the infected host cell, either by that cell being stably transformed with the essential gene, transiently transformed with the essential gene, or having that gene supplied on a helper vims. In a recombinant vaccinia vims or raccoon poxvims genome of the present invention, an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen can be located in any suitable non-essential gene or intergenic region. Preferred non-essential genes include a thymidine kinase gene, a hemagglutination gene, an anti-inflammatory gene, and an A-type inclusion gene. Preferred anti-inflammatory genes include a soluble cytokine receptor gene, a serpin gene, a complement receptor gene and an immunoglobulin receptor gene. Particularly preferred non-essential genes are the thymidine kinase genes of raccoon poxvims and vaccinia vims. Another recombinant molecule of the present invention comprises a recombinant plasmid which includes an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen operatively linked to a eukaryotic transcription control region. A recombinant plasmid of the current invention has utility as a genetic immunization vaccine to protect an animal against plague. A preferred recombinant plasmid contains an origin of replication for propagation in a bacterial host (e.g., Escherichia colϊ), a mode of selection, such as an antibiotic resistance gene, and a suitable cloning site for isolated nucleic acid molecules of the present invention. Any suitable eukaryotic transcription control region can be used, such as, but not limited to, those disclosed herein. Particularly preferred transcription control regions include, but are not limited to, a HCMV immediate-early promoter (preferably in conjunction with intron-A), a Rous sarcoma vims long terminal repeat, and an SV40 early promoter. The incorporation of polyadenylation sequences, for example, bovine growth hormone or SV40 polyadenylation sequences, is also preferred. A preferred recombinant plasmid can also include an enhancer region, for example, a HCMV intron-A sequence or an EMCV-IRES sequence.
A recombinant molecule of the present invention is a molecule that can include at least one of any isolated nucleic acid molecule that encodes an antigen from Yersinia, Pasteurella, or Francisella, operatively linked to at least one eukaryotic transcription control region capable of effectively regulating expression of the nucleic acid molecule(s) in the cell to be transformed, examples of which are disclosed herein. Preferred recombinant molecules include at least one of the following nucleic acid molecules: nYpFl(a)sec544, nYpFl(b)sec544, nYpFlsec5!0, nYpFlanc576, nYpFlanc513, nYpF 1 mat474ι nYpF 1 mat450, and nYpF 1 mat^ . Particularly preferred recombinant molecules of the present invention include vRCN-pl l-nYpFl(a)sec544) vRCN-pl 1- nYpF 1 anc576, vRCN-p 11 -nYpF 1 mat474, pCMV-nYpF 1 (b)sec544, pCMV-nYpF 1 anc576, and pCMV-nYpFlmat474. The term "vRCN" refers to a recombinant raccoon poxvims genome and the term "pCMV" refers to a recombinant plasmid comprising the HCMV immediate-early transcription control region. Details regarding the production of recombinant molecules of the present invention are disclosed herein. Another embodiment of the present invention is a recombinant vims. A recombinant vims of the present invention includes any animal vims comprising a suitable recombinant molecule, i.e. a recombinant vims genome, of the present invention. Suitable recombinant vimses of the present invention include poxvimses, herpesviruses, alphavimses (for example Sindbis vims), picomaviruses (for example, poliovims or mengovims), retrovimses, adenovimses, and adeno-associated vimses. A preferred recombinant vims of the present invention comprise a poxvims, for example, an orthopoxvims, a parapoxvirus, an entomopoxvims, or an avipoxvims (i.e., fowlpox). A more preferred recombinant vims comprises a recombinant orthopoxvims, particularly a vaccinia vims or a raccoon poxvims. An example of a more preferred embodiment of the present invention is a recombinant raccoon poxvims (RCN) comprising a nucleic acid molecule encoding an FI antigen of Yersinia pestis operatively linked to a vaccinia vims pi 1 promoter, as disclosed in Examples 1 and 2 below. Particularly preferred recombinant vimses of the present invention comprise recombinant raccoon poxvimses RCN:p 11 -nYpF 1 (a)sec544> RCN:p 11 -nYpF 1 anc576, and RCN:p 11 -nYpF 1 mat474-, with RCN:pl l-nYpFl(a)sec544 being the more preferred. One embodiment of the present invention is an attenuated recombinant vims. As used herein, an attenuated vims is a vims that results in less pathogenicity than its wild- type counterpart when used to infect an animal. A preferred attenuated vims of the present invention causes little or no pathogenicity when used to infect an animal. An attenuated recombinant vims can be produced by inactivating a viral gene that, due to that gene's inactivation, results in an attenuated vims. Methods to inactivate a gene are disclosed herein. An attenuated recombinant vims can be identified by exposing animals to the vims and measuring clinical signs, such as fever, lesions, or viremia, in those animals compared to similar animals exposed to the wild-type vims. Clinical signs to measure vary with each individual vims, and are known to one skilled in the art. Suitable viral genes to inactivate in order to produce an attenuated recombinant vims include any gene that when inactivated leads to an attenuated vims. A preferred attenuated recombinant vims of the present invention is a vims having a recombinant genome in which a heterologous nucleic acid molecule, i.e., one encoding a Yersinia, Pasteurella, or Francisella antigen, is inserted into a viral gene, the insertion resulting in an attenuated vims. A particularly preferred attenuated recombinant vims of the present invention is an attenuated recombinant vaccinia vims or raccoon poxvims. A particularly preferred method of attenuation of a recombinant vaccinia vims or raccoon poxvims is by insertion of a heterologous nucleic acid molecule into the thymidine kinase (tk) locus. An attenuated recombinant vims of the present invention, particulary an attenuated recombinant raccoon poxvims, has utility, for example, as a therapeutic composition to protect an animal from plague. While not being bound by theory, the inventors believe that a recombinant raccoon poxvims need not be further attenuated for use as a live viral vaccine in most animals due to the low pathogenicity of wild-type RCN. See, for example, Esposito, et al., 1989, Vaccines 89, Cold Spring Harbor Labs Press, 403-408. Another embodiment of the present invention is a recombinant cell comprising a eukaryotic host cell transformed with at least one of any recombinant molecule of the present invention. Suitable and preferred recombinant molecules with which to transform cells are disclosed herein. The terms "transform" or "transformed", as used in the present invention, refer to any way in which a recombinant molecule can be inserted into a cell. Transformation techniques include, but are not limited to, transfection, viral infection with a recombinant vims, viral transduction with a recombinant vims, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
Suitable host cells to transform include any cell that can be transformed with a recombinant molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins useful in the production of a multivalent vaccines). Host cells of the present invention can be any cell capable of producing at least one antigen of the present invention. Preferred host cells primarily include mammalian cells. Most preferred host cells include BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell line), CRFK cells (normal cat kidney cell line), BSC-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvims), COS (e.g., COS-7) cells, and Vero cells. Particularly preferred host cells are BHK cells, BSC-1 cells, MDCK cells, CRFK cells, CV-1 cells, COS cells, Rat-2 cells, Vero cells, and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NLH/3T3 cells, LMTK31 cells and/or HeLa cells.
A recombinant cell of the present invention can include any eukaryotic host cell transformed with at least one of any recombinant molecule of the present invention. A preferred recombinant cell includes at least one isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen operatively linked to at least one eukaryotic transcription control region capable of effectively regulating expression of the nucleic acid molecule(s) in that cell; particularly preferred nucleic acid molecules to include are nYpFl(a)sec544, nYpFl(b)sec544, nYpFlsec510, nYpFlanc576, nYpFlanc513, nYpFlmat474, nYpFlmat450, and/or nYpFlmat^. A more preferred recombinant cell includes one or more of the following recombinant molecules: vRCN-pl 1- nYpFl(a)sec544, vRCN-pl l-nYpFlanc576, vRCN-pl l-nYpFlmat474, pCMV- nYpF 1 (b)sec544, pCMV-nYpF 1 anc576, and pCMV-nYpF 1 mat474. Particularly preferred recombinant cells of the present invention include BSC-1 :RCN:pll-nYpFl(a)sec544, BSC-1 :RCN-pl l-nYpFlanc576, BSC-l:RCN-pl l-nYpFlmat474, as well as any animal cells comprising pCMV-nYpFl(a)sec544, pCMV-nYpFlanc576, and pCMV-nYpFlmat474. One embodiment of the present invention is a therapeutic composition that, when administered to an animal in an effective manner, is capable of protecting that animal from plague. Therapeutic compositions of the present invention include a recombinant molecule comprising an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen operatively linked to a eukaryotic transcription control region. Suitable therapeutic compositions include recombinant animal vims genomes, recombinant vimses, recombinant plasmids and recombinant cells as disclosed herein. In order to protect an animal from plague, a therapeutic composition of the present invention is administered to the animal in an effective manner prior to infection in order to prevent disease, reduce disease symptoms and/or prevent transmission of the disease from asymptomatic carriers (i.e., as a preventative vaccine). Therapeutic compositions of the present invention can be administered to any animal susceptible to such therapy, preferably to mammals, and more preferably to cats, primates, rodents, ungulates, bears, dogs, camels, and pigs. Preferred animals to protect against plague include domestic cats, humans, bobcats, cougars, domestic dogs, coyotes, foxes, rock squirrels, ground squirrels, prairie dogs, black footed ferrets, domestic ferrets, pronghorn antelope, badgers, bears, wild boars, domestic pigs, camels, chipmunks, red deer, mule deer, fishers, foxes, gerbils, martens, urban mice, wild mice, polecats, rabbits, urban rats, wild rats, tree squirrels, and voles. Even more preferred animals to protect against plague include domestic cats, domestic dogs, humans, rock squirrels, California ground squirrels, prairie dogs, domestic ferrets, and black-footed ferrets. As used herein, the term plague refers to the group of diseases most normally caused by the bacterium Yersinia pestis, but also, in some cases, similar diseases caused by other species within the genera Yersinia, Pasteurella, or Francisella. As such, plague includes, but is not limited to, diseases such as bubonic plague, septicemic plague, pneumonic plague, urban plague, rat plague, wild rodent plague, sylvatic plague, campestral plague, high plains plague, disseminated intravascular coagulopathy, la peste bubonique, The Pest, The Black Plague, and The Black Death.
Another embodiment of the present invention is a therapeutic composition to protect an animal from plague that also includes at least one additional isolated nucleic acid molecule encoding an antigen from a pathogen other than Yersinia, Pasteurella or Francisella, operatively linked to one or more eukaryotic transcription control regions, such that a multivalent therapeutic composition is produced. Such a multivalent therapeutic composition can be produced by combining one or more additional isolated nucleic acid molecules into a recombinant molecule of the present invention, or by combining one or more recombinant molecules with a recombinant molecule of the present invention. When administered to an animal, such a multivalent therapeutic composition is able to direct the expression of one or more antigens in the cells of that animal such that the animal is protected from diseases caused by other infectious agents in addition to Yersinia, Pasteurella or Francisella. Examples of multivalent therapeutic compositions include, but are not limited to, a Yersinia, Pasteurella or Francisella antigen of the present invention plus one or more antigens protective against one or more other infectious agents, such as, but not limited to: vimses (e.g., calicivimses, distemper vimses, hepatitis vimses, herpesviruses, immunodeficiency vimses, infectious peritonitis vimses, leukemia vimses, panleukopenia vimses, parvovimses, picomavimses, rabies vimses, other cancer-causing or cancer-related vimses); bacteria (e.g., Leptospira, Rochalimaea); fungi and fungal- related microorganisms (e.g., Candida, Cryptococcus, Histoplasma); and other parasites (e.g., Babesia, Cryptosporidium, Eimeria, Encephalitozoon, Hepatozoon, Isospora, Microsporidia, Neospora, Nosema, Plasmodium, Pneumocystis, Toxoplasma, as well as helminth parasites). Therapeutic compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosol, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non- liquid formulation, the excipient can comprise dextrose, human semm albumin, preservatives, etc., to which sterile water or saline can be added prior to administration. In one embodiment of the present invention, a therapeutic composition can include an adjuvant. Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of cytokines and chemokines (e.g., granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (LL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I (IGLF), transforming growth factor beta, RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), macrophage inflammatory proteins (e.g., MLP-1 alpha and MLP-1 beta), and Leishmania elongation initiating factor (LEIF)); bacterial components (e.g., endotoxins, in particular superantigens, exotoxins and cell wall components); aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins, viral coat proteins; block copolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™, Inc. Norcross, GA), Ribi adjuvants (Ribi ImmunoChem Research, Inc., Hamilton, MT); and saponins and their derivatives (e.g., Quil A (Superfos Biosector A/S, Denmark). Protein adjuvants of the present invention can be delivered in the form of the protein themselves or of nucleic acid molecules encoding such proteins using the methods described herein. In one embodiment of the present invention, a therapeutic composition can include a carrier. Carriers include compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, vimses, other cells, oils, esters, and glycols. While not being bound by theory, an advantage of a therapeutic composition comprising a recombinant vims is that a carrier is usually not required.
