WO2011056829A1 - Vaccin oral destiné à protéger les animaux contre la peste - Google Patents

Vaccin oral destiné à protéger les animaux contre la peste Download PDF

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WO2011056829A1
WO2011056829A1 PCT/US2010/055229 US2010055229W WO2011056829A1 WO 2011056829 A1 WO2011056829 A1 WO 2011056829A1 US 2010055229 W US2010055229 W US 2010055229W WO 2011056829 A1 WO2011056829 A1 WO 2011056829A1
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rcn
seq
vaccine
recombinant
homology
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PCT/US2010/055229
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English (en)
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Tonie Ellen Rocke
Jorge Emilio Osorio
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United States Department Of The Interior
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    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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 novel recombinant raccoon poxvirus vaccines against plague having improved efficacy that may be orally administered and used for large scale vaccination.
  • the present invention further relates to a novel truncated form of the V gene of Y. pestis (V307), and a recombinant raccoon poxvirus construct containing V307 therein (RCN-V307).
  • Vaccines of the present invention may include either (1 ) RCN-V307 administered with a recombinant raccoon poxvirus construct containing F1 therein - designated herein as ((RCN-F1 ) + (RCN-V307)), or (2) a single recombinant raccoon poxvirus construct containing both F1 and V307 therein (designated herein as RCN-(F1 +V307)).
  • the vaccines may be administered orally, and preferably in the form of a bait.
  • Plague caused by Y. pestis, is a zoonotic disease transmitted primarily by fleas that has recently re-emerged in numerous parts of the world. To reduce this public health threat and to protect against the potential use of Y. pestis as a bioweapon, development of novel plague vaccines for both humans and animals has been the focus of extensive research in recent years. [1 -3]
  • the capsular F1 antigen (17.5 kDa) and the secreted V antigen (35 kDa) are natural virulence factors produced by Y. pestis [4, 5] and have been shown to be highly immunogenic, conferring a degree of protection equivalent to that of live plague vaccines such as the EV76 vaccine [6].
  • Vaccines based on these two antigens and a recombinant protein fusion of F1 and V have been developed [7], providing distinct advantages in comparison to the previous bacterial vaccines. However, like most proteins, they are usually weakly immunogenic when administered in the absence of an appropriate adjuvant or when administered via the oral route.
  • RCN Raccoon poxvirus
  • rRCN vaccines have been administered to a variety of mammalian species, including mice, rats, rabbits, raccoons, skunks, bobcats, cats, dogs and sheep [9-12], prairie dogs [13] and black-footed ferrets (Rocke, unpublished data) with no reported side effects.
  • rRCN vaccines have induced protective immune responses against rabies in raccoons, dogs, cotton rats, rabbits, bobcats, and foxes [9, 12, 14], and protective immune responses in domestic cats against feline panleukopenia virus, feline caliciviruses, and feline infectious peritonitis [1 1 , 15].
  • RCN-F1 encephalomyocarditis virus
  • IVS internal ribosomal entry site
  • tPA tissue plasminogen activator
  • the prior art teaches an orally administered RCN-F1 vaccine, administered as a bait, as having limited efficacy against Y. pestis challenge.
  • the present invention provides for such a vaccine.
  • the present invention relates to novel recombinant raccoon poxvirus vaccines against plague having improved efficacy that may be orally administered, and may be suitably used for large-scale vaccination of free- ranging wildlife populations, and in particular black-tailed prairie dogs and black- footed ferrets.
  • the present invention includes a novel truncated form of the V gene of Yersinia pestis (Y. pestis) (V307), and an antigen expressed by V307 or an epitope thereof that is capable of eliciting the desired immune response.
  • the present invention further includes a recombinant raccoon poxvirus construct having the truncated form of the V gene of Y. pestis inserted therein (RCN- V307).
  • the RCN-V307 is used in combination with a recombinant raccoon poxvirus construct that expresses the F1 -antigen of Y. pestis (RCN-F1 ) to form a recombinant raccoon poxvirus vaccine suitable for oral administration - ((RCN- F1 ) + (RCN-V307)).
  • the present invention further includes a single recombinant raccoon poxvirus construct having both the truncated form of the V gene of V. pestis (V307) and the F1 gene of Y. pestis inserted therein - (RCN-(F1 +V307)).
  • This construct expresses both the truncated V-antigen of Y. pestis and the F1 antigen of Y. pestis.
  • the RCN-(F1 +V307) construct may be used as an oral vaccine to protect and/or treat animals against plague.
  • the vaccines herein are used to protect and/or treat an animal against plague.
  • the vaccines when administered to an animal before its exposure to plague, induce protective immunity against plague.
  • These vaccines may be administered orally in any form, including incorporation into a bait or animal feed.
  • the vaccines herein may further be used in a veterinary setting or other medical treatment environment, and orally administered and/or administered via injection to an animal susceptible to plague.
