US20130251737A1 - Modified Mareks Disease Virus, and Vaccines Made Therefrom - Google Patents

Modified Mareks Disease Virus, and Vaccines Made Therefrom Download PDF

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US20130251737A1
US20130251737A1 US13/841,684 US201313841684A US2013251737A1 US 20130251737 A1 US20130251737 A1 US 20130251737A1 US 201313841684 A US201313841684 A US 201313841684A US 2013251737 A1 US2013251737 A1 US 2013251737A1
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mdv
virus
birds
vaccinated
vaccine
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Joyce Pritchard
Teshome Mebatsion
Michel Bublot
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Boehringer Ingelheim Animal Health USA Inc
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Merial LLC
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Assigned to MERIAL LIMITED reassignment MERIAL LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BUBLOT, MICHEL, MEBATSION, TESHOME, PRITCHARD, JOYCE
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Priority to US16/262,774 priority patent/US11510978B2/en
Priority to US18/058,619 priority patent/US20230310589A1/en
Assigned to MERIAL, INC. reassignment MERIAL, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MERIAL LIMITED
Assigned to Boehringer Ingelheim Animal Health USA Inc. reassignment Boehringer Ingelheim Animal Health USA Inc. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MERIAL, INC.
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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
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A61K39/255—Marek's disease virus
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
    • C12N2710/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

  • the present invention relates generally to viral vaccines and methods of using the same. More particularly, the present invention relates to novel vaccines for protecting chickens against infection with Marek's disease virus, and having improved safety and efficacy over existing vaccines.
  • Marek's disease a highly prevalent and important lymphoproliferative disease of chickens, is controlled in commercial chickens by live virus vaccines consisting of attenuated or naturally avirulent MD-related herpesviruses.
  • vaccination programs have been considered effective overall, the poultry industry continues to experience losses due to MD.
  • MD virus Given the tendency of MD virus to become more virulent with time (e.g. by reversion to more virulent form) coupled with the economic pressures confronting the poultry industry, there remains a strong incentive to develop safer and more efficacious products that will protect better in the face of early challenge with very virulent field strains without causing adverse side effects (e.g. thymic dystrophy).
  • serotype 1 the oncogenic form responsible for the disease, including high- and low-virulence MD virus and their attenuated variants
  • serotype 2 a non-oncogenic MD virus
  • serotype 3 herpesvirus of turkeys (HVT).
  • An early MD vaccine consists of the serotype 3 virus originally isolated from turkeys as reported in Witter et al. [Am. J. vet. Res. 31:525-538 (1970)] and Okazaki et al. [U.S. Pat. No. 3,642,574].
  • HVT HVT
  • Vaccines produced from the naturally avirulent SB-1 strain [Schat et al., J. Natl. Cancer inst. 60:1075-1082 (1978) and U.S. Pat. No. 4,160,0241, an isolate of a serotype 2 MD virus] have been licensed in the united States since 1984.
  • the SB-1 strain is poorly protective against the highly virulent MDV strains. It is usually used in combination with HVT as a bivalent vaccine since the two viruses together produce greater protection than does either one alone [Schat et a/., Avian Pathol. 11:593-606 (1982); Witter, Avian Pathol. 11:49-62 (1982), the contents of which are incorporated by reference herein].
  • the SB-1+HVT bivalent vaccine represents greater than 50% of the United States market for MD vaccines at present and is considered to be among the most efficacious of the various MD products available. However, sporadic losses occur despite its use.
  • CVI988/C Another MD vaccine produced from strain CVI988 clone C (CVI988/C) has been licensed for commercial use in the United States. This vaccine was derived from a mildly virulent serotype 1 MD virus attenuated by serial passage in tissue culture and has been reported by De Boer et al. [Avian Dis. 30:276-283 (1986)]. A further passaged derivative of CVI988/C, identified as CVI988/C/R6, has also been described by De Boer et al. [Advances in Marek's Disease Research, pp. 405-4 3 (1988)].
  • CVI988/Rispens which has been in commercial use in other countries for a number of years, was found to be highly effective against challenge with several very virulent MD virus strains by Witter et al. [4th Intl. Symp. Marek's Disease, pp. 315-319 (1992)].
  • Md11 a very virulent serotype 1 MD field isolate
  • Md11/75C An experimental vaccine derived from Md11, a very virulent serotype 1 MD field isolate, was reported by Witter, supra. Md11 was attenuated by serially passaging 75 times in cell culture, and the resultant vaccine was designated Md11/75C. This vaccine has been shown to provide good protection against challenge with Md5 and most other highly virulent MD viruses tested; but it was less efficacious against challenge with the JM/102W strain, a prototype MD virus effectively protected against by HVT and SB-1 vaccines. Furthermore, its efficacy was consistently lower in chicks with HVT antibody.