One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).
Some preferred therapeutic compositions of the present invention include at least a portion of a recombinant vims genome, comprising a recombinant vims vaccine. Preferred recombinant vims genomes include those based on alphavimses, poxvimses, adenovimses, adeno-associated vimses, picomavimses, herpesvimses, and retrovimses, with those based on poxvimses being particularly preferred.
A therapeutic composition comprising a recombinant vims of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule produces attenuated vims. A number of recombinant viruses can be used, including, but not limited to, those based on alphavimses, poxvimses, adenovirases, adeno-associated vimses, picomavimses, herpesvimses, and retrovimses. Preferred recombinant vims vaccines are those based on poxvimses.
A therapeutic composition comprising a genetic immunization (i.e., naked nucleic acid) vaccine of the present invention includes an isolated nucleic acid molecule of the present invention operatively linked to a eukaryotic transcription control region in a recombinant molecule of the present invention. A genetic immunization vaccine of the present invention can comprise one or more nucleic acid molecules of the present invention in the form of, for example, a dicistronic recombinant molecule. Any suitable eukaryotic transcription control region can be used. Particularly preferred transcription control regions include the HCMV intermediate early promoter (preferably in conjunction with intron- A), Rous sarcoma vims long terminal repeat, and tissue-specific transcription control regions, as well as transcription control regions endogenous to viral vectors if viral vectors are used. The incorporation of polyadenylation sequences and enhancers are also preferred. A recombinant cell vaccine of the present invention includes recombinant eukaryotic cells of the present invention that express one or more Yersinia, Pasteurella, or Francisella antigens of the present invention. Preferred recombinant cells for this embodiment include BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinant cells. Recombinant cell vaccines of the present invention can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about 108 to about 1012 cells per kilogram body weight. Recombinant cell vaccines can comprise whole cells, cells stripped of cell walls or cell lysates.
The present invention also includes methods to protect an animal from plague using a therapeutic composition of the current invention. According to this embodiment, a recombinant molecule of the present invention can be administered to an animal in a fashion to enable the recombinant molecule to enter one or more cells of the animal, such that an antigen encoded by an isolated nucleic acid molecule contained therein is expressed into a protective antigen in the animal. Recombinant molecules of the present invention can be delivered to an animal by a variety of methods including, but not limited to, (a) administering a genetic immunization vaccine, e.g., naked DNA or RNA molecules, such as is taught, for example, in Wolff et al., ibid., or (b) administering a nucleic acid molecule packaged as a recombinant vims vaccine or as a recombinant cell vaccine (i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle).
Genetic immunization vaccines of the present invention can be administered by a variety of methods. Suitable delivery methods include, for example, intramuscular injection, subcutaneous injection, intradermal injection, intradermal scarification, particle bombardment, oral application, and nasal application, with intramuscular injection, intradermal injection, intradermal scarification and particle bombardment being preferred. A preferred single dose of a genetic immunization vaccine ranges from about 1 nanogram (ng) to about 1 milligram (mg), depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Examples of administration methods are disclosed, for example, in U.S. Patent No. 5,204,253, by Bruner, et al., issued April 20, 1993, PCT Publication No. WO 95/19799, published July 27, 1995, by McCabe, and PCT Publication No. WO 95/05853, published March 2, 1995, by Carson, et al. Genetic immunization vaccines of the present invention can be contained in an aqueous excipient (e.g., phosphate buffered saline) alone or with a carrier (e.g., lipid-based vehicles), or it can be bound to microparticles (e.g., gold particles).
When administered to an animal, a recombinant vims vaccine of the present invention infects cells within the immunized animal and directs the production of a Yersinia, Pasteurella, or Francisella antigen that is capable of protecting the animal from plague. For example, a recombinant vims vaccine comprising an isolated nucleic acid molecule encoding a Yersinia, Pasteurella, or Francisella antigen of the present invention is administered according to a protocol that results in the animal producing a sufficient immune response to be protected from a plague challenge. A preferred single dose of a recombinant vims vaccine of the present invention is from about 1 x 104 to about 1 x 108 vims plaque forming units (pfu) per animal. Administration protocols are well-known to those skilled in the art, with subcutaneous, intramuscular, intradermal, intranasal, and oral administration routes being preferred. A particularly preferred method of administration, especially in cats, for a recombinant vims vaccine of the present invention is by oral delivery. Since RCN, for example, has been shown to be effective in cats when administered orally, the induction of a strong mucosal response is a possibility. As such, a preferred therapeutic composition to administer to an animal is a recombinant RCN comprising an isolated nucleic acid molecule encoding the FI antigen of Yersinia pestis. A particularly preferred therapeutic composition comprises RCN:pl l-nYpFlsec544.
The efficacy of a therapeutic composition of the present invention to protect an animal from plague can be tested in a variety of ways including, but not limited to, detection of protective antibodies, detection of cellular immunity within the treated animal, or challenge of the treated animal with Yersinia, Pasteurella, or Francisella (preferably Yersinia pestis) to determine whether the treated animal is resistant to disease. In one embodiment, therapeutic compositions can be tested in a target animal and then serum from that vaccinated animal can be transferred to animal models such as mice, to test for protection by passive immunity. Such techniques are known to those skilled in the art. Preferred embodiments of the present invention include (a) a recombinant raccoon poxvims genome that includes an isolated nucleic acid molecule encoding a Yersinia pestis antigen operatively linked to a poxvims transcription control region, (b) a recombinant raccoon poxvims including such a recombinant genome, (c) a recombinant cell including such a recombinant genome, (d) a recombinant plasmid that includes an isolated nucleic acid molecule encoding a Yersinia pestis antigen operatively linked to a eukaryotic transcription control region, and (e) a recombinant cell that includes such a recombinant plasmid. A particularly preferred eukaryotic transcription control region is the human cytomegalovirus (HCMV) immediate-early promoter. Other preferred embodiments include therapeutic compositions comprising a recombinant raccoon poxvims genome, a recombinant raccoon poxvims, a recombinant cell, and/or a recombinant plasmid as described above.
The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.
Examples It is to be noted that the Examples include a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook, et al., ibid., Ausubel, et al., ibid., and related references. Example 1:
This example discloses the production of a recombinant raccoon poxvirus capable of expressing a secreted Y. pestis FI antigen.
A. Recombinant molecule pKB3poly-nYpFl(a)sec544, containing a nucleic acid molecule encoding the FI antigen of Yersinia pestis operatively linked to a vaccinia vims pi 1 late promoter transcription control region was produced in the following manner. The pKB3poly poxvims shuttle vector was created by modifying a region of plasmid pKB3 (P,,-type) plasmid (described in U.S. Patent No. 5,348,741, by Esposito et al., issued September 20, 1994) such that the initiation codon linked to the pi 1 promoter was mutated and additional unique polylinker restriction sites were added. The resulting poxvims vector, referred to as pKB3poly, requires the insert DNA to provide the ATG initiation codon when inserted downstream of the pi 1 promoter. The pKB3poly vector was designed such that foreign DNA cloned into the polylinker region of pKB3poly vector will recombine into the thymidine kinase (tk) gene of a wild-type orthopoxvims.
Plasmid YPRl (Simpson, et al., ibid., obtained from the National Institutes of Health Rocky Mountain Laboratories, Hamilton, MT) was used as a template for PCR amplification of the FI nucleic acid molecule using sense primer EJH031 5' ACG
CGCGTCGACG AGGTAATATA TGAAAAAAAT CAG 3'; denoted herein as SEQ ID NO: 14 (Sail site in bold) and antisense primer EJH032 5' CGCGGATCCC TATATGGATT ATTGGTTAGA TACGG 3'; denoted herein as SEQ LD NO: 15 (BamHl site in bold). These primers were synthesized based on a published Y. pestis FI nucleotide sequence, available in Galyov, et al., ibid. The PCR amplified product was digested with restriction endonucleases Sail and BamHl and gel purified, resulting in a double stranded nucleic acid molecule of about 544 base-pairs denoted herein as nYpFl(a)sec544 the sequence of which is denoted herein as SEQ ED NO:l. It is to be noted that the PCR fragment amplified from plasmid YPRl was not derived from the same strain of Yersinia pestis as the published sequence and, as such, may comprise an allelic or strain variant of the published sequence. SEQ ED NO:l contains an open reading frame of about 510 nucleotides, assuming a start codon extending from about nucleotide 17 through about nucleotide 19 of SEQ ID NO:l and a termination codon extending from about nucleotide 527 through about 529 of SEQ ED NO:l. The coding region is denoted herein as nYpFlsec510, the coding strand of which is presented herein as SEQ ED NO:3. SEQ ED NO:3 encode a full-length F 1 protein of about 170 amino acids, denoted herein as PYpFlsec170_ the sequence of which is presented herein as SEQ ED NO:2. As disclosed in Galyov, et al., ibid., PYpFlsec170 includes an N-terminal signal peptide sequence of about 21 amino acids extending from about amino acid 1 to about amino acid 21 of SEQ ED NO:2. In its native milieu, this signal peptide directs the secretion of F 1 across the inner and outer Y. pestis membranes where it is assembled into a capsule around the bacterial cell. The mature form of PYpFlsec170 is represented by PYpFlmat149, having the amino acid sequence SEQ ID NO:21. PYpFlmat149 is encoded by nucleic acid molecule nYpFlmatψ,,, having the coding strand nucleotide sequence represented by SEQ ID NO:22, assuming a first codon extending from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:22.
The PCR-amplified fragment comprising nYpFl(a)sec544 was ligated into BamHl and S /1-digested and gel-purified pKB3poly transfer vector, resulting in recombinant molecule pKB3poly-nYpFl(a)sec544. Plasmid DNA comprising pKB3poly- nYpFl(a)sec544 was purified using Qiagen columns (available from Qiagen, Chatsworth, CA).
B. A recombinant raccoon poxvims capable of expressing Y. pestis FI antigen was produced as follows. BSC-1 African green monkey kidney cells (obtained from American Type Culture Collection (ATCC), Rockville, MD) were infected at a multiplicity of infection (MOI) of 0.05 with wild type raccoon poxvims RCN CDC/V71- I-85A (obtained from Dr. Joseph Esposito; Esposito et al., 1985, Virology 143, 230-251) and were then transfected with pKB3poly-nYpFl(a)sec544 plasmid DNA by calcium phosphate precipitation to form recombinant cell BSC1 :RCN:pl l-nYpFl(a)sec544 by recombination of pKB3poly-nYpFl(a)sec544 with the wild-type RCN DNA at the tk locus. The resulting recombinant vims, denoted RCN:pl l-nYpFl(a)sec544, was plaque purified twice in RAT-2 rat embryo, thymidine kinase mutant cells (available from
ATCC) in the presence of bromodeoxyuridine (BUDR, available from Sigma Chemical Company, St. Louis, MO) to select for tk" recombinants. A tk" recombinant vims was plaque purified a third time on BSC-1 cells without BUDR. Example 2:
This example demonstrates expression of cellular and secreted forms of Y. pestis F 1 antigen in RCN:p 11 -nYpF 1 (a)sec544-infected cells.
Expression of 7 pestis FI antigen in RCN:pl l-nYpFl(a)sec544-infected cells and its secretion from the cells was monitored by the following method. BSC-1 cells were plated into 6 well polystyrene dishes in about 2 ml of MEM medium (available from Life Technologies, Inc., Gaithersburg, MD) containing 5 % fetal bovine serum (FBS) per well. The cells were allowed to grow overnight at 37°C with 5% CO2. The medium was removed from the cells and replaced with about 2ml of MEM containing 1.0 % FBS. The cells were then infected with RCN:pl l-nYpFl(a)sec544 at an MOI of approximately 0.025 pfu/cell and were further incubated for about 2 days at 37°C, 5% CO2 until 100% cytopathic effect (CPE) was observed. The culture was harvested by scraping the infected cells into the medium. The culture was centrifuged at 6000 RPM in a table-top centrifuge for 6 min. at room temperature.