  • Figure 1 is a diagram of vaccine constructs described herein. These include (1 ) RCN-IRES-tPA-YpF1 (designated herein as RCN-F1 ), (2) RCN/Vfull, (3) RCN/Vt (also designated herein as RCN-V307 or RCN-Vt (30 7)) , and (4) RCN/F1 (also designated herein as RCN-(F1 +V307) or RCN-(F1 +Vt (30 7)) , wherein both F1 and V307 are present in the same RCN vector).
  • RCN-IRES-tPA-YpF1 designated herein as RCN-F1
  • RCN/Vfull also designated herein as RCN-V307 or RCN-Vt (30 7)
  • RCN/F1 also designated herein as RCN-(F1 +V307) or RCN-(F1 +Vt (30 7)
  • Figure 2 is a table that sets forth vaccine treatments and dosages administered to AJ mice via intramuscular inoculation and their response to challenge with Yersinia pestis (C092; original strain obtained from US Army Institute of Infectious Diseases) at various dosages.
  • Median survival time (MST) is the day that 50% of the animals succumbed to infection.
  • Figure 3 is a photograph of a Western blot of RCN-V307, and RCN-Vfull infected vera cell monolayers.
  • Figure 4 is a graph of the hazard ratio and 95% confidence intervals for rRCN vaccine treatments administered to mice i.m. either singly or in combination in relation to RCN-F1 at a dosage of 1 x 10 7 . Letters in parenthesis indicate the dosages administered.
  • Figure 5 is a graph of least squares means log anti-F1 titer for rRCN vaccine treatments administered to mice i.m. either singly or in combination. Letters indicate the dosages administered.
  • Figure 6 is a graph of least squares mean log anti-V titer for rRCN vaccine treatments administered to mice i.m. either singly or in combination. Letters indicate the dosages administered.
  • Figure 7 is a graph of the geometric mean serum IgG antibody titers against F1 and V antigens in black-tailed prairie dogs that were vaccinated against plague by consuming RCN-F1 and RCN-V307 vaccine-laden baits or by parenteral injection of F1 -V fusion protein.
  • Mean pre-challenge titers of all vaccinates were higher than controls (P ⁇ 0.05).
  • Mean titers of animals injected with F1 -V fusion proteins were higher (P ⁇ 0.001 ) than those that consumed RCN vaccines in baits.
  • Figure 9 is a diagram showing the IRES-F1 construct.
  • Figure 10 is a sequence listing for the full amino acid and nucleotide sequences for Y. pestis LcrV (SEQ ID NOS: 1 and 2 respectively).
  • Figure 11 is a sequence listing for the amino acid and nucleotide sequences for a novel shortened Y. pestis LcrV protein (SEQ ID NOS: 3 and 4 respectively).
  • Figure 12 is a sequence listing for the nucleotide sequence of the IRES-FI/Vt fragment (SEQ ID NO: 5).
  • Figure 13 is a sequence listing of is a sequence listing for the amino acid and nucleotide sequences for a shortened Y. pestis LcrV protein with a frame shift mutation at the C-terminal (SEQ ID NOS: 6 and 7 respectively).
  • the present invention relates to vaccines having improved efficacy for the treatment and/or protection of animals, and particularly of animals in the wild, against plague.
  • the vaccines of the present invention are recombinant raccoon poxvirus vaccines. They use recombinant raccoon poxviruses as vectors to deliver the F1 gene of Y. pestis (F1 ) and a novel truncated form of the LcrV (V) gene of Y. pestis to an animal susceptible to plague.
  • F1 F1
  • V LcrV
  • FIG. 10 SEQ ID NOS: 1 and 2
  • FIG. 1 1 SEQ ID NOS: 3 and 4
  • Vaccines of the present invention may comprise (1 ) a recombinant RCN construct containing F1 therein, and a separate recombinant RCN construct containing V307 therein, wherein these recombinant constructs are both administered to an animal susceptible to plague (designated as (RCN-F1 )+(RCN- V307)); or (2) a single recombinant RCN construct containing both F1 and V307 therein (designated as RCN-(F1 +V307) or RCN(F1 +Vt (3 o7)) -
  • the sequence listing of the combination F1/V307 fragment also designated F1 /Vt( 3 o7)
  • FIG. 12 SEQ ID NO: 5
  • the invention includes methods of preventing and/or treating an animal susceptible to plague, as well as a kit containing the vaccine constructs described herein, and instructions for use thereof.
  • an administration route “selected from the group consisting of” refers to one or more of the routes in that group, including combinations thereof.
  • immunize, immunized and immunization refer to the ability to elicit an immune response in an animal.
  • An immune response can be humoral or cell-mediated, or a combination thereof. Methods to measure an immune response are known to those skilled in the art; examples of some of such methods are disclosed herein.
  • an immunized animal is protected from disease caused by the agent against which the animal is being immunized.