  • Md11/75C/R2/23 (or R2/23) was found by Witter et al. [Avian Dis., 35:877-891 (1991)] to possess the highly protective nature of the parent strain without its residual pathogenicity.
  • RM1 A recombinant Marek's disease virus, referred to as RM1, having the long terminal repeats of reticuloendotheliosis virus stably integrated into the repeat short (RS) regions of its genome was also described.
  • This strain was generated at the USDA-ARS-ADOL from a pathogenic serotype 1 Marek's disease virus strain JM [Witter et al., 1997, Avian Dis., 41:407-421, and Jones et al., 1996, J. Virology, 70(4):2460-2467i.
  • JM pathogenic serotype 1 Marek's disease virus strain JM [Witter et al., 1997, Avian Dis., 41:407-421, and Jones et al., 1996, J. Virology, 70(4):2460-2467i.
  • the RM1 strain has been shown to provide a level of protection similar or superior to that of CVI988, it has also been associated with residual pathogenicity, causing thymic
  • the present invention is based, in part, upon the production and use of vaccines comprising Marek's Disease Viruses (MDV) originally disclosed in application number U.S. Ser. No. 10/623,891 (published as US2005/0019348A1, to Reddy et al.), the contents of which, as well as the application's entire prosecution history, are both herein incorporated by reference in their entirety.
  • MDV Marek's Disease Viruses
  • CVRM2 was a heterogeneous population of recombinant and parental MDV, and when they isolated and subsequently administered to avians a pure clonal line of CVRM2 (hereinafter referred to as “RN1250”), they obtained safety and efficacy results far exceeding those a skilled person would have expected on reading Reddy et al.
  • FIG. 1 shows a schematic organization of MDV genome, which contains a unique long (UL) region flanked by inverted repeat (IRS), terminal repeat long (TRL), internal repeat long (IRL), and a unique short region (US), and is flanked by two inverted repeats, internal repeat short (IRS) and terminal repeat short (TRS). Also shown is a schematic representation of the overlapping cosmid clones generated to rescue an infectious virus from a highly virulent strain of MDV;
  • FIG. 2 depicts generation of the B40-Pac cosmid used to generate the CVRM vaccine
  • FIG. 3 illustrates PCR-based diagnostic of recombinant MDV; presented are the PCR primers, expected product sizes, and agarose gel image, which indicates the CVRM2 was not clonal, but in fact a dual population of recombinant and parental MDV. Lanes: 1) ladder; 2) negative; 3) Rispens; 4) GA 22; 5) Rismavac; 6) RB1B; 7) RN1250; 8) positive (original mixed population);
  • FIG. 4 presents PCR confirmation of RN1250 MSV, RN1250 x+5, and BP5.
  • PCR reactions with all primer pairs resulted in the expected PCR product and banding pattern, thus there was no evidence for presence of the parental Rispens virus among RN1250 MSV, RN1250 x+5, and BP5 isolates.
  • an “immunological response” to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest.
  • an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.
  • the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.
  • animal is intended mammals, birds, and the like.
  • Animal or host as used herein includes mammals and human.
  • the animal may be selected from the group consisting of equine (e.g., horse), canine (e.g., dogs, wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wild cats, other big cats, and other felines including cheetahs and lynx), ovine (e.g., sheep), bovine (e.g., cattle), porcine (e.g., pig), avian (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g., prosimian, tarsier, monkey, gibbon, ape), ferrets, seals, and fish.
  • the term “animal” also
  • Cloning The selection and propagation of (a) genetic material from a single individual, (b) a vector containing one gene or gene fragment, or (c) a single organism containing one such gene or gene fragment.
  • Cloning vector A plasmid, virus, retrovirus, bacteriophage, cosmid, artificial chromosome (bacterial or yeast), or nucleic acid sequence which is able to replicate in a host cell, characterized by one or a small number of restriction endonuclease recognition sites at which the sequence may be cut in a predetermined fashion, and which may contain an optional marker suitable for use in the identification. of transformed cells, e.g., tetracycline resistance or ampicillin resistance.
  • a cloning vector may or may not possess the features necessary for it to operate as an expression vector.
  • Expression The process undergone by a structural gene to produce a polypeptide. Expression requires transcription of DNA, post-transcriptional modification of the initial RNA transcript, and translation of RNA.
  • Expression Cassette A nucleic acid sequence within a vector which is to be transcribed, and a promoter to direct the transcription.