The supernatant and cells were prepared for western blot analysis as follows. The cell pellet was washed in PBS and resuspended in 50 μl of lx loading buffer (125 mM Tris, pH 6.8, 4% SDS, 0.05% Bromophenol blue, 20% glycerol, and 10% β- mercaptoethanol.). The cell lysate was heated to 95°C for 5 min., and then filtered through a 0.45 μm filter unit, for example, an Ultrafree-MC™ 0.45 μm filter unit, available from Millipore Corp., Bedford, MA. An about 1.9 cm2 equivalent of the filtered sample (about 10 μl) was analyzed by western blot as described below. The supernatant from the infected cells (about 2 ml) was centrifuged at 14,000 rpm for 5 min. at room temperature in a microcentrifuge and was then concentrated to 100 μl in an ultrafiltration device with a 10-kD molecular weight cutoff, for example, a Microcon- 10™ unit, available from Amicon, Inc., Beverly, MA, according to the manufacturer's instructions. The supernatant was combined with an equal volume of 2X loading buffer and heated to 95°C for 5 min. An about 1.9 cm2 equivalent of the prepared concentrated supernatant (about 20 μl) was analyzed by western blot as described below. The cell lysate and concentrated supernatant fractions of the RCN:pl 1- nYpFl(a)sec544-infected BSC-1 cells, prepared as described above, FI antigen purified from 7 pestis (obtained from the Centers for Disease Control (CDC), Fort Collins, CO), and appropriate wild-type vims infected-cell controls were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blot analysis using monospecific polyclonal rabbit anti-Fl antigen antiserum (obtained from the CDC). Both the RCN:pl l-nYpFl(a)sec544 infected-cell lysate and supernatant fractions revealed a eukaryotic version of FI antigen that migrated as a doublet with apparent molecular weights ranging from about 17 kD to about 22 kD. Upon comparing the cm2- adjusted equivalent amounts of the cell and supernatant fractions analyzed on the western blot, it appeared that the supernatant fraction contained about 4 times more FI antigen than the infected cell lysate fraction. While not being bound by theory, this result suggests that FI antigen is secreted from the RCN:pl l-nYpFl(a)sec544-infected BSC-1 cells, which implies that the bacterial signal segment contained on nYpFl(a)sec544 is functional in eukaryotic cells (resulting in an antigen equivalent to PYpFlmat149). The 17-kD band migrated at a position very similar, if not identical, to that of FI antigen purified from Yersinia pestis. As deduced from the nucleotide sequence, the predicted native size of 7. pestis FI, with the signal peptide removed, is 15.5 kD (Galyov, et al., ibid). Native-expressed FI antigen is known to be glycosylated, which accounts for its larger observed size of 17 kD; see, for example Bennett, et al., 1974, J. Bacteriol., 117, 48-55. Since the RCN-expressed FI antigen (the lower and more abundant band of the doublet) ran at an apparent molecular weight similar, if not identical, to native FI, it was probably also post-translationally modified. While not being bound by theory, the protein migrating with a 22-kD apparent molecular weight could represent another form of post-translational modification. A third, larger immunoreactive protein band, migrating at a molecular weight significantly greater than 90 kD but less than 250 kD was observed in the infected-cell lysate fraction (but not in the supernatant fraction). While not being bound by theory, this band could represent a multimeric form of FI (common in 7. pestis, see, for example, Bennett, et al., ibid.) or, alternatively, could represent FI protein bound tightly to a cellular or viral protein. Example 3:
This example discloses the immunization of mice with recombinant vims RCN:pl l-nYpFl(a)sec544, and the generation of antibodies in the immunized animals. A. Vims stocks for immunization of mice were prepared as follows. BSC-1 cells were seeded into 40-225 cm2 flasks in MEM containing 5 % FBS and incubated at 37°C for 2-3 days until a confluent monolayer was formed. The cells were infected at an MOI of 0.01 pfu/cell with RCN:pl l-nYpFl(a)sec544. The vims stock was treated with 0.125mg/ml trypsin for 15 min. at 37°C just prior to infecting the cells. The infected cells were incubated for 36-48 hrs. at 37°C until 100% CPE was obtained. The infected cells were detached from the flasks with sterile glass beads and the culture was centrifuged at 5000 rpm for 15 min in a table-top centrifuge at room temperature. The infected cells were resuspended in 30 ml of cold 10 mM Tris buffer, pH 9.0 and homogenized with 40 strokes in a dounce homogenizer on ice. The homogenized sample was centrifuged at 300 x g for 5 min at 5-10°C in a tabletop centrifuge. The supernatant was saved on ice. The pellet was resuspended in 10 ml of cold 10 mM Tris, pH 9.0, and homogenized with 20 strokes in a dounce homogenizer on ice. The sample was centrifuged as before and the supernatant was removed and combined with the first supernatant. The combined supernatant was sonicated on ice for 3 pulses at 6 watts of 15 seconds each with a hand held sonicator, for example, a VirSonic60™ sonicator (available from The VirTis Co., Inc., Gardiner, NY). The supernatant was then layered onto three 13-ml cushions of 36% sucrose (in 10 mM Tris, pH 9.0) and centrifuged for 80 min. at 32,900 x g, at 4°C using, for example, a Model J2-21M ultracentrifuge fitted with a JA-20 rotor, available from Beekman Instruments, Inc., Fullerton CA, to pellet the vims particles. The pelleted vims was resuspended in 4 ml of cold 1 mM Tris, pH 9.0 and sonicated on ice with 2 pulses of 15 seconds each. The vims was aliquotted and stored at -70°C. Prior to vaccinating animals, an aliquot was thawed and titered by plaque assay, using techniques familiar to those skilled in the art.
B. To evaluate the immunogenicity in mice of recombinant vims RCN:pl 1- nYpFl(a)sec544 expressing the 7 pestis FI capsular antigen, four groups of AJ mice, all about three weeks old, were immunized by injection into the footpad, as follows. Group 1, consisting of 60 mice, received about 1 X 108 plaque forming units (pfu) of RCNηpll- nYpFl(a)sec544 in about 30 μl of diluent (1 mM Tris, pH 9.0). Group 2, consisting of 36 mice, received about 1 μg of FI protein purified from 7 pestis (obtained from the CDC) also in about 30 μl of diluent. Group 3, consisting of 36 mice, received about 1 X 108 pfu of a control RCN vims, RCN-lacZ, in about 30 μl of diluent. RCN-lacZ comprises the gene encoding E. coli beta-galactosidase driven by the vaccinia p7.5 promoter, which was inserted into the tk locus of RCN by a method similar to that described in Example IB above. Group 4, consisting of 36 mice, received about 30 μl of diluent.
C. The response to FI antigen by the immunized mice was measured by enzyme-linked immunosorbent assay (ELISA) assay as follows. Blood was collected from all immunized mice 5 days prior to infection and at days 10, 20, 30, 37 and 58 post-infection. Serum samples were prepared by methods well known to those skilled in the art. The serum samples were tested for anti-Fl antibodies using an ELISA for total IgM/IgG, performed as follows. Individual wells of 96-well ELISA plates were coated with purified FI antigen (about 1.0 μg in about 50 μl carbonate buffer, pH 9.6 per well), and were incubated overnight at 4°C or at 37°C for 2 hours. Plates were washed with Tris-buffered saline with 0.1% Tween-20 (TBST), and then blocked with 200 μl of blocking buffer (0.03% bovine serum albumin in TBST), by incubating at 37°C for 1 hour or overnight at 4°C. Following the blocking step and further washing, the mouse serum samples (1 :40 and 1:640 dilutions in 50 μl total volume, diluted in TBST) were added to duplicate wells on the plates. Known negative and positive-control mouse serum samples, diluted as the test serum samples, were also added on each plate. The plates were incubated at 37°C for 1 hour. After another wash, horseradish peroxidase labeled goat anti -mouse conjugate (50 μl of a 1:2000 dilution, available from Jackson ImmunoResearch Lab., Inc., West Grove, PA) was added to each well, and incubated at 37°C for 1 hour. The plates were then washed and 50 μl of peroxidase substrate was added to each well (using, for example, the ABTS™ peroxidase substrate available from Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD), and incubated for 15 minutes at room temperature. Finally, the reaction was stopped by the addition of 50 μl of stop solution (1% SDS). The ELISA plates were read on an ELISA plate reader set at 405 nm. The average and standard deviations (SD) of the negative controls at 1 :40 and 1 :640 dilutions were calculated, and any specimen that was the average +3SD was considered positive. The mean O.D. and standard deviations of the ELISA for serum samples taken on days 20, 30, and 37 are summarized in Table 1.
Table 1
Figure imgf000037_0001
The ELISA results demonstrate that the RCN:pl l-nYpFl(a)sec544-iιrιmunized mice (Group 1) produced antibodies against 7 pestis FI antigen that, at least by day 37, approached the antibody levels produced by mice immunized with purified FI antigen (Group 2). Example 4:
This Example discloses the production of recombinant plasmids encoding various forms of the 7 pestis FI antigen. A. Eukaryotic expression vector pPVXC was produced as follows. Vector pRc/RSV (available from Lnvitrogen Corp., San Diego, CA) was cleaved by restriction enzyme Pvuϊl, and the 2963 -base pair PvwII fragment was gel purified. That fragment was self-ligated to form vector pRc/RSV (Pvu), which contains a Rous Sarcoma Vims (RSV) long terminal repeat, a multiple cloning site, a bovine growth hormone polyadenylation sequence, a bacterial origin of replication, and an ampicillin resistance gene. Expression vector pPVXC was produced by introducing a HindllUSspl fragment containing the HCMV intermediate early promoter and first intron (i.e., intron- A) into pRc/RSV(Pvu) that had been cleaved by H dIII and Nrul. This manipulation removed sequences encoding the RSV long terminal repeat from pRc/RSV( vw). B. A recombinant plasmid, denoted herein as pCMV-nYpFl(b)sec544, in which a nucleic acid molecule encoding a full-length FI protein is operatively linked to the ΗCMV immediate-early transcription control region, was produced as follows. Nucleic acid molecule nYpFl(b)sec544, which encodes PYpFlsec,70(i.e., SEQ ID NO:2), was produced by PCR amplification of that molecule from plasmid YPRl (described in Example 1) using forward primer JO-1, having nucleic acid sequence 5'GGCAAGCTTG AGGTAATATA TGAAAAAAAT CAG 3', represented herein as SEQ ID NO: 16 (HindlH site in bold); and reverse primer JO-2 having nucleic acid sequence 5' GGCGAATTCC TATATGGATTA TTGGTTAGAT ACGG 3', represented herein as SEQ ED NO: 17 (EcoRI site in bold). These primers were synthesized based on a published 7. pestis FI nucleotide sequence (Galyov, et al., ibid.) as described in Example 1. The only differences between YpFl(a)sec544 and YpFl(b)sec544 are the 5' and 3' restriction enzyme sites. The PCR amplified product was digested with restriction endonucleases HindR and EcoRI and gel purified, resulting in a double stranded nucleic acid molecule of about 544 base-pairs denoted herein as nYpF 1 (b)sec544ι the sequence of the coding strand of which is denoted herein as SΕQ LD NO:4. The coding sequence, open reading frame and the mature processed protein encoded by SΕQ LD NO:4 are all identical to those described in Example 1 for SEQ ID NO: 1. It is to be noted that the PCR fragment amplified from plasmid YPRl was not derived from the same strain of Yersinia pestis as the published sequence and, as such, may comprise an allelic or strain variant of the published sequence. Recombinant molecule pCMV-nYpFl(b)sec544 was produced by ligating nucleic acid molecule nYpFl(b)sec544 into pPVXC that had been cleaved by Hindu! and EcoRI and gel purified.