  • immunization of an animal with a recombinant raccoon poxvirus expressing proteins from plague LcrV and F1 genes will protect that animal from plague.
  • Such an animal preferably displays (i.e., has) a protective antibody liter. Particularly preferred are high plague antibody liters, as measured by Enzyme- linked immunosorbent assay (ELISA; see examples).
  • a recombinant raccoon poxvirus of the present invention can be formulated in an excipient that the animal to be treated can tolerate.
  • the present invention includes administration of a composition comprising a recombinant raccoon poxvirus, wherein the composition further comprises an excipient.
  • excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous solutions physiologically balanced salt solutions.
  • Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran.
  • Excipients can also include various amounts of additives, such as substances that enhance isotonicity and chemical stability.
  • buffers examples include phosphate buffer, bicarbonate buffer and Tris buffer.
  • Standard formulations can either be liquid injectable or solids which can be taken up in a suitable liquid as a suspension or solution for injection.
  • the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
  • Oral methods of delivery can consist of solids as described in further detail herein.
  • the recombinant raccoon poxvirus can also include an adjuvant and/or a carrier.
  • an adjuvant and/or a carrier are not required to produce an efficacious vaccine, and in some cases, the advantages of the poxviruses of the present invention would be precluded by the use of some adjuvants.
  • adjuvants or carriers are not precluded by the present invention.
  • Adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen.
  • Suitable adjuvants include, but are not limited to, other bacterial cell wall components; aluminum-based salts, calcium-based salts; silica; polynucleotides, toxoids; serum proteins; other viral coat proteins; other bacterial-derived preparations; block copolymer adjuvants, and saponins and their derivatives.
  • Carriers are typically 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 formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters and glycols.
  • sequence homology or “homology” means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences.
  • sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred.
  • Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or 60 polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M.
  • a recombinant raccoon poxvirus of the present invention includes not only a recombinant raccoon poxvirus genome but also an envelope and core in which the genome is packaged.
  • the viral envelope and core are preferably a raccoon poxvirus envelope and a raccoon poxvirus core, encoded at least in part by raccoon poxvirus genes, thereby imparting to the recombinant raccoon poxvirus the host range of a natural raccoon poxvirus isolate.
  • the present invention also includes recombinant raccoon poxviruses having envelopes and/or cores that have been modified to, for example, alter (e.g., broaden, narrow, or completely change) the host range of the recombinant raccoon poxvirus genome.
  • modifications can be accomplished by one skilled in the art by, for example, modifying raccoon poxvirus envelope and/or core genes and/or replacing such genes with those of another virus.
  • Altered genes can be located on the raccoon poxvirus genome itself and/or in the genome of the cell in which the recombinant virus is produced.
  • a recombinant raccoon poxvirus genome of the present invention is a raccoon poxvirus genome in which nucleotides have been deleted, inserted, substituted or inverted using recombinant techniques known to those skilled in the art such that the recombinant raccoon poxvirus genome is no longer the same as a natural genome.
  • a recombinant raccoon poxvirus genome of the present invention includes a (i.e., one or more) heterologous nucleic acid molecule.
  • a heterologous nucleic acid molecule is a nucleic acid molecule isolated from a source other than raccoon poxvirus.
  • a heterologous nucleic acid molecule of the present invention encodes a protein, such as a heterologous antigen, that is, a non-raccoon poxvirus antigen.
  • a heterologous nucleic acid molecule of the present invention is operatively linked to a transcription control region, meaning that the heterologous nucleic acid molecule is expressed as an RNA and/or protein by the recombinant viral genome upon infection into a cell.
  • the transcription control region can be endogenous to the genome or the heterologous nucleic acid molecule can be operatively linked to an exogenous transcription control region to form a recombinant molecule.
  • Such a recombinant molecule can be introduced into the genome by methods known to those skilled in the art, for example by homologous recombination.
  • a suitable transcription control sequence is any sequence that allows for transcription of a heterologous nucleic acid molecule of the present invention. Examples include, but are not limited to, poxvirus promoters such as pll, p7.5, and synthetic promoters, as well as other viral promoters such as CMV promoters.
  • a suitable location for a heterologous nucleic acid molecule or recombinant molecule of the present invention is in an essential region, a nonessential region, or an intergenic region of the raccoon poxvirus genome.
  • Examples include, but are not limited to, a thymidine kinase gene, a hemaglutinin gene, a serpin gene, a cytokine receptor gene, and an interferon receptor gene.
  • One embodiment of the present invention is a recombinant raccoon poxvirus that also comprises a nucleic acid molecule encoding an immunomodulator.
  • Suitable immunomodulators include compounds that enhance certain immune responses as well as compounds that suppress certain immune responses.
  • Compounds that enhance the immune response include compounds that preferentially enhance humoral immunity as well as compounds that preferentially enhance cell-mediated immunity. Suitable compounds can be selected depending on the desired outcome.