  • the expression cassette may contain one or more unrelated DNA sequences encoding one or more peptides of interest.
  • Expression control sequences are DNA sequences involved in any way in the control of transcription or translation and must include a promoter. Suitable expression control sequences and methods of making and using them are well known in the art.
  • a replicon such as a plasmid, virus, retrovirus, bacteriophage, cosmid, artificial chromosome (bacterial or yeast), or nucleic acid sequence which is able to replicate in a host cell, characterized by a restriction endonuclease recognition site at which the sequence may be cut in a predetermined fashion for the insertion of a heterologous DNA sequence.
  • An expression vector has a promoter positioned upstream of the site at which the sequence is cut for the insertion of the heterologous DNA sequence, the recognition site being selected so that the promoter will be operatively associated with the heterologous D ⁇ umlaut over ( ⁇ ) ⁇ A sequence.
  • a heterologous DNA sequence is “operatively associated” with the promoter in a cell when RNA polymerase which binds the promoter sequence transcribes the coding sequence into mRNA which is then in turn translated into the protein encoded by the coding sequence.
  • nucleic acid and “polynucleotide” refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids.
  • polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches.
  • the sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component.
  • Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support.
  • the polynucleotides can be obtained by chemical synthesis or derived from a microorganism.
  • genes are used broadly to refer to any segment of polynucleotide associated with a biological function.
  • genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression.
  • gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.
  • an “isolated” biological component refers to a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.
  • Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.
  • a partially purified polypeptide preparation is one in which the polypeptide is more enriched than the polypeptide is in its natural environment. That is the polypeptide is separated from cellular components.
  • substantially purified is intended that such that at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, or more of the cellular components or materials have been removed.
  • a polypeptide may be partially purified.
  • partially purified is intended that less than 60% of the cellular components or material is removed. The same applies to polynucleotides.
  • the polypeptides disclosed herein can be purified by any of the means known in the art.
  • Variants include allelic variants.
  • allelic variant refers to a polynucleotide or a polypeptide containing polymorphisms that lead to changes in the amino acid sequences of a protein and that exist within a natural population (e.g., a virus species or variety). Such natural allelic variations can typically result in 1-5% variance in a polynucleotide or a polypeptide.
  • Allelic variants can be identified by sequencing the nucleic acid sequence of interest in a number of different species, which can be readily carried out by using hybridization probes to identify the same gene genetic locus in those species. Any and all such nucleic acid variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity of gene of interest, are intended to be within the scope of the invention.
  • the term “derivative” or “variant” refers to a polypeptide, or a nucleic acid encoding a polypeptide, that has one or more conservative amino acid variations or other minor modifications such that (1) the corresponding polypeptide has substantially equivalent function when compared to the wild type polypeptide or (2) an antibody raised against the polypeptide is immunoreactive with the wild-type polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous.
  • the term “variant” further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein.
  • conservative variation denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue.
  • particularly preferred substitutions will generally be conservative in nature, as described above.
  • a “vector” refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered to a target cell, either in vitro or in vivo.
  • the heterologous polynucleotide may comprise a sequence of interest for purposes of prevention or therapy, and may optionally be in the form of an expression cassette.
  • a vector needs not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors and viral vectors.
  • recombinant means a polynucleotide with semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.
  • Heterologous means derived from a genetically distinct entity from the rest of the entity to which it is being compared.
  • a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide.
  • a promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.
  • the polynucleotides of the invention may comprise additional sequences, such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, 5′UTR, 3′UTR, transcription terminators, polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.
  • additional sequences such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, 5′UTR, 3′UTR, transcription terminators, polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.
  • Operably Encodes or Associated each refer to the functional linkage between a promoter and nucleic acid sequence, wherein the promoter initiates transcription of RNA corresponding to the DNA sequence.
  • a heterologous DNA sequence is “operatively associated” with the promoter in a cell when RNA polymerase which binds the promoter sequence transcribes the coding sequence into mRNA which is then in turn translated into the protein encoded by the coding sequence.
  • a promoter A DNA sequence within a larger DNA sequence defining a site to which RNA polymerase may bind and initiate transcription.
  • a promoter may include optional distal enhancer or repressor elements.
  • the promoter may be either homologous, i.e., occurring naturally to direct the expression of the desired nucleic acid, or heterologous, I.e., occurring naturally to direct the expression of a nucleic acid derived from a gene other than the desired nucleic acid.
  • a promoter may be constitutive or inducible.