C. A recombinant plasmid, identified herein as pCMV-nYpFlanc576, capable of expressing an FI antigen fused to a eukaryotic membrane anchor domain, was produced as follows. Nucleic acid molecule nCgGanc192, which encodes the membrane anchoring domain of the canine herpesvirus (CHV) glycoprotein gG gene was produced by PCR amplification from CHV viral DNA using forward primer JO-3, having a sequence 5' GGGATGACGT CGTCGGTTAT AATAATTGTA ATACCC 3', represented herein as SΕQ LD NO: 18 (Tthl 111 site in bold), and reverse primer JO-4 having nucleic acid sequence 5'GGCGAATTCT TAAATATCAT AAAAATTTAA TTTCTGGGG 3', represented herein as SΕQ ID NO: 19 (EcoRI site in bold). The PCR amplified product was digested with restriction endonucleases Tthl 1 II and EcoRI and gel purified, resulting in a double stranded nucleic acid molecule of about 192 base-pairs denoted herein as nCgGanc192, the coding strand sequence of which is denoted herein as SΕQ ID NO:5. SΕQ LD NO:5 comprises nucleotides about 1072-1248 of SΕQ LD NO:9 in pending U.S. Patent Application Serial No. 08/602,010, ibid. Translation of SEQ ED NO:5 yields a protein of about 61 amino acids, denoted herein as PCgGanc61, the amino acid sequence of which is presented in SEQ ED NO:6. Recombinant molecule pCMV- nYpFlanc576 was produced by digesting pCMV-nYpFl(b)sec544 (produced as described in Example 4B) with Tth 1111 and EcoRI, gel purifying the larger restriction fragment from this digest, and ligating this fragment with nCgGanc192. This manipulation resulted in the first 418 nucleotides of nYpFl(b)sec544 being fused in-frame with nCgGanc192. The fusion produced coding region nYpFlanc576, the coding strand sequence of which is denoted herein as SΕQ LD NO:7. Translation of SΕQ ΕD NO:7 yields a protein of about 192 amino acids, denoted PYpFlanc192, the amino acid sequence of which is presented in SΕQ LD NO:8, assuming an initiation codon extending from about nucleotide 1 through about nucleotide 3 of SΕQ LD NO:7. PYpFlanc192 includes an N-terminal signal peptide sequence of about 21 amino acids extending from about amino acid 1 to about amino acid 21 of SΕQ ΕD NO:8. The mature form of PYpFlanc192 is represented by PYpFlanc171, having the amino acid sequence SEQ ID NO:10. PYpFlancI71 is encoded by nucleic acid molecule nYpFlanc513, having the coding strand sequence represented by SEQ ED NO:9.
D. A recombinant plasmid, denoted herein as pCMV-nYpFlmat474, capable of expressing a non-secreted form of FI antigen, was produced as follows. Nucleic acid molecule nYpFlmat474, which encodes a non-secreted form of the 7 pestis FI antigen, was produced by PCR amplification of that molecule from plasmid YPRl (described in Example 1) using forward primer JO-5, having nucleic acid sequence 5' CCCAAGCTTA TGGACGATTT AACTGCAAGC ACC 3', represented herein as SEQ ID NO:20 (Hindlϊl site in bold); and reverse primer JO-2 having nucleic acid sequence 5' GGCGAATTCC TATATGGATT ATTGGTTAGA TACGG 3', represented herein as SEQ ED NO: 17 (EcoRI site in bold). The PCR amplified product was digested with restriction endonucleases Hindlll and EcoRI and gel purified, resulting in a double stranded nucleic acid molecule of about 474 base-pairs, denoted herein as nYpFlmat474ι the coding strand nucleotide sequence of which is denoted herein as SΕQ LD NO:l 1. Translation of SΕQ ID NO: 11 yields a protein of about 150 amino acids, denoted PYpFlmat!50, the amino acid sequence of which is presented in SΕQ LD NO:12, assuming a start codon spanning from about nucleotide 7 to about nucleotide 9 of SEQ ID NO:l 1 and a stop codon spanning from about nucleotide 457 to about nucleotide 459 of SEQ ID NO:ll. The coding region of PYpFlmat150is referred to herein as nYpFlmat450, the coding strand sequence of which is represented in SEQ ED NO:13. Recombinant molecule pCMV-nYpFlmat474 was produced by ligating nucleic acid molecule nYpFlmat474 into pPVXC that has been cleaved by HindR and EcoRI and gel purified. Example 5:
This example demonstrates the production of a recombinant protein from a recombinant plasmid of the present invention.
Transient expression of PYpFlsec170 from recombinant plasmid pCMV-nYpFl(b)sec5 4 in baby hamster kidney cells (BHK, available from ATCC) was performed as follows. Briefly, six-well polystyrene tissue culture plates were seeded with about 3 x 105 BHK cells/well in 2 ml of MEM NEAA Earle's salts (available from Irvine Scientific, Santa Ana, CA), supplemented with 100 mM L-glutamine and 5% FBS (complete growth media). Cells were grown to about 80% confluence (about 48 hr). The recombinant plasmid to be transfected, produced as described in Example 4B, was purified using Qiagen columns (available from Qiagen) per manufacturer's instructions. Using polystyrene plates, about 0.5 μg of recombinant plasmid pCMV-nYpFl(b)sec544 was mixed with about 100 μl OptiMEM medium (available from LTI). About 10 μl Lipofectamine (available from LTI) was mixed with about 500 μl OptiMEM. The recombinant plasmid mixture was then added to the Lipofectamine mixture and incubated at room temperature for about 30 min. After incubation, about 500 μl OptiMEM was added and the entire mixture was overlaid onto the BHK cells that had been rinsed with OptiMEM. Cells were incubated for 4 hours at 37°C, 5% CO2, 90% relative humidity. The transfection mixture was then removed and replaced with about 1 ml of OptiMEM.
Transfected cells were incubated at 37°C, 5% CO2, 90% relative humidity for about 24 hr or about 48 hr, at which times the cell supematants and cells were harvested separately. The media was removed, the cells were washed twice with about 2 ml PBS and were then scraped off the plate in about 1.5 ml PBS. The cells were then pelleted by centrifugation, the PBS was removed and the cells were frozen at -70°C. The cell- supernatants were frozen without any further manipulations.
Cell and supernatant samples were subjected to SDS PAGE and immunoblot analyses by methods similar to those described in Example 2 above, except that 10 μl samples of supernatant were assayed without being concentrated. Rabbit anti-Fl antigen antiserum, as described in Example 2, was immunoreactive with antigens expressed by the cells and supernatants harvested about 48 hours after transfection with plasmid pCMV-nYpFl(b)sec541. Example 6: This example discloses the production of a recombinant plasmid encoding a secreted form of the 7 pestis FI antigen.
A. Eukaryotic expression vector pPVXC-tPA was produced as follows. A double-stranded cassette comprising the tissue plasminogen activator (t-PA) signal peptide sequence (see, for example, Wang, RF and Mullins, JI, 1995, Gene 153 (2), 197-202) was constmcted by annealing two complementary synthetic ohgonucleotides: JO-6 having nucleic acid sequence 5'AGCTTCAATC ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC AGTCTTCGTT TCGGCCGGCC CGGGAT3' (partial Hindlϊl and EcoRV sites underlined, t-PA initiation codon in bold, N el site in double underline); and JO-7 having nucleic acid sequence 5'ATCCCGGGCC GGCCGAAACG AAGACTGCTC CACACAGCAG CAGCACACAG CAGAGCCCTC TCTTCATTGC ATCCATGATT GA3' (partial EcoRV and Hindlϊl sites underlined, Nael site in double underline). These ohgonucleotides were annealed by methods known by those skilled in the art to produce an about 82-base-pair cassette with a 4-nucleotide overhang on the 5' end. This cassette comprises the coding strand encoding the t-PA signal peptide sequence, ntPA69, extending from nucleotide 11 to nucleotide 79 of JO-6. Translation of ntPArø yields a protein of about 23 amino acids, denoted herein as PtPA23. The resulting double- stranded cassette was cloned into the pPVXC plasmid (described in Example 4A) which had been previously digested with Hindlϊl and EcoRV and gel purified. The resulting expression vector, pPVXC-tPA, contains the t-PA signal peptide sequence followed by an Nαel restriction site into which a protein coding sequence may be inserted in-frame. B. A recombinant plasmid, denoted herein as pCMV-ntPA/YpFlsec534, in which a nucleic acid molecule encoding the mature FI protein fused in-frame with the t-PA signal peptide sequence is operatively linked to the HCMV immediate-early transcription control region, was produced as follows. The nucleic acid molecule nYpFlmat481 which encodes the mature FI protein was produced by PCR amplification from recombinant plasmid pCMV-nYpFl(b)sec544 (described in Example 4B) using forward primer JO-8, having nucleic acid sequence 5'GGCGCCGGCG CAGATTTAAC TGCAAGCACC3' (Nαel site in bold), and reverse JO-9 having nucleic acid sequence 5' GGCCTCGAGC GGAATTCTTA GGATCCTTGG TTAGATACTG TTACGG 3' (Xhol site in bold, stop codon underlined). The resulting PCR product was digested with restriction endonucleases Nαel and Xhol and gel purified, resulting in a double-stranded nucleic acid molecule of about 481 base pairs denoted herein as nYpFlmat481. This sequence comprises a region of SEQ JD ΝO:l extending from nucleotide 63 to nucleotide 512, which encodes a portion of SEQ ED NO:2 extending from amino acid 14 through amino acid 163. Recombinant molecule pCMV- ntPA/YpFlmat534 was produced by ligating nucleic acid molecule nYpFlmat481 into pPVXC-tP A that had been digested with Nael and Xhol and gel purified. This manipulation results in nYpFlmat481 being fused in-frame with ntPA^. The fusion produces coding region ntPA/YpFlsec534. Translation of ntPA/YpFlsec534 yields a protein of about 178 amino acids, denoted herein as PtPA/YpFlsec178. Example 7:
This Example demonstrates the production of recombinant protein from recombinant plasmid pCMV-ntPA/YpFlsec534 .
Transient expression of PtPA/YpFlsec178 from recombinant plasmid pCMV- ntPA/YpFlsec534, produced as described in Example 6, by transfection into CHO cells, was performed as described in Example 5. Cell and supernatant samples were subjected to SDS PAGE followed by western blot analysis by methods similar to those described in Example 2, but with some slight modifications. The cell pellets were quantified by spectrophotometric absorbance at 600nm. The cells were then centrifuged and resuspended in a volume of 2X loading buffer so that 20 μl were equivalent to 0.1 O.D. units. Gels were loaded with 5 μl of cell sample per lane (0.025 OD units). Supematants were concentrated approximately 67-fold with Microcon-10™ unit, and gels were loaded with 15 μl concentrated supernatant sample per lane. Rabbit anti-Fl antigen antiserum, as described in Example 2, was immunoreactive with antigens of about 18 kD expressed by both the cells and supernatants harvested about 48 hours after transfection with plasmid pCMV-ntP A/YpF 1 sec534.
The expression of PtPA/YpFlsec178 was also detected by immunofluorescence following transient transfection of BHK cells. Briefly, six- well polystyrene tissue culture plates were seeded with about 3xl05 BHK cells per well in 2 ml of MEM NEAA Earle's salts (available from Irvine Scientific, Santa Ana, CA), supplemented with 100 nM L-glutamine and 5% FBS (complete growth media). Recombinant molecule pCMV- nfPA/YpFlsecS34 was purified using Qiagen® Maxiprep columns, according to the manufacturer's instructions. The purified recombinant plasmid (1.0 μg) was mixed with 100 μl of OptiMEM® media and incubated for 10 min at room temperature. A mixture of 10 μl of Lipofectamine® and 100 μl of OptiMEM® media (reagents available from Life Technologies Inc. (LTI) was incubated for 10 min at room temperature. The recombinant plasmid mixture was then added to the Lipofectamine mixture and incubated at room temperature for 30 min. After incubation, 800 μl of OptiMEM was added, and the entire mixture was overlaid onto BHK cells that had been rinsed with OptiMEM. Cells were incubated for 5 hours at 37°C, 5% CO2, and 90% relative humidity. The transfection mixture was then removed and replaced with 2 ml of DMEM containing 10% FBS and incubated for about 24 to 48 hours. For immunofluorescence assays, transfected cells were rinsed three times with IX PBS and then fixed in a Methanol/ Acetone (50/50) solution for 5 min on ice. Fixed cells were rinsed three times with PBS. Rabbit anti-Fl antigen antiserum (1 :50 dilution in PBS) was added, and the cells were incubated for 1 hr at 37°C. Following three rinses with PBS, a secondary a FITC-conjugated anti-rabbit antibody (available from Kirkegaard & Perry, Gaithersburg, MD), diluted 1:25 in 0.25% Evans Blue/PBS was added and incubated for 1 hour at 37°C. Cells were then rinsed three times, overlaid with 50% glycerol and examined with a fluorescence microscope. Rabbit anti-Fl antigen antiserum was immunoreactive with antigens expressed by cells transfected with plasmid pCMV-ntPA/YpFlsec534. Example 8:
This example describes the immunization of mice with recombinant molecules of the present invention and the generation of antibodies in the immunized animals.