  • Suitable immunomodulators include, but are not limited to, cytokines, chemokines, superantigens, and other immunomodulators as well as compounds that induce the production of cytokines, chemokines and other immunomodulators.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • CSF colony stimulating factor
  • EPO erythropoietin
  • interleukin 2 IL-2
  • interleukin-3 IL-3
  • interleukin 4 IL- 4
  • interleukin 5 IL-5
  • interleukin 6 IL-6
  • IL-7 interleukin 7
  • IL-8 interleukin 10
  • IL-12 interleukin 12
  • interferon gamma interferon gamma inducing factor I
  • TGF- ⁇ transforming growth factor beta
  • RANTES regulated upon activation, normal T-cell expressed and presumably secreted
  • macrophage inflam-matory proteins e.g., MIP-1 alpha and MIP-1 beta
  • LIF Leishmania elongation initialing factor
  • a suitable composition for administering to animals in accordance with the present invention can include one or more recombinant raccoon poxviruses, each including one or more heterologous nucleic acid molecules, as disclosed herein.
  • a composition of the present invention can be multivalent, that is, either delivering one or more antigens derived from a single source or antigens from multiple sources, such as those disclosed herein.
  • the term "vaccine” means a composition which, when administered to an animal, stimulates an immune response against an antigen.
  • a vaccine may comprise a protein, recombinant protein, DNA or RNA which upon introduction into a host, is able to provoke the immune response including but not limited to the production of antibodies, cytokines and other cellular responses.
  • a vaccine may be used for the prevention of or prophylaxis against a disease, to include the passive or active immunization with antibodies or vaccines of the invention such that the disease or infection does not occur.
  • active immunity to infection we mean the ability of an organism to produce specific responses such as production of cytokines, lymphokines, antibodies or other substances, or cellular capacity to inhibit or retard infection in response to a contact with an antigen.
  • passive immunity to infection we mean the transfer to a host of the specific antibodies or other substances or cells capable of inhibiting or retarding infection.
  • Heterologous nucleic acid molecules, recombinant molecules, recombinant raccoon poxvirus genomes, recombinant raccoon poxviruses and compositions of the present invention can be produced by methods known lo those skilled in the art.
  • the raccoon pox virus used is the same as that having ATCC designation VR-838TM, classification: Poxviridae, Orthopoxvirus; Strain: Herman; Original Source: Isolated by Y.F. Herman from respiratory tract of raccoon with no clinical symptoms, Maryland, USA, 1964; depositor: J.H. Nakana).
  • RCN-F1 construct is well known in the art. A detailed description of an RCN-F1 construct of the type that may be employed herein is set forth in Mencher et al. [13], Osorio, et al. [1 6], and Rocke et al. [31 ] all of which are incorporated herein in their entirety. These references teach that experiments were conducted wherein RCN-F1 incorporated into baits were offered to black-tailed prairie dogs, and that immunized prairie dogs had higher survival rates (56% and 38%, in [13] and [31 ] respectively) than unimmunized control animals (12% and 1 1 %, respectively).
  • FIG 1 1 The nucleic acid sequence, and amino acid sequence corresponding to V307 is set forth herein as FIG 1 1 (SEQ ID NOS: 3 and 4).
  • Overheim et al. identifies numerous truncated forms of the LcrV gene and how to make them, it does not teach the specific V307 gene of the present invention.
  • the novel RCN-vectored vaccine construct containing the novel V307 therein is designated herein as RCN-V307 or RCN-Vt (3 o7)-
  • V307 Methods employed herein in making the RCN-vectored constructs, and the truncated form of the V gene (V307) can be broadly described as follows:
  • RCNV raccoon poxvirus
  • Raccoon poxvirus vector created by modifying the vaccinia virus thymidine kinase (tk) gene-containing pKB3 plasmid are known in the art (Esposito et al., 1988 [43]).
  • the known constructs include an additional unique polylinker restriction sites, and vaccinia tk sequences replaced by RCNV tk sequences.
  • the resulting pTK vector was designed such that foreign DNA cloned into the polylinker region is flanked by RCNV tk sequences and can recombine into the f/ gene of a wild-type RCNV.
  • the plasmid pCD1 was used as a template for PCR amplification of the full length LcrV sequence of Y. pestis (FIG. 10 and SEQ ID NOS: 1 and 2).
  • the PCR amplified product was cloned into a pre-existing plasmid pTK-IRES- tPA-YpF1 [16] such that LcrV sequence replaced YpF1 sequence.
  • the resulting plasmid pTK/Vfull displayed the full length LcrV sequence, flanked by the RCNV tk sequences, and preceded by the secretory signal of tissue plasminogen activator (tPA) and the internal ribosomal entry site (IRES) of encephalomyocarditis virus.
  • the operon is driven by the vaccinia virus promoter P1 1 (late class).