  • a vaccine is defined herein in its broad sense to refer to any type of biological agent in an administrable form capable of stimulating a protective immune response in an animal inoculated with the vaccine.
  • the present invention provides Recombinant Marek's disease virus (MDV), into which has been inserted via homologous recombination, a long terminal repeat (LTR) derived from a reticuloendotheliosis virus (REV). These recombinants are effective to elicit an immune response in an avian to Marek's disease virus without causing a significant degree of pathogenicity in the avian.
  • LTR long terminal repeat
  • REV reticuloendotheliosis virus
  • avians are chickens.
  • CVRM-2 was produced by authors of Reddy et al., as described therein, and as summarized in FIGS. 1 and 2 of this disclosure.
  • instant Applicants performed careful PCR-based analysis to confirm the identity/integrity of the virus isolate ( FIG. 3 presents the agarose gel resolution of the amplified products).
  • FIG. 3 presents the agarose gel resolution of the amplified products.
  • Applicants found to their surprise the sample was not a clonal isolate, but was in fact a combined population of recombinant CVRM2 and the parental MDV strain.
  • Applicants then performed the necessary plaque purification to obtain a pure isolate consisting only of CVRM2 (and not the parental MDV Rispens strain).
  • the new, clonal isolate is referred to as “RN1250” throughout this disclosure.
  • the recombinant MDV of this invention may be produced by modification of MDV serotype 1 strain CVI988, or any of its clones or serially passaged strains, which are collectively referred to herein as strains “CVI988/X”.
  • CVI988/X includes, but is not limited to, the previously described original low-passage strain, CVI988/Rispens (Rispens et al., 1972, Avian Dis., 16:106-125 and 126-138), strain CVI988 clone C (CVI988/C) (De Boer, U.S. Pat. No. 4,673,572, and De Boer et al., 1986, Avian Dis.
  • the invention provides for a novel, recombinant, attenuated MDV strain, which is produced replacing a portion of the native CVI988/X sequence with exogenous DNA, which comprises a long terminal repeat (LTR) sequence from a reticuloendotheliosis virus (REV).
  • LTR long terminal repeat
  • REV reticuloendotheliosis virus
  • recombination of strain CVI988 was effected using MDV serotype 1 strain RM1 as a source of the exogenous LTRs (RM1 is a recombinant MDV into which REV LTRs had been integrated).
  • RM1 is a recombinant MDV into which REV LTRs had been integrated.
  • the REV LTR was excised from the purified RM1 viral DNA by Pac 1 digestion and inserted into a shuttle vector, B40, prepared from a very virulent strain of Marek's disease virus, Md5.
  • the resultant recombinant vector, B40-Pac was used for insertion of the LTRs into the Marek's disease virus strain CVI988.
  • the purified viral DNA of MDV strain CVI988 was co-transfected into chicken or duck embryonic fibroblast (CEF or DEF) cells with Not I-digested recombinant vector.
  • Recombinant viruses having the RM1 LTRs integrated into their genome replicated more quickly than the parental CVI988 strain. Without being bound by theory, it is believed that this increased rate of replication is the result of the insertion of the reticuloendotheliosis virus LTR into the genome of the MDV upstream of the ICP4 gene.
  • the MDV CVI988/X may be modified via the addition of isolated reticuloendotheliosis virus LTRs from sources other than the RM1 strain.
  • the insertion site of the LTR in the RM1 strain of MDV has been shown to be between IRL and IRS of the genome (Jones et al., 1996, Retroviral insertional activation in a herpesvirus: transcriptional activation of US genes by an integrated long terminal repeat in a MDV clone, J. Virology, 70(4):2460-2467). This corresponds approximately to position 152,745 of the Md5 strains of Marek's disease virus.
  • This region is located within a 1,704 base pair long EcoR1 fragment (nucleotides 152, 198-153, 902) of serotype 1 Md5 (Tulman et al., 2000, The genome of a very virulent Marek's disease virus, J. Virology, 74d7):7980-7988).
  • This 1,704 bp EcoR1 fragment can be cloned into a plasmid vector lacking Drain restriction endonuclease site and used as a transfer vector for introduction of any LTR in to the MDV genome.
  • This EcoRX fragment has a unique DraIII restriction site located 10 bp upstream of the LTR location in RM1.
  • the LTRs can be inserted into the DraIII site of the 1,704 base pair EcoR1 fragment to generate the LTR transfer vector, in order to generate recombinant MDV with LTR insertions, the transfer vector should be linearized with EcoR1, extracted with phenol and chloroform and precipitated with ethanol. Co-transfection of the linearized transfer vector along with DNA from any serotype 1 MDV strain into permissible cells in culture will result in the introduction of LTR sequences into the MDV genome by homologous recombination.