A. Plasmid DNA for immunizations was produced as follows. Plasmid DNAs described below were purified using Qiagen® megacolumns, per manufacturer's instructions. The endotoxin level of each preparation was measured by QCL-1000 kit (available from Biowhittaker, Walkersville, MD) prior to animal immunization, and was found to be 0.043 Endotoxin Units/ug of DNA, an acceptable level for animal immunizations. B. Mice were immunized with plasmid DNA using the following method. Four groups of BALB/c mice of about three weeks of age were immunized by intramuscular injection as follows. Group 1, consisting of 5 mice, received about 100 μg of pPVXC plasmid DNA (produced as described in Example 4) in about 30 μl of diluent (TE). Group 2, consisting of 5 mice, received about 100 μg of pCMV-nYpFl(b)sec544 plasmid DNA (produced as described in Example 4) in about 30 μl of diluent (TE). Group 3, consisting of 5 mice, received about 100 μg of pCMV-ntPA/YpFlsec534 plasmid DNA (produced as described in Example 6) in about 30 μl of diluent (TE). Group 4, consisting of 5 mice, received about 1 μg of FI protein purified from 7 pestis (as described in Example 2) also in about 30 μl of diluent. C. The immune response to FI antigen in the immunized mice was measured by enzyme-linked immunosorbent assay (ELISA) as follows. Blood was collected from all immunized mice 5 days prior to infection and at days 10, 20, 30, 40 post-vaccination. Serum samples were prepared by methods well known to those skilled in the art. The semm samples were tested for anti-Fl antibodies using an ELISA for total IgM/IgG, performed as described in Example 3C. The antibody titers, geometric means, and standard deviations of the ELISA for serum samples taken on days -5 (i.e., 5 days prior to infection), 10, 20, 30, and 40 are summarized in Table 1. Table 1
Mouse # Group Day -5 Day 10 Day 20 Day 30
Group 1 : 1 CMV <40 <40 <40 <40
2 CMV <40 40 <40 40
3 CMV <40 <40 <40 40
4 CMV <40 <40 <40 40
5 CMV <40 <40 <40 40
Group 2: 1 CMV/F1 <40 <40 <40 <40
2 CMV/F1 <40 <40 <40 <40
3 CMV/F1 <40 <40 <40 <40
4 CMV/F1 <40 <40 <40 <40
5 CMV/F1 <40 <40 <40 <40
Group 3: 1 CMV/tPA-F1 <40 <40 640 2,560
2 CMV/tPA-F1 <40 40 160 2,560
3 CMV/tPA-F1 <40 160 640 10,240
4 CMVAPA-F1 <40 2,560 10,240 10,240
5 CMV PA-F1 <40 <40 640 2,560
Geom Mean 1.98 2.87 3.63
St. Deviat 0.78 0.65 0.32
Group 4:1 F1 <40 10,240 10,240 40,960
2 F1 <40 10,240 2,560 2,560
3 F1 <40 640 640 10,240
4 F1 <40 640 640 2,560
5 F1 <40 10,240 640 2,56
Geom Mean 3.47 3.13 3.74
St. Dev 0.65 0.53 0.53
The results show that the pCMV-ntPA/YpFlsec534 plasmid was highly immunogenic in mice inducing high levels of antibodies in all immunized mice. At day 30 post- vaccination, anti-Fl antibody titers in mice vaccinated with the pCMV-ntPA/YpFlsec534 plasmid were equivalent to those detected in animals vaccinated with the FI protein. Example 9:
This example discloses the production of a recombinant raccoon poxvims containing the EMCV LRES and a fused 7 pestis tPA-Fl antigen.
A. Eukaryotic expression vector pCITE-tPA was constmcted as follows. A double-stranded cassette comprising the tissue plasminogen activator (t-PA) signal peptide sequence (Wang et al, ibid.) was constmcted by annealing two complementary synthetic ohgonucleotides: JO- 10, having nucleotide sequence 5'ATGCAATGAA GAGAGGGCTC TGCTGTGTGC TGCTGCTGTG TGGAGCAGTC TTCGTTTCTG CCGGCCCGGG TATACG3': and JO-11 having nucleotide sequence 5' GATCCGTATA CCCGGGCCGG CAGAAACGAA GACTGCTCCA CACAGCAGCA GCACACAGCA GAGCCCTCTC TTCATTGCAT3'. The annealed sequence contains three blunt cutting restriction sites: Nael (double underline); Smal (bold); and Bstl 1071 (single underline); located at the 3' end of the annealed signal sequence. The annealed cassette lacks the t-PA initiation codon at the 5' end, and is designed to blunt ligate to the scl site in pCITE-4a (available from Novagen), thus utilizing the EMCV ERES preferential ATG. This changes the first amino acid from a lysine to a glutamic acid, which is a conservative change and the protein retains its original hydrophobicity. The annealed cassette was ligated into the pCITE 4a+ plasmid that had been previously digested with Mscl and BamHl and gel purified. The resulting nucleic acid molecule was designated pCITE-tPA.
B. Recombinant molecule pCMV-LRES-tPA containing the tPA signal peptide sequence operatively linked to a CMV promoter and EMCV ERES was constructed as follows. A DNA fragment containing IRES-tP A, denoted herein as nLRES-tP A, was PCR amplified from pCITE-tPA using forward primer JO- 12 having nucleotide sequence 5'AGGCGCGCCG 7XO4CGTTAT TTTCCACCAT ATTGCCG3'( scI site in bold, Sail site in italics), and reverse primer JO- 13 having nucleotide sequence 5'CGAATTCGG rCCGTATACC3' (EcoRI site in bold, BamHl site in italics, and Z?stl l071 site underlined). Recombinant molecule pCMV-LRΕS-tPA was produced by ligating nucleic acid molecule nLRΕS-tPA that had been digested with Ascl and EcoRI into a modified pCMV-intA vector. This modified pCMV-intA vector was created by annealing, using techniques known to those skilled in the art, the following two complementary synthetic ohgonucleotides: JO-14 having nucleotide sequence 5 'AGCTTGGCGC GCCG3 ' (Htπdlll site in italics, Ascl site in bold, first base of BamHl site underlined), and JO- 15 having nucleotide sequence 5'GATCCGGCGC GCCA3' (BamH site underlined, Ascl site in bold, first base of HwdLTI site underlined). The annealed ohgonucleotides were ligated into the pCMV-intA plasmid that had been digested with Hindϊϊl and BamHl and gel purified. C. Recombinant molecule pCMV-IRES-ntPA YpFlsec525, containing a nucleic acid molecule encoding the mature FI antigen of 7 pestis fused in- frame with the t-PA signal peptide sequence and operatively linked to a CMV promoter and EMCV IRES, was produced as follows. Nucleic acid molecule nYpFlmat468 was PCR amplified from pCMV-nYpFl(b)sec544 (described in Example 4B) using forward primer JO-8 and reverse primer JO- 16, having nucleic acid sequence 5'CGGAATTCTT AGG^rCCTTG GTTAGATACG GTTACGG3' (EcoRI site in bold, stop codon underlined, BamHl site in italics). The resulting PCR product was digested with restriction endonucleases NgoMI and EcoRI and gel purified, resulting in a double-stranded molecule of 468 base pairs denoted herein as nYpFlmat468. Recombinant molecule pCMV-ΕRΕS- ntPA/YpFlsec525 was produced by ligating nucleic acid molecule nYpFlmat468 into the pCMV-IRΕS-tPA vector that had been digested with NgoMl and EcoRI and gel purified. D. A recombinant raccoon poxvims (RCΝV) capable of expressing 7 pestis FI antigen was produced as follows. Recombinant cell Vero:RCΝ:IRΕS-ntPA/ΥpFlsec525 containing nucleic acid molecule ntPA/YpFlsec525 operatively linked to a vaccinia vims pi 1 late promoter transcription control region and EMCV ERES was produced in the following manner. Recombinant molecule pCMV-IRES-ntPA/YpFlsec525 was digested with Sail and EcoRI to generate a fragment containing nucleic acid molecule ntPA/YpFlsec525 operatively linked to a CMV promoter and ΕMCV IRES. This fragment was then cloned into a RCN transfer vector that had been digested with Sail and EcoRI and gel purified. The resulting plasmid was recombined into a raccoon pox vims in Vero cells as described in Example IB to form recombinant cell
Vero:RCN:IRES-ntPA/YpFlsec525. The resulting recombinant vims, denoted RCN:ERES-ntPA/YpFlsec525, was plaque purified as described in Example IB. Example 10:
This example demonstrates enhanced expression of 7 pestis FI antigen in cells infected with a recombinant raccoon pox vims of the present invention.
Expression of 7 pestis FI antigen in RCN:ERES-ntPA/YpFlsec525-infected cells was monitored by the following method. Vero cells were plated at 7 xlO cells/well in six well dishes with MEM+5%FBS one day prior to infection. Cells were infected in duplicate at an MOI of approximately 0.5 with vimses that had pre-treated with trypsin (1 mg/ml) for 15 min at 37°C. Upon infection, the media was changed to MEM without FBS, and the infected cells were incubated for about 24 to 48 hr. The vimses used included: (a) wild type raccoon poxvims RCN CDC/V71-1-85 A (described in Example IB); (b)RCN:pl l-nYpFl(a)sec544 (produced as described in Example IB); and (c) RCN:IRES-ntPA YpFlsec525. The infected cells were harvested by washing cells into media, and recovering the cells by centrifugation at 10,000 rpm for 5 min at room temperature. The supernatants and cells were prepared for western blot analysis as described in Example 2. About 10 μl of each cell fraction and 30 μl of each concentrated media sample were loaded on a 4-20% SDS-PAGE gel and run for 1 hr at 200 V. The separated proteins were transferred to nitrocellulose using a Bio-Rad transfer apparatus at 100 V for 1 hr. The filter was subjected to western blot analysis using polyclonal rabbit anti-Fl antigen antisemm (described in Example 2). Filters were scanned and analyzed for density with a NIH image program. Analysis of the results, in comparison with a known quantity of FI antigen, indicated that the presence of the EMCV ERES motif led to an about two-fold increase in FI protein production; that is, cells infected with RCN:ERES-ntPA/YpFlsec525 produced about twice as much protein as did cells infected with RCN:p 11 -nYpF 1 (a)sec544. Example 11 :
This example discloses the production of recombinant mengoviruses containing several forms of the 7 pestis FI antigen.
A. Recombinant molecule pMV-nYpFlmat450, encoding the mature FI antigen in frame with the polyprotein coding region of mengovims, was prepared as follows. The mature FI coding region was amplified from recombinant molecule pCMV-LRES- ntPA/YpFlsec525 (produced as described in Example 9C) using forward primer JO-17, having the nucleotide sequence 5'GGGGCTAGCA GATTTAACTG CAAGCACCAC and reverse primer JO- 18 having the nucleotide sequence 5'GGGGCTAGCT GGTTAGATAC GGTTACGGTT ACAGCAGC (Nhel sites shown in bold). The amplified fragment was purified using QIAquick™ PCR purification kit (available from Qiagen Inc., Valencia, CA) as recommended by manufacturer. The PCR-amplified fragment was re-circularized by ligation, digested with restriction endonuclease Nhel, and gel purified, resulting in a double-stranded nucleic acid molecule of about 450 base pairs denoted herein as nYpFlmat450. Recombinant molecule pMV-nYpFlmat450 was produced by ligating nucleic acid molecule nYpFlmat450 into menogvims plasmid pCoCe (available from the University of Wisconsin, Madison, WI; see also U.S. Patent No. 5,229,111, by Duke et al, issued July 20, 1993) that had been digested with Nhel and gel purified. This manipulation resulted in nYpFlmat450 being fused in-frame with the sequence encoding the mengovims polyprotein. B. Recombinant molecule pMV-ntPA/YpFlsecsl9 , encoding the mature FI antigen fused with the t-PA signal peptide sequence, and in frame with the polyprotein coding region of mengovims, was prepared as follows. The mature FI coding region fused to the t-PA signal peptide sequence was amplified from recombinant molecule pCMV-LRES-ntPA/YpFlsec525 (produced as described in Example 9C) using forward primer JO- 14, having the nucleotide sequence 5 ' GGGGCTAGCC GATGCAATGA AGAGAGGGCT CT 3' and reverse primer JO-13 (Nhel site shown in bold). The amplified fragment was purified and digested with restriction endonuclease Nhel as described in Example 11 A, resulting in a double-stranded nucleic acid molecule of about 519 base pairs denoted herein as ntPA/YpF 1 sec519. Recombinant molecule pMV- ntPA/YpFlsec519 was produced by ligating nucleic acid molecule ntPA-YpFlsec519 into the mengovims pCoCe plasmid described in Example 11 A that had been digested with Nhel and gel purified. This manipulation resulted in ntPA/YpFlsec519 being fused in- frame with the sequence encoding the mengovims polyprotein.