  • the combination of IRES and tPA before a sequence has been shown to strongly increase its expression [16].
  • F1 and LcrV from Y. pestis have been shown to induce a protective immunity against a challenge with plague.
  • F1 -V fusion protein is now considered as the standard for vaccination against plague in animals and humans.
  • the goal of the pTK/F1 /Vt( 3 07) construct is to combine both F1 and the novel truncated LcrV in a RCN virus, conferring a better protection.
  • the F1 gene was isolated together with IRES and tPA from the preexisting plasmid pTK-IRES-tPA-YpF1 by PCR.
  • the truncated LcrV gene Vt (3 o7) was isolated together with tPA from the plasmid pTK/Vt( 3 07) also by PCR.
  • the isolated IRES-tPA-F1 sequence was cloned in pTK-sE/L(-/+) immediately after the sE/L promoter oriented in the positive direction and the isolated tPA-Vt( 3 07) sequence was cloned immediately after the sE/L promoter oriented in the negative direction.
  • the resulting pTK/F1 /Vt( 3 07) shuttle vector then comprises both IRES-tPA-F1 and tPA-Vt( 30 7) sequences back-to-back, driven by sE/L promoters (see FIG. 9).
  • the removal of the IRES before the tPA-Vt (3 07) sequence is necessary due to interferences that occur during the RNA transduction process when two IRES are present in a same transcript.
  • flasks of BSC-1 African green monkey kidney cells were infected with wild-type raccoon poxvirus and were then transfected with one of the plasmid shuttles, pTK-Vfull, pTK-Vt (3 07) or pTK-F1 -Vt (30 7) using LIPOFECTAMIN (Gibco, Grand Island, NY), but it is understood that other suitable transfection reactions may be used in this process.
  • the resulting recombinant viruses were plaque purified three times in RAT-2 thymidine kinase mutant rat embryo cells in the presence of bromodeoxyuridine (BrdU) to select for tk recombinants.
  • RCN-Vfull also referred to herein as recombinant RCN constructs
  • RCN-V307 also referred to herein as recombinant RCN constructs
  • RCN-(F1 +V307) or RCN-(F1 +Vt (3 o7)) was previously described.
  • Suitable excipients include those well known in the art that would be suitable for oral administration (i.e., in the form of baits, pills, elixirs (liquid), etc.), and those suitable for injection, such as phosphate buffered saline. Selection of a suitable excipient is well within the skill of the art.
  • mice were vaccinated via injection using both constructs, RCN-F1 and RCN-V307. Survival of these vaccinated mice upon plague challenge was significantly enhanced over mice that had been vaccinated with either construct alone (see Example 1 )
  • LcrV low calcium response V gene
  • the different forms of LcrV used to design the RCN-vectored vaccines are designated herein as Vfull (FIG. 10 and SEQ ID NOS: 1 and 2) and V307 (the novel, truncated V307 gene - FIG. 1 1 and SEQ I D NOS: 3 and 4).
  • Vfull and V307 were then each inserted into separate RCN vectors (resulting in RCN-V307 and RCN-Vfull). They were then each individually tested in mice in combination with RCN-F1 via simultaneous injections - ((RCN-Vfull + RCN-F1 ) and (RCN- V307 + RCN-F1 )).
  • Rat embryonic fibroblasts [Rat-2 (ATCC # CRL-1764)] and African green monkey kidney epithelial cells [BSC-1 (ATCC # CCL-26) and Vera (ATCC # CCL-18)] were maintained at 37 Q C and 5% C0 2 in Medium 199 supplemented with 0.01 g/L L-glutamine and 5% fetal bovine serum (FBS) and were used for culturing virus.
  • Rat embryonic fibroblasts [Rat-2 (ATCC # CRL-1764)] and African green monkey kidney epithelial cells [BSC-1 (ATCC # CCL-26) and Vera (ATCC # CCL-18)
  • BSC-1 African green monkey kidney epithelial cells
  • Vera ATCC # CCL-18
  • Raccoon poxvirus Herman strain [8], having ATCC designation VR-838, was mixed 1 :1 with trypsin-versene solution (0.05% trypsin; 0.02% EDTA in Earle's Balanced Salt Solution) and incubated for 15 min at 37°C to release infectious particles from aggregates that may have formed upon storage prior to inoculation into cells.
  • RCN-V constructs various versions of the Y. pestis IcrV gene (Vfull and V307) were cloned into the polylinker region of the pTK shuttle vector [16] so that the introduced DNA and upstream c/ ' s-acting elements (the p1 1 late promoter of vaccinia virus and the EMCV-IRES) were flanked by RCN thymidine kinase gene (tk) sequences.
  • the introduced genes (lacking their native translation initiation codons) were inserted in-frame behind the tPA secretory signal sequence (aa 2-23).