C. Recombinant molecule pMV-ntPA/YpFlanc705 , encoding the mature FI antigen fused with the t-PA signal peptide sequence and the CHV gG membrane anchor sequence (described in Example 4C), and in frame with the polyprotein coding region of mengovims, was prepared as follows. The mature FI coding region fused to the t-PA signal peptide sequence was amplified from recombinant molecule pCMV-IRES- ntPA/YpFlsec525 as in Example 1 IB using forward primer JO-14, and reverse primer JO-15 having nucleotide sequence 5'CGGAATTCTT AGGATCCTTG
GTTAGATACG GTTACGG-3' (BamHl site shown in bold). An about 196 bp fragment was then amplified from CHV genomic DΝA (described in Example 4C) using forward primer JO- 16 having nucleotide sequence 5'CGGGATCCAA TGGTTATAAT AATTGTAATA CCC -3' (BamHl site shown in bold), and reverse primer JO-17 having nucleotide sequence 5'AACGCTAGCA GAATATCATA AAATAATAAT TTCTG-3' (Nhel site shown in bold). These two resulting PCR- amplified fragments were purified and digested with BamHl. The fragments were then ligated in the presence of polynucleotide kinase, digested with Nhel, and gel purified, producing nucleic acid molecule ntPA/YpFlanc705. Recombinant molecule pMV- ntPA/YpFlanc705 was produced by ligating nucleic acid molecule ntPA/YpFlan^os into the mengovims pCoCe plasmid described in Example 11 A that had been digested with Nhel and gel purified. This manipulation resulted in ntPA/YpFlanc705 being fused in- frame with the sequence encoding the mengovims polyprotein. Example 12:
This example describes the production of recombinant mengoviruses from the recombinant molecules described in Example 11.
A. Recombinant mengovims RNA was produced from recombinant molecules pMV-nYpFlmat45o, pMV-ntPA/YpFlsec519, andpMV-ntPA/YpFlanc705 using in vitro transcription, as follows. Two μg of each recombinant molecule was linearized with BamHl (pMV-nYpFlmat450 and pMV-ntPA/YpFlsec519) or H dlLI (pMV- ntPA/YpF 1 anc705) in a 50 μl reaction mix. Each DNA was extracted once with phenol/CΗCl3 and precipitated. RNA from each recombinant molecule was then synthesized by in vitro transcription using MEGAscript™ kit (available from Ambion Inc., USA) in 20 μl of reaction volume containing 1 μg of each template DNA essentially as described by manufacturer, producing mengovims RNA molecules rMV- nYpFlmat450, rMV-ntPA/YpFlsec519, and rMV-ntPA/YpFlanc705. The yield of each mengovims RNA was evaluated by running an aliquot of RNA on 1% non-denaturing agarose gel.
B. Recombinant mengo vimses were produced by RNA electroporation into HeLa cells, as follows. HeLa cells were grown in a T225 cm2 tissue culture flask to -80% confluency in D-MEM- 10%, which is D-MEM media supplemented with 10% FBS, 2 mM glutamine, 100 μg/ml streptomycin, 100 units/ml penicillin, IX MEM vitamins mixture (all reagents available from LTI). The cells were trypsinized using a standard protocol and resuspended in D-MEM- 10%. The cells were washed three times in ice-cold OPTI-MEM I media (available from LTI) and resuspended in 500 μl aliquots of 5x106 cells for each RNA sample. Approximately 2 μg of each mengovims RNA, produced as described in Example 12 A, was added to an aliquot of cells and the mixtures were immediately subjected to two pulses of electrical discharge in BTX-500 electroporation device (Electro cell manipulator 600, BTX Inc., Santa Clara, CA) with the following settings: 400 V, 800 μF, 13 ohms. After incubation for 5-10 min at room temperature, the transfected recombinant cells, denoted herein as HeLa:MV- nYpFlmat450 , HeLa:MV-ntPA/YpFlsec519, and HeLa:MV-ntPA/YpFlanc705 were resuspended in 10 ml of OPTI-MEM supplemented with 1% of FBS and were transferred into tissue culture flasks. Infectious vimses, denoted herein as MV- nYpFlmat450, MV-ntPA/YpFlsec5ι9, and MV-ntPA YpFlanc705 were collected after complete CPE was observed, at approximately 2 days post electroporation. The infected cells were lysed by two freeze-thaw cycles followed by clarification of cell lysates by centrifugation for 15 min at 3,000 rpm in GP8R refrigerated centrifuge (Forma Scientific Inc.).
Sequence Listing (1) GENERAL INFORMATION:
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(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 227-7400
(B) TELEFAX: (617) 742-4214 (2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 544 nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURES: (A) NAME/KEY: CDS
(B) LOCATION: 17..529
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:
GTCGACGAGG TAATAT ATG AAA AAA ATC AGT TCC GTT ATC GCC ATT GCA 49 Met Lys Lys He Ser Ser Val He Ala He Ala 1 5 10
TTA TTT GGA ACT ATT GCA ACT GCT AAT GCG GCA GAT TTA ACT GCA AGC 97 Leu Phe Gly Thr He Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser 15 20 25
ACC ACT GCA ACG GCA ACT CTT GTT GAA CCA GCC CGC ATC ACT CTT ACA 145 Thr Thr Ala Thr Ala Thr Leu Val Glu Pro Ala Arg He Thr Leu Thr 30 35 40
TAT AAG GAA GGC GCT CCA ATT ACA ATT ATG GAC AAT GGA AAC ATC GAT 193 Tyr Lys Glu Gly Ala Pro He Thr He Met Asp Asn Gly Asn He Asp 45 50 55 ACA GAA TTA CTT GTT GGT ACG CTT ACT CTT GGC GGC TAT AAA ACA GGA 241 Thr Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly 60 65 70 75
ACC ACT AGC ACA TCT GTT AAC TTT ACA GAT GCC GCG GGT GAT CCC ATG 289 Thr Thr Ser Thr Ser Val Asn Phe Thr Asp Ala Ala Gly Asp Pro Met 80 85 90
TAC TTA ACA TTT ACT TCT CAG GAT GGA AAT AAC CAC CAA TTC ACT ACA 337 Tyr Leu Thr Phe Thr Ser Gin Asp Gly Asn Asn His Gin Phe Thr Thr 95 100 105
AAA GTG ATT GGC AAG GAT TCT AGA GAT TTT GAT ATC TCT CCT AAG GTA 385 Lys Val He Gly Lys Asp Ser Arg Asp Phe Asp He Ser Pro Lys Val 110 115 120
AAC GGT GAG AAC CTT GTG GGG GAT GAC GTC GTC TTG GCT ACG GGC AGC 433 Asn Gly Glu Asn Leu Val Gly Asp Asp Val Val Leu Ala Thr Gly Ser 125 130 135 CAG GAT TTC TTT GTT CGC TCA ATT GGT TCC AAA GGC GGT AAA CTT GCA 481 Gin Asp Phe Phe Val Arg Ser He Gly Ser Lys Gly Gly Lys Leu Ala 140 145 150 155
GCA GGT AAA TAC ACT GAT GCT GTA ACC GTA ACC GTA TCT AAC CAA TAA 529 Ala Gly Lys Tyr Thr Asp Ala Val Thr Val Thr Val Ser Asn Gin 160 165 170 TCCATATAGG GATCC 544
(2) INFORMATION FOR SEQ ID Nθ:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 170 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2:
Met Lys Lys He Ser Ser Val He Ala He Ala Leu Phe Gly Thr He 1 5 10 15
Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala 20 25 30
Thr Leu Val Glu Pro Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala 35 40 45 Pro He Thr He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val 50 55 60
Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser 65 70 75 80
Val Asn Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr 85 90 95
Ser Gin Asp Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys 100 105 110
Asp Ser Arg Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu 115 120 125 Val Gly Asp Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val 130 135 140
Arg Ser He Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr 145 150 155 160
Asp Ala Val Thr Val Thr Val Ser Asn Gin 165 170
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 510 nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
ATGAAAAAAA TCAGTTCCGT TATCGCCATT GCATTATTTG GAACTATTGC AACTGCTAAT 60
GCGGCAGATT TAACTGCAAG CACCACTGCA ACGGCAACTC TTGTTGAACC AGCCCGCATC 120
ACTCTTACAT ATAAGGAAGG CGCTCCAATT ACAATTATGG ACAATGGAAA CATCGATACA 180 GAATTACTTG TTGGTACGCT TACTCTTGGC GGCTATAAAA CAGGAACCAC TAGCACATCT 240
GTTAACTTTA CAGATGCCGC GGGTGATCCC ATGTACTTAA CATTTACTTC TCAGGATGGA 300
AATAACCACC AATTCACTAC AAAAGTGATT GGCAAGGATT CTAGAGATTT TGATATCTCT 360
CCTAAGGTAA ACGGTGAGAA CCTTGTGGGG GATGACGTCG TCTTGGCTAC GGGCAGCCAG 420
GATTTCTTTG TTCGCTCAAT TGGTTCCAAA GGCGGTAAAC TTGCAGCAGG TAAATACACT 480 GATGCTGTAA CCGTAACCGT ATCTAACCAA 510
(2) INFORMATION FOR SEQ ID Nθ:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 544 nucleotides
(B) TYPE: nucleic acid (C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURES:
(A) NAME/KEY: CDS (B) LOCATION: 17..529
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
AAGCTTGAGG TAATAT ATG AAA AAA ATC AGT TCC GTT ATC GCC ATT GCA 49 Met Lys Lys He Ser Ser Val He Ala He Ala 1 5 10 TTA TTT GGA ACT ATT GCA ACT GCT AAT GCG GCA GAT TTA ACT GCA AGC 97 Leu Phe Gly Thr He Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser 15 20 25
ACC ACT GCA ACG GCA ACT CTT GTT GAA CCA GCC CGC ATC ACT CTT ACA 145 Thr Thr Ala Thr Ala Thr Leu Val Glu Pro Ala Arg He Thr Leu Thr 30 35 40
TAT AAG GAA GGC GCT CCA ATT ACA ATT ATG GAC AAT GGA AAC ATC GAT 193 Tyr Lys Glu Gly Ala Pro He Thr He Met Asp Asn Gly Asn He Asp 45 50 55
ACA GAA TTA CTT GTT GGT ACG CTT ACT CTT GGC GGC TAT AAA ACA GGA 241 Thr Glu Leu Leu Val Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly 60 65 70 75
ACC ACT AGC ACA TCT GTT AAC TTT ACA GAT GCC GCG GGT GAT CCC ATG 289 Thr Thr Ser Thr Ser Val Asn Phe Thr Asp Ala Ala Gly Asp Pro Met 80 85 90 TAC TTA ACA TTT ACT TCT CAG GAT GGA AAT AAC CAC CAA TTC ACT ACA 337 Tyr Leu Thr Phe Thr Ser Gin Asp Gly Asn Asn His Gin Phe Thr Thr 95 100 105 AAA GTG ATT GGC AAG GAT TCT AGA GAT TTT GAT ATC TCT CCT AAG GTA 385 Lys Val He Gly Lys Asp Ser Arg Asp Phe Asp He Ser Pro Lys Val 110 115 120
AAC GGT GAG AAC CTT GTG GGG GAT GAC GTC GTC TTG GCT ACG GGC AGC 433 Asn Gly Glu Asn Leu Val Gly Asp Asp Val Val Leu Ala Thr Gly Ser 125 130 135
CAG GAT TTC TTT GTT CGC TCA ATT GGT TCC AAA GGC GGT AAA CTT GCA 481 Gin Asp Phe Phe Val Arg Ser He Gly Ser Lys Gly Gly Lys Leu Ala 140 145 150 155 GCA GGT AAA TAC ACT GAT GCT GTA ACC GTA ACC GTA TCT AAC CAA TAA 529 Ala Gly Lys Tyr Thr Asp Ala Val Thr Val Thr Val Ser Asn Gin 160 165 170
TCCATATAGG AATTC 544
(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 192 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1..186
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 : GAC GTC GTC GGT TAT AAT AAT TGT AAT ACC CAT ATA AAG GTA ATT GGA 48 Asp Val Val Gly Tyr Asn Asn Cys Asn Thr His He Lys Val He Gly 1 5 10 15
TTT GGA ACA ATT ATC TTT ATT ATT TTA TTT TTT GTT GCT GTG TTT TTT 96 Phe Gly Thr He He Phe He He Leu Phe Phe Val Ala Val Phe Phe 20 25 30
TGT GGA TAT ACT TGT GTA TTA AAC TCT CGT ATT AAA ATG ATT AAC CAT 144 Cys Gly Tyr Thr Cys Val Leu Asn Ser Arg He Lys Met He Asn His 35 40 45
GCT TAT ATA CAA CCC CAG AAA TTA AAT TTT TAT GAT ATT TAA GAATTC 192 Ala Tyr He Gin Pro Gin Lys Leu Asn Phe Tyr Asp He 50 55 60
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Asp Val Val Gly Tyr Asn Asn Cys Asn Thr His He Lys Val He Gly 1 5 10 15
Phe Gly Thr He He Phe He He Leu Phe Phe Val Ala Val Phe Phe 20 25 30
Cys Gly Tyr Thr Cys Val Leu Asn Ser Arg He Lys Met He Asn His 35 40 45 Ala Tyr He Gin Pro Gin Lys Leu Asn Phe Tyr Asp He 50 55 60
(2) INFORMATION FOR SEQ ID Nθ:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 576 nucleotides (B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURES: (A) NAME/KEY: CDS
(B) LOCATION: 1..576
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
ATG AAA AAA ATC AGT TCC GTT ATC GCC ATT GCA TTA TTT GGA ACT ATT 48 Met Lys Lys He Ser Ser Val He Ala He Ala Leu Phe Gly Thr He 1 5 10 15
GCA ACT GCT AAT GCG GCA GAT TTA ACT GCA AGC ACC ACT GCA ACG GCA 96 Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala 20 25 30
ACT CTT GTT GAA CCA GCC CGC ATC ACT CTT ACA TAT AAG GAA GGC GCT 144 Thr Leu Val Glu Pro Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala 35 40 45
CCA ATT ACA ATT ATG GAC AAT GGA AAC ATC GAT ACA GAA TTA CTT GTT 192 Pro He Thr He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val 50 55 60 GGT ACG CTT ACT CTT GGC GGC TAT AAA ACA GGA ACC ACT AGC ACA TCT 240 Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser 65 70 75 80 GTT AAC TTT ACA GAT GCC GCG GGT GAT CCC ATG TAC TTA ACA TTT ACT 288 Val Asn Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr 85 90 95
TCT CAG GAT GGA AAT AAC CAC CAA TTC ACT ACA AAA GTG ATT GGC AAG 336 Ser Gin Asp Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys 100 105 110
GAT TCT AGA GAT TTT GAT ATC TCT CCT AAG GTA AAC GGT GAG AAC CTT 384 Asp Ser Arg Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu 115 120 125 GTG GGG GAT GAC GTC GTC GGT TAT AAT AAT TGT AAT ACC CAT ATA AAG 432 Val Gly Asp Asp Val Val Gly Tyr Asn Asn Cys Asn Thr His He Lys 130 135 140
GTA ATT GGA TTT GGA ACA ATT ATC TTT ATT ATT TTA TTT TTT GTT GCT 480 Val He Gly Phe Gly Thr He He Phe He He Leu Phe Phe Val Ala 145 150 155 160
GTG TTT TTT TGT GGA TAT ACT TGT GTA TTA AAC TCT CGT ATT AAA ATG 528 Val Phe Phe Cys Gly Tyr Thr Cys Val Leu Asn Ser Arg He Lys Met 165 170 175
ATT AAC CAT GCT TAT ATA CAA CCC CAG AAA TTA AAT TTT TAT GAT ATT 576 He Asn His Ala Tyr He Gin Pro Gin Lys Leu Asn Phe Tyr Asp He 180 185 190
(2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 192 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Lys Lys He Ser Ser Val He Ala He Ala Leu Phe Gly Thr He 1 5 10 15
Ala Thr Ala Asn Ala Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala 20 25 30
Thr Leu Val Glu Pro Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala 35 40 45 Pro He Thr He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val 50 55 60
Gly Thr Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser 65 70 75 80 Val Asn Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr 85 90 95
Ser Gin Asp Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys 100 105 110 Asp Ser Arg Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu 115 120 125
Val Gly Asp Asp Val Val Gly Tyr Asn Asn Cys Asn Thr His He Lys 130 135 140
Val He Gly Phe Gly Thr He He Phe He He Leu Phe Phe Val Ala 145 150 155 160
Val Phe Phe Cys Gly Tyr Thr Cys Val Leu Asn Ser Arg He Lys Met 165 170 175
He Asn His Ala Tyr He Gin Pro Gin Lys Leu Asn Phe Tyr Asp He 180 185 190 (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS: '
(A) LENGTH: 513 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double (D)' TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1..513 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
GCA GAT TTA ACT GCA AGC ACC ACT GCA ACG GCA ACT CTT GTT GAA CCA 48 Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val Glu Pro 1 5 10 15
GCC CGC ATC ACT CTT ACA TAT AAG GAA GGC GCT CCA ATT ACA ATT ATG 96 Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala Pro He Thr He Met 20 25 30
GAC AAT GGA AAC ATC GAT ACA GAA TTA CTT GTT GGT ACG CTT ACT CTT 144 Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu 35 40 45 GGC GGC TAT AAA ACA GGA ACC ACT AGC ACA TCT GTT AAC TTT ACA GAT 192 Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe Thr Asp 50 55 60
GCC GCG GGT GAT CCC ATG TAC TTA ACA TTT ACT TCT CAG GAT GGA AAT 240 Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin Asp Gly Asn 65 70 75 80 AAC CAC CAA TTC ACT ACA AAA GTG ATT GGC AAG GAT TCT AGA GAT TTT 288 Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser Arg Asp Phe 85 90 95
GAT ATC TCT CCT AAG GTA AAC GGT GAG AAC CTT GTG GGG GAT GAC GTC 336 Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val 100 105 110
GTC GGT TAT AAT AAT TGT AAT ACC CAT ATA AAG GTA ATT GGA TTT GGA 384 Val Gly Tyr Asn Asn Cys Asn Thr His He Lys Val He Gly Phe Gly 115 120 125 ACA ATT ATC TTT ATT ATT TTA TTT TTT GTT GCT GTG TTT TTT TGT GGA 432 Thr He He Phe He He Leu Phe Phe Val Ala Val Phe Phe Cys Gly 130 135 140
TAT ACT TGT GTA TTA AAC TCT CGT ATT AAA ATG ATT AAC CAT GCT TAT 480 Tyr Thr Cys Val Leu Asn Ser Arg He Lys Met He Asn His Ala Tyr 145 150 155 160
ATA CAA CCC CAG AAA TTA AAT TTT TAT GAT ATT 513
He Gin Pro Gin Lys Leu Asn Phe Tyr Asp He 165 170
(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val Glu Pro 1 5 10 15
Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala Pro He Thr He Met 20 25 30 Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu 35 40 45
Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe Thr Asp 50 55 60
Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin Asp Gly Asn 65 70 75 80
Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser Arg Asp Phe 85 90 95
Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val 100 105 110 Val Gly Tyr Asn Asn Cys Asn Thr His He Lys Val He Gly Phe Gly 115 120 125
Thr He He Phe He He Leu Phe Phe Val Ala Val Phe Phe Cys Gly 130 135 140 Tyr Thr Cys Val Leu Asn Ser Arg He Lys Met He Asn His Ala Tyr 145 150 155 160
He Gin Pro Gin Lys Leu Asn Phe Tyr Asp He 165 170
(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 474 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 7..459
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: AAGCTT ATG GCA GAT TTA ACT GCA AGC ACC ACT GCA ACG GCA ACT CTT 48 Met Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu 1 5 10
GTT GAA CCA GCC CGC ATC ACT CTT ACA TAT AAG GAA GGC GCT CCA ATT 96 Val Glu Pro Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala Pro He 15 20 25 30
ACA ATT ATG GAC AAT GGA AAC ATC GAT ACA GAA TTA CTT GTT GGT ACG 144 Thr He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr 35 40 45
CTT ACT CTT GGC GGC TAT AAA ACA GGA ACC ACT AGC ACA TCT GTT AAC 192 Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn 50 55 60
TTT ACA GAT GCC GCG GGT GAT CCC ATG TAC TTA ACA TTT ACT TCT CAG 240 Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin 65 70 75 GAT GGA AAT AAC CAC CAA TTC ACT ACA AAA GTG ATT GGC AAG GAT TCT 288 Asp Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser 80 85 90
AGA GAT TTT GAT ATC TCT CCT AAG GTA AAC GGT GAG AAC CTT GTG GGG 336 Arg Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly 95 100 105 110 GAT GAC GTC GTC TTG GCT ACG GGC AGC CAG GAT TTC TTT GTT CGC TCA 384 Asp Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val Arg Ser 115 120 125
ATT GGT TCC AAA GGC GGT AAA CTT GCA GCA GGT AAA TAC ACT GAT GCT 432 He Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala 130 135 140
GTA ACC GTA ACC GTA TCT AAC CAA TAA TCCATATAGG AATTC 474
Val Thr Val Thr Val Ser Asn Gin 145 150 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 150 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12 :
Met Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu 1 5 10
Val Glu Pro Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala Pro He 15 20 25 30
Thr He Met Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr 35 40 45
Leu Thr Leu Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn 50 55 60 Phe Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin 65 70 75
Asp Gly Asn Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser 80 85 90
Arg Asp Phe Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly 95 100 105 110
Asp Asp Val Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val Arg Ser 115 120 125
He Gly Ser Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala 130 135 140 Val Thr Val Thr Val Ser Asn Gin 145 150
(2) INFORMATION FOR SEQ ID NO: 13:
( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH: 450 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
ATGGCAGATT TAACTGCAAG CACCACTGCA ACGGCAACTC TTGTTGAACC AGCCCGCATC 60
ACTCTTACAT ATAAGGAAGG CGCTCCAATT ACAATTATGG ACAATGGAAA CATCGATACA 120
GAATTACTTG TTGGTACGCT TACTCTTGGC GGCTATAAAA CAGGAACCAC TAGCACATCT 180 GTTAACTTTA CAGATGCCGC GGGTGATCCC ATGTACTTAA CATTTACTTC TCAGGATGGA 240
AATAACCACC AATTCACTAC AAAAGTGATT GGCAAGGATT CTAGAGATTT TGATATCTCT 300
CCTAAGGTAA ACGGTGAGAA CCTTGTGGGG GATGACGTCG TCTTGGCTAC GGGCAGCCAG 360
GATTTCTTTG TTCGCTCAAT TGGTTCCAAA GGCGGTAAAC TTGCAGCAGG TAAATACACT 420
GATGCTGTAA CCGTAACCGT ATCTAACCAA 450 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
ACGCGCGTCG ACGAGGTAAT ATATGAAAAA AATCAG 36
(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CGCGGATCCC TATATGGATT ATTGGTTAGA TACGG 35
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE : primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GGCAAGCTTG AGGTAATATA TGAAAAAAAT CAG 33
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GGCGAATTCC TATATGGATT ATTGGTTAGA TACGG 35
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 bases (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GGGATGACGT CGTCGGTTAT AATAATTGTA ATACCC 36
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 bases
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GGCGAATTCT TAAATATCAT AAAAATTTAA TTTCTGGGG 39 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 bases
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
CCCAAGCTTA TGGCAGATTT AACTGCAAGC ACC 33
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 149 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val Glu Pro 1 5 10 15
Ala Arg He Thr Leu Thr Tyr Lys Glu Gly Ala Pro He Thr He Met 20 25 30
Asp Asn Gly Asn He Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu 35 40 45
Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe Thr Asp 50 55 60
Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gin Asp Gly Asn 65 70 75 80 Asn His Gin Phe Thr Thr Lys Val He Gly Lys Asp Ser Arg Asp Phe
85 90 95
Asp He Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val 100 105 110
Val Leu Ala Thr Gly Ser Gin Asp Phe Phe Val Arg Ser He Gly Ser 115 120 125
Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val Thr Val 130 135 140
Thr Val Ser Asn Gin 145 (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 447 nucleotides
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: GCAGATTTAA CTGCAAGCAC CACTGCAACG GCAACTCTTG TTGAACCAGC CCGCATCACT 60
CTTACATATA AGGAAGGCGC TCCAATTACA ATTATGGACA ATGGAAACAT CGATACAGAA 120
TTACTTGTTG GTACGCTTAC TCTTGGCGGC TATAAAACAG GAACCACTAG CACATCTGTT 180
AACTTTACAG ATGCCGCGGG TGATCCCATG TACTTAACAT TTACTTCTCA GGATGGAAAT 240
AACCACCAAT TCACTACAAA AGTGATTGGC AAGGATTCTA GAGATTTTGA TATCTCTCCT 300
AAGGTAAACG GTGAGAACCT TGTGGGGGAT GACGTCGTCT TGGCTACGGG CAGCCAGGAT 360
TTCTTTGTTC GCTCAATTGG TTCCAAAGGC GGTAAACTTG CAGCAGGTAA ATACACTGAT 420
GCTGTAACCG TAACCGTATC TAACCAA 447
While the various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications are adaptations are within the scope of the present invention, as set forth in the following claims.

Claims

What is claimed is:
1. A recombinant molecule comprising an isolated nucleic acid molecule that encodes an antigen selected from the group consisting of a Yersinia antigen, a Pasteurella antigen, and a Francisella antigen, said nucleic acid molecule being operatively linked to a eukaryotic transcription control region.