  • BSC-1 cells at 80% confluence were infected at a multiplicity of infection (MOI) of 0.06 with wild type RCN (Herman strain, ATCC designation VR-838). Infected cells were then transfected in serum-free Opti- MEM (Invitrogen, Carlsbad, CA) with a mixture containing 4 g of plasmid DNA [16] and 10 pL of LIPOFECTAMINE 2000 (Invitrogen, Carlsbad, CA) per well of a 6-well plate, according to the instructions provided by the manufacturer. The cell medium was replaced 5 h later.
  • MOI multiplicity of infection
  • mice received the empty vector virus (RCN-TK-) and was used as a negative control. All animals were boosted (same formulations, dosage, and route) on day 28 post-initial inoculation. Four weeks after the boost, all animals were challenged with the C092 isolate of Y. pestis [provided by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID)]. Each group of 12 mice was subdivided into groups of animals and each received either 70,000 cfu (3,500 LD50), 700,000 cfu (35,000 LD50), or 7,000,000 cfu (350,000 LD50) of Y. pestis.
  • mice were vaccinated IM with the vaccine combinations listed in Table 1 of FIG 2.
  • One group of mice in each experiment received RCN-TK- and served as the negative control.
  • the animals were boosted with the same formulations and dosages on day 28 PI.
  • Four weeks after the boost the animals were challenged with Y. pestis via i.d. injection and monitored for morbidity and mortality for 21 days as described above.
  • a fourth experiment was conducted to confirm results of the previous experiments.
  • Groups of 6-8 week old AJ mice (8 mice per group) were immunized and boosted as described above with single (RCN-F1 , RCN-Vfull, RCN-V307) or combined ((RCN-F1 + RCN-Vfull), (RCN-F1 + RCN-V307), (RCN- F1 + RCN-TK-)) vaccines.
  • a control group of 4 mice received RCN-TK- only. All constructs were inoculated at a dosage level of 5 x 10 7 pfu, the level that gave the best protection in experiment 3. Plague-induced mortality was verified in selected mice in each experiment by isolation of Y.
  • Antibody titers to Y. pestis F1 and V were determined using ELISA with antigens supplied by USAMRIID as described previously [16]. Serum samples were serially diluted 4-fold from 1 :160 to 1 :163,840 and test samples were run in duplicate. Titers of 1 :160 were considered negative. The highest dilution that was positive (exceeded the mean of four negative control samples by three standard deviations) was considered the endpoint and its reciprocal value recorded as the titer.
  • the SAS least squares means (population marginal means) for the treatments and pair-wise significance tests for each of them were computed using the SAS PDIFF option.
  • the least squares means along with +/- two standard errors computed from the treatment groups pooled across experiments.
  • Antibody titers were compared between treatment groups using analysis of variance with additive experiment and treatment effects. Antibody titers to F1 were significantly elevated (P ⁇ 0.0001 ) in all treatment groups that received RCN-F1 alone or in combination with another RCN construct compared to controls (TK-) and vaccinates that did not receive the RCN-F1 construct (data not shown). Population marginal means for anti-F1 titers of all treatment groups that received RCN-F1 alone or in combination with another RCN construct are shown in FIG 5.
  • Anti-V antibody titers were significantly elevated (P ⁇ 0.0001 ) in all treatment groups that received RCN-Vfull or RCN-V307 alone or in combination with RCN-F1 compared to controls (TK-) and vaccinated groups that received RCN-F1 or RCN-F1 +RCN-TK " (data not shown).
  • TK- controls
  • vaccinated groups that received RCN-F1 or RCN-F1 +RCN-TK
  • the anti-V titers of groups that received RCN-V307 at a dosage of 5 x 10 7 pfu alone or in combination with an equivalent dosage of RCN-F1 were not significantly different (P > 0.05).
  • groups that received RCN-V307 at a dosage of 1 x 10 7 pfu or in combination with RCN-F1 at 1 x 10 7 pfu had a lower mean anti-V titer than the RCN-Vfull group (P ⁇ 0.001 ).
  • mice [16] and prairie dogs [13] immunized with an rRCN-vectored vaccine expressing Y. pestis F1 antigen (RCN-F1 ) were protected against plague challenge.
  • the RCN-F1 vaccine provided full protection in vaccinated mice upon challenge with 100,000 cfu of Y. pestis and partial protection (56%) of orally immunized prairie dogs challenged with 130,000 cfu.
  • RCN-Vfull in combination with RCN- F1 was not as effective as administration of RCN-V307 in combination with RCN- F1 .
  • full length LcrV antigen has been shown to trigger the release of interleukin 10 (IL-10) by host immune cells, a cytokine that suppresses innate immune functions [18, 20].
  • IL-10 interleukin 10
  • the elevated IL-10 suppresses the release of pro-inflammatory cytokines, such as tumor necrosis factor alpha and gamma interferon, altering the defense mechanisms required to combat the pathogenesis of plague [18, 20].
  • the RCN-V307 construct developed in this study contains a similar deletion of 19 aa at the C-terminus of the LcrV gene (308-326).
  • the increased protective efficacy of the RCN-V307 construct compared to RCN-Vfull is believed to have resulted from removal of the immunosuppressive region located within the truncated sequence.
  • F1 antigen is a capsule-like protein expressed by Y. pestis at 37 C, after the bacteria begins multiplying in eukaryotic tissues, but not at 28 ° C [21 ], the approximate body temperature of fleas and the temperature we used to grow our Y. pestis challenge inoculum.
  • Antibody to V antigen in vaccinated mice that also had anti-F1 antibody clearly improved their response to i.d. challenge in our study, perhaps by blocking the immunomodulatory activity of Y. pestis LcrV [18], and/or by blocking the delivery of Yops in infected macrophage-like cells [24].
  • nearly all (23/24) of the animals vaccinated with single RCN-V constructs died upon Y. pestis challenge, even at our lowest challenge dose of 3,500 LD50s (70,000 cfu), suggesting anti-V antibody alone was insufficient to protect the animals.
  • Other studies have demonstrated the suitability of the LcrV antigen as a vaccine against F1 + and F1 " Y.
  • the RCN-V307 vaccine construct significantly increased protection against plague in mice.
  • the best survival and highest anti-F1 and anti-V antibody titers in vaccinated mice were derived from a combination of 5 x 10 7 pfu for each virus, a feasible dosage from a production standpoint.
  • a dual injection vaccine is not a desired or suitable final product for vaccinating animals in the field such as free-ranging prairie dogs, such a vaccine is useful for the treatment of domestic animals.
  • a combination vaccine vectored by RCN that contains both F1 and V antigens is a better alternative than single vaccines containing F1 or V alone.
  • the animals were dusted with carbaryl prior to shipment, and, upon arrival at NWHC, they were inspected for external parasites (none were found), injected with an ANTHELMINTIC (200 ⁇ g/kg of Ivermectin, Merck & Co., Inc, West Point, PA), then treated with 200 ⁇ of ADVANTAGE FLEA CONTROL (Imidacloprid; Bayer HealthCare, Shawnee Mission, KS), via external application to the skin on the back of the neck.
  • Electronic microchip identification units (Avid Identification Systems, Inc., Folsom, LA) were inserted SC into each animal between the scapula. Prairie dogs were group-housed in isolation rooms with approximately 180 ft 2 of floor space.
  • BETA CHIPS Northwestern Productions Corporation, Warrensburg, NY covered the floor; and for shelter, custom-made stainless steel nest boxes with connecting lengths of PVC pipe were provided.
  • An alfalfa-based pelleted food approximately 50 g per animal per day
  • fresh vegetables broccoli, carrot, green beans, and sweet potato chunks
  • RCN-IRES-tPA-YpF1 (designated as RCN-F1 herein)
  • RCN-IRES-tPA-YpV307 was prepared using the V gene truncated at amino acid position 307.
  • each vaccine construct was created by replacing the thymidine kinase (TK) gene of RCN with the gene for Y. pestis F1 or V307 and associated regulatory elements (a poxvirus promoter, an internal ribosome entry site, and the secretory leader from tissue plasminogen activator).
  • TK thymidine kinase
  • Virus stocks were thawed and diluted to 5 X 10 7 TCID 50 /ml in Hank's medium (Gibco BRL, Carlsbad, CA) supplemented with 5% glycerin (Sigma, St. Louis, MO) immediately before adding to sweet potato gelatin baits. Preparation of baits and validation of vaccine titer was described in Mencher et al.
  • Y. pestis challenge Three weeks after consumption of the final vaccine-laden bait and ten weeks after the injectable vaccine administration, animals were challenged with the CO92 wild type isolate of Y. pestis (provided by USAMRIID). Stock aliquots of the bacteria, prepared and quantified as previously described (Osorio et al.[16]) were diluted 1000-fold in sterile saline. A volume of 0.2 ml of this solution was administered to each prairie dog by SC injection in the right hip region. Plate counts of the challenge inoculum verified that a dose of approximately 70,000 cfu (3,500 mouse intradermal LD 50 ) was given to each prairie dog, and concurrent mouse tests confirmed its virulence.
  • tissue samples from lung, liver, spleen, and skin or any visible abscess at the site of inoculation were cultured in brain heart infusion broth (Difco, Lawrence, Kansas) and on blood agar plates (Becton-Dickinson, Franklin Lakes, NJ) at 28°C for up to 72 hr. Plague-induced mortality was verified by isolating Y. pestis specific DNA sequences from bacterial colonies using PCR and primers specific for the Y. pestis F1 gene [7]. DNA fragments were fractionated and directly visualized using standard techniques.