2. The recombinant molecule of Claim 1 , wherein said antigen is selected from the group consisting of Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Pasteurella multocida, and Francisella tularensis antigens.
3. The recombinant molecule of Claim 1 , wherein said isolated nucleic acid molecule encodes a Yersinia pestis antigen.
4. The recombinant molecule of Claim 1 , wherein said isolated nucleic acid molecule encodes a Yersinia pestis antigen selected from the group consisting of an FI antigen, a V antigen, a pesticin antigen, a W antigen, a pH6 antigen, a superoxide dismutase antigen, a Yersinia outer protein antigen, high-molecular weight iron-regulated membrane protein antigen, a murine toxin antigen, and a hemin storage protein antigen.
5. The recombinant molecule of Claim 1 , wherein said isolated nucleic acid molecule encodes a Yersinia pestis antigen selected from the group consisting of an FI antigen and a V antigen. 6. The recombinant molecule of Claim 1, wherein said isolated nucleic acid molecule encodes a Yersinia pestis FI antigen. 7. The recombinant molecule of Claim 1, wherein said isolated nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of nYpFl(a)sec544, nYpFl(b)sec544, nYpFlsec510, nYpFlanc576, nYpFlanc513, nYpFlmat474, nYpFlmat45o, and nYpFlmatw; and a nucleic acid molecule comprising a variant of a nucleic acid molecule selected from the group consisting of nYpFl(a)sec544, nYpFl(b)sec544, nYpFlsec510, nYpFlanc576, nYpFlanc513, nYpFlmat474, nYpFlmat450, and nYpFlmat^. 8. The recombinant molecule of Claim 1, wherein said isolated nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO:22; and a nucleic acid molecule comprising a variant of a nucleic acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, and SEQ ID NO:22.
9. The recombinant molecule of Claim 1 , wherein said isolated nucleic acid molecule encodes an antigen selected from the group consisting of: an antigen selected from the group consisting of PYpFlsecI70, PYpFlanc192, PYpFlanc171, PYpFlmat150ι PYpFlmat149; and an antigen encoded by a variant of a nucleic acid molecule encoding an antigen selected from the group consisting of PYpFlsec170, PYpFlanc192, PYpFlanc171, PYpFlmat150_PYpFlmatI49.
10. The recombinant molecule of Claim 1, wherein said isolated nucleic acid molecule encodes an antigen selected from the group consisting of: an antigen having an amino acid sequence selected from the group consisting of SEQ ID
NO:2, SEQ ID O:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:21; and an antigen encoded by a variant of a nucleic acid molecule encoding an antigen having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:21. 11. The recombinant molecule of Claim 1 , wherein said recombinant molecule comprises an animal virus genome.
12. The recombinant molecule of Claim 11, wherein said eukaryotic transcription control region is heterologous to said genome.
13. The recombinant molecule of Claim 11 , wherein said eukaryotic transcription control region is endogenous to said genome and is located at a position selected from the group consisting of a natural position within said genome and a non- natural position within said genome.
14. The recombinant molecule of Claim 11, wherein said animal virus genome is selected from the group consisting of a poxvirus genome, a herpesvirus genome, an alphavirus genome, a picornavirus genome, a retrovirus genome, an adenovirus genome, and an adeno-associated virus genome.
15. The recombinant molecule of Claim 11 , wherein said animal virus genome is selected from the group consisting of an orthopoxvirus genome, a parapoxvirus genome, an entomopoxvirus genome, and an avipoxvirus genome.
16. The recombinant molecule of Claim 11 , wherein said eukaryotic transcription control region is selected from the group consisting of an early/late poxvirus promoter, a late poxvirus promoter, a CMV immediate-early promoter, and an SV40 promoter.
17. The recombinant molecule of Claim 11 , wherein said eukaryotic transcription control region is selected from the group consisting of an early/late poxvirus promoter and a late poxvirus promoter.
18. The recombinant molecule of Claim 11 , wherein said genome is an orthopoxvirus genome selected from the group consisting of a raccoon poxvirus genome and a vaccinia virus genome.
19. The recombinant molecule of Claim 11 , wherein said eukaryotic transcription control region is selected from the group consisting of a p7.5 promoter, a pi 1 promoter and a pSYN promoter.
20. The recombinant molecule of of Claim 11, wherein said isolated nucleic acid molecule is located in a region of said genome selected from the group consisting of a non-essential gene and an intergenic region. 21. The recombinant molecule of of Claim 11, wherein said isolated nucleic acid molecule is located in a non-essential gene of an orthopoxvirus genome, said non-essential gene being selected from the group consisting of a thymidine kinase gene, a hemagglutination gene, an anti-inflammatory gene, and an A-type inclusion gene. 22. The recombinant molecule of Claim 21 , wherein said anti-inflammatory gene is selected from the group consisting of a soluble cytokine receptor gene, a serpin gene, a complement receptor gene, and an immunoglobulin receptor gene. 23. The recombinant molecule of Claim 1 , wherein said recombinant molecule comprises vRCN-pl l-nYpFl(a)sec544. 24. A recombinant virus comprising a recombinant molecule as set forth in Claim 11.
25. The recombinant virus of Claim 24, wherein said virus is attenuated.
26. A recombinant virus of Claim 24, wherein said recombinant virus comprises RCN:p 11 -nYpF 1 (a)sec544.
27. A recombinant cell comprising a recombinant molecule, said recombinant molecule comprising an animal virus genome as set forth in Claim 11.
28. A recombinant cell comprising a recombinant virus as set forth in Claim 24.
29. The recombinant cell of Claim 28, wherein said cell comprises BSC- 1 :RCN:p 1 lnYpF 1 (a)sec544.
30. The recombinant molecule of Claim 1 , wherein said molecule comprises a recombinant plasmid.
31. The recombinant molecule of Claim 30, wherein said eukaryotic transcription control region comprises a promoter selected from the group consisting of a human cytomegalovirus immediate-early promoter, a simian virus 40 early promoter, and a Rous sarcoma virus long terminal repeat promoter. 32. The recombinant molecule of Claim 30, wherein said eukaryotic transcription control region comprises a polyadenylation region selected from the group consisting of a bovine growth hormone polyadenylation region and an SV40 polyadenylation region.
33. The recombinant molecule of Claim 30, wherein said eukaryotic transcription control region comprises a transcription enhancer region.
34. The recombinant molecule as set forth in Claim 30, wherein said recombinant molecule is selected from the group consisting of pCMV-nYpFl(b)sec544, pCMV-nYpFlanc576, and pCMV-nYpFlmat474.
35. A recombinant cell comprising a recombinant molecule of Claim 30. 36. The recombinant cell of Claim 35, wherein said recombinant molecule is selected from the group consisting of pCMV-nYpFl(b)sec544, pCMV-nYpFlanc576, and pCMV-nYpFlmat474. 37. A recombinant cell of Claim 35, wherein said recombinant molecule comprises a nucleic acid molecule that expresses an antigen selected from the group consisting of: an antigen selected from the group consisting of PYpFlsec170,
PYpFlanc192, PYpFlanc171, PypFlmat150, andPYpFlmat149; and an antigen encoded by a variant of a nucleic acid molecule encoding an antigen selected from the group consisting of PYpFlsec170, PYpFlanc192, PYpFlanc171, PYpFlmat150j and PYpFlmat149.
38. An isolated nucleic acid molecule encoding a Yersinia pestis FI antigen fused, in-frame, with a eukaryotic membrane anchor domain.
39. The isolated nucleic acid molecule of Claim 38, wherein said eukaryotic membrane anchor domain is selected from the group consisting of a vesicular stomatitis virus glycoprotein membrane anchor domain, a respiratory syncytial virus G protein membrane anchor domain, a herpesvirus glycoprotein membrane anchor domain, an immunoglobulin membrane anchor domain, and a glycosyl phosphotidylinositol membrane anchor domain.
40. The isolated nucleic acid molecule of Claim 38, wherein said membrane anchor domain is a canine herpesvirus glycoprotein membrane anchor domain selected from the group consisting of a glycoprotein G membrane anchor domain, a glycoprotein E membrane anchor domain and a glycoprotein I membrane anchor domain.
41. The isolated nucleic acid molecule of Claim 38, wherein said nucleic acid molecule comprises nYpFlanc576.
42. An isolated nucleic acid molecule comprising nYpFlanc576. 43. A recombinant raccoon poxvirus genome comprising an isolated nucleic acid molecule encoding a Yersinia pestis FI antigen operatively linked to a poxvirus transcription control region. 44. The recombinant genome of Claim 43, wherein said recombinant genome comprises vRCN-p 11 -nYpF 1 sec544. 45. A recombinant raccoon poxvirus comprising a recombinant raccoon poxvirus genome, said genome comprising an isolated nucleic acid molecule encoding a Yersinia pestis FI antigen operatively linked to a poxvirus transcription control region. 46. The recombinant raccoon poxvirus of Claim 45, wherein said virus comprises RCN:pl l-nYpFl(a)sec544.
47. A recombinant cell comprising a recombinant raccoon poxvirus genome, said genome comprising an isolated nucleic acid molecule encoding a Yersinia pestis FI antigen operatively linked to a poxvirus transcription control region.
48. The recombinant cell of Claim 47, wherein said recombinant cell comprises vRCN-pl l-nYpFl(a)sec544.
49. The recombinant cell of Claim 47, wherein said recombinant cell comprising BSC-1 :RCN:pl l-nYpFl(a)sec544.
50. A recombinant plasmid comprising an isolated nucleic acid molecule encoding a Yersinia pestis FI antigen operatively linked to a eukaryotic transcription control region.
51. The recombinant plasmid of Claim 50, wherein said recombinant plasmid is selected from the group consisting of pCMV-nYpFl(b)sec544, pCMV- nYpFlanc576, and pCMV-nYpFlmat474.
52. A recombinant cell comprising a recombinant plasmid, said plasmid comprising an isolated nucleic acid molecule encoding a Yersinia pestis FI antigen operatively linked to a eukaryotic transcription control region.
53. A therapeutic composition to protect an animal from plague comprising a recombinant molecule, said recombinant molecule comprising an isolated nucleic acid molecule that encodes an antigen selected from the group consisting of a Yersinia antigen, a Pasteurella antigen, and a Francisella antigen, said nucleic acid molecule being operatively linked to a eukaryotic transcription control region.
54. The therapeutic composition of Claim 53, wherein said recombinant molecule is selected from the group consisting of a recombinant animal virus genome and a recombinant plasmid.
55. The therapeutic composition of Claim 53, wherein said recombinant molecule enters the cells of said animal, such that said antigen is expressed in said cells.
56. The therapeutic composition of Claim 53, wherein said therapeutic composition comprises a recombinant virus. 57. The therapeutic composition of Claim 53, wherein said therapeutic composition comprises a recombinant plasmid.
58. The therapeutic composition of Claim 53, wherein said composition further comprises a component selected from the group consisting of an excipient, an adjuvant, and a carrier.
59. The therapeutic composition of Claim 53, wherein plague comprises a disease selected from the group consisting of bubonic plague, septicemic plague, pneumonic plague, urban plague, rat plague, wild rodent plague, sylvatic plague, campestral plague, high plains plague, la peste bubonique, The Pest, The Black Plague, and The Black Death.
60. A method to protect an animal from plague comprising administering to said animal a therapeutic composition comprising a recombinant molecule, said molecule comprising an isolated nucleic acid molecule that encodes an antigen selected from the group consisting of a Yersinia antigen, a Pasteurella antigen, and a Francisella antigen, said nucleic acid molecule being operatively linked to a eukaryotic transcription control region. 61. The method of Claim 60, wherein said recombinant molecule is selected from the group consisting of a recombinant animal virus genome and a recombinant plasmid. 62. The method of Claim 60 comprising administering to said animal a recombinant virus. 63. The method of Claim 60 comprising administering to said animal a recombinant plasmid. 64. The method of Claim 62 wherein said therapeutic composition is administered by a method selected from the group consisting of oral, subcutaneous injection, intradermal injection, and intramuscular injection. 65. The method of Claim 62, wherein said therapeutic composition is administered orally. 66. The method of Claim 63, wherein said therapeutic composition is administered by a method selected from the group consisting of injection, oral application, nasal application, particle bombardment, or intradermal scarification.
7. The method of Claim 66, wherein said injection is administered by a method selected from the group consisting of intradermal injection and intramuscular injection.
PCT/US1997/022617 1996-12-04 1997-12-04 Recombinant plague vaccine WO1998024912A2 (en)

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