  • Antibody titers to Y. pestis F1 antigen were determined using ELISA with F1 antigen and V antigens supplied by USAMRIID, as described previously (Rocke et al. 2008b [31 ]). Briefly, serum samples were serially diluted fourfold from 1 :160 to 1 :40,960; test samples were run in duplicate. Each plate also contained four replicates of a negative control serum sample and two replicates of a positive control serum sample. A horseradish-peroxidase labeled anti-prairie dog IgG custom-prepared by Bethyl Laboratories (Montgomery, TX) was diluted 1 :100 and used as the secondary antibody.
  • Titers ⁇ 1 :160 were considered negative and recorded as 1 :40.
  • the highest dilution that was positive (exceeded the mean of four negative control samples by three standard deviations) was considered the endpoint and its reciprocal value recorded as the titer.
  • the preferred embodiments of this invention are compositions and methods of vaccine production that produce antibody response and survival to challenge of prairie dogs orally vaccinated against plague with RCN-based vaccines in comparison to controls and animals that received an injectable vaccine.
  • Prairie dogs consumed > 75% of the RCN vaccine-laden bait offered to them. No adverse reactions were observed in any of the animals that consumed these or that received the injectable F1 -V vaccine.
  • IgG titers to F1 and V antigens increased significantly (df 14, P ⁇ 0.001 ) from a mean pre-challenge titer of 1 :738 and 1 :843 respectively to a mean post-challenge titer of 1 :16,708 and 1 :7648.
  • mean post-challenge titers of animals that received F1 -V fusion vaccine injections did not rise significantly (df 6, P >0.05) over their peak pre-challenge values.
  • Anti-F1 antibody was detected in the one surviving control animal at a very high titer (1 :163,480), but no anti-V antibody was detected.
  • nucleic acid molecules of the present invention inserted into the RCN vector - F1 , V307, and (F1 +V307) - may be readily prepared by methods known in the art, for example, by directly synthesizing the nucleic acid sequence using methods and equipment known in the art such as automated oligonucleotide synthesizers, PCR technology, recombinant DNA techniques, and the like.
  • the vaccines of the present invention may be incorporated/placed into baits that are able to maintain vaccine stability. Those baits that are suitable for field use could be used to broadcast the vaccine-laden baits from planes or other vehicles. One having ordinary skill in the art with knowledge of the present invention will be able to identify baits suitable for use herein.
  • the vaccine within the scope of the present invention is primarily described with reference to being suitable for oral administration, and in particular for delivery to animals found in the wild in the form of a bait, the route of vaccine administration/delivery is not intended to be so limited.
  • administration of the RCN-vectored vaccine via injection was found to significantly improve survival of plague-challenged mice.
  • the RCN-vectored vaccine of the present invention may be administered orally or by injection.
  • the vaccine may be incorporated into any carrier suitable for delivery of said vaccine to an animal - i.e., to include carriers suitable for injection or oral administration (capsule, pill, liquid, etc.). Selection of a suitable carrier is well within the skill of the art.
  • the invention also has significant veterinary application, and may be administered in an office setting to animals susceptible to plague.
  • RCN was used as the vector in these studies, other poxviruses could act as suitable vectors for insertion and delivery of F1 , V307 and F1 -V307. Moreover, although the showings herein relate to use of a specifically identified RCN vector, one having ordinary skill in the art will recognize that, and be able to select, other RCN vectors, if available, that may be suitably employed herein as well.
  • V307 and Vt (3 o7) are used interchangeably herein.

Abstract

La présente invention concerne un vaccin contre la peste destiné aux animaux, comprenant un poxvirus recombinant de raton laveur comportant au moins une séquence d'acide nucléique hétérologue traduisant pour une protéine antigénique de Y. pestis. Ladite séquence d'acide nucléique hétérologue du vaccin comprend une forme tronquée inédite du gène V de Y. pestis, ou une combinaison quelconque associant ladite séquence inédite et la séquence traduisant pour l'antigène F1 de Y. pestis. L'invention concerne également un vaccin oral contenant un poxvirus recombinant de raton laveur et pouvant être administré à des animaux sauvages sous la forme d'un appât ou d'un aliment.
PCT/US2010/055229 2009-11-03 2010-11-03 Vaccin oral destiné à protéger les animaux contre la peste WO2011056829A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006060728A2 (fr) * 2004-12-02 2006-06-08 University Of Chicago Procedes et compositions impliquant des proteines lcrv
WO2009009759A2 (fr) * 2007-07-11 2009-01-15 Fraunhofer Usa, Inc. Antigènes yersinia pestis, compositions de vaccins, et méthodes associées

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006060728A2 (fr) * 2004-12-02 2006-06-08 University Of Chicago Procedes et compositions impliquant des proteines lcrv
WO2009009759A2 (fr) * 2007-07-11 2009-01-15 Fraunhofer Usa, Inc. Antigènes yersinia pestis, compositions de vaccins, et méthodes associées

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