  • recombinant MDV having the RETV LTRs may be prepared from any MDV, including other CVI988/X strains, using the deposited CVRM-2, provided the CVRM-2 is plaque purified to ensure the virus is RN1250, and not a combination of RN1250 and the parental MDV strain.
  • REV LTRs are suitable for use herein.
  • Numerous suitable reticuloendotheliosis viral LTRs have been isolated and described, and include but are not limited to those described by Kost et al. (1993, Retrovirus insertion into herpesvirus: characterization of a Marek's disease virus harboring a solo LTR, Virology, 192:161-169), Ridgway (1992, REV LTR elements are efficient promoters in cells of various species and tissue origin, including human lymphoid cells, Gene, 121:213-218), Boerkoel and Kung [1992, Transcriptional interaction between retroviral long terminal repeats (LTRs): mechanism of 5′ LTR suppression and 3′ LTR promoter activation of c-myc in avian B-cell lymphomas, J.
  • LTRs long terminal repeats
  • the REV LTR nucleic acid sequences disclosed herein, or their biologically functional equivalents, can be used in accordance with the present invention.
  • the phrase “biologically functional equivalents” as used herein denotes nucleic acid sequences exhibiting the same or similar biological activity/immunoprotective activity as the above-mentioned reticuloendotheliosis viral LTR nucleic acid sequences (i.e., when introduced into the CVI988 MDV host in a functionally operable manner they elicit a protective immune response without causing a significant degree of pathogenicity in the chicken).
  • nucleic acid sequences described herein can be altered by base substitutions, insertions, additions, or deletions to produce biologically functionally equivalent nucleic acids that retain promoter or enhancer activity.
  • variants of the genomic DNAs or cDNAs should possess more than 75% homology, preferably more than 85% homology, and most preferably more than 95% homology, to the naturally occurring REV LTRs discussed herein.
  • the vaccine of the recombinant Marek's disease virus of the invention may be prepared as a cell-free preparation, or in the preferred embodiment, as a cell-associated preparation.
  • a cell-associated vaccine can be prepared directly from In vitro culture of the live viral agents in a suitable growth medium, such as chicken embryo fibroblasts as described by Witter (U.S. Pat. No. 4,895,718, the contents of which are incorporated by reference herein).
  • a suitable growth medium such as chicken embryo fibroblasts as described by Witter (U.S. Pat. No. 4,895,718, the contents of which are incorporated by reference herein).
  • a suitable growth medium such as chicken embryo fibroblasts as described by Witter (U.S. Pat. No. 4,895,718, the contents of which are incorporated by reference herein).
  • to prepare cell-free virus inocula cells from infected host tissue or cell culture are sonicated or otherwise disrupted as previously described. The cellular debris is removed by centrifugation and the centrifu
  • the vaccine may be prepared from the killed virus or from immunogenic components separated from the virus, although such processing would incur significantly greater costs.
  • a subunit vaccine can be prepared by separating from the killed virus one or more purified viral proteins identified as having immunogenic properties.
  • the viral agent is prepared for administration by formulation in an effective immunization dosage with a pharmaceutically acceptable carrier or diluent, such as physiological saline or tissue culture medium.
  • a pharmaceutically acceptable carrier or diluent such as physiological saline or tissue culture medium.
  • effective immunization dosage is defined as being that amount which will induce immunity in a chicken against challenge by a virulent strain of Marek's disease virus, immunity is considered as having been induced in a population of chickens when the level of protection for the population is significantly higher than that of an unvaccinated control group (measured at a confidence level of at least 80%, preferably measured at a confidence level of 95%).
  • Po protective index
  • the vaccine will contain at least about 200 PFU (plaque-forming units) of the virus, and preferably between about 2000 and 5000 PFU.
  • the vaccine can be effectively administered any time after the chicken attains immunocompetence, which is at about the 18th day of incubation (3 days prehatch); but it is normally administered by inoculation within 24-48 hours after hatching.
  • the recombinant viral DNA may be administered as a DNA vaccine as described by Tischer et al. (2002, J. Gen. Virology, 83:2367-2376, the contents of which are incorporated by reference herein).
  • vaccinal efficacy can be enhanced by combining the recombinant Marek's disease viruses of the invention with other viral agents into bivalent or polyvalent vaccines.
  • the pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle may be a water-in-oil emulsion.
  • the water-in-oil emulsion may be a water/oil/water (W/O/W) triple emulsion.
  • the pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle may be an oil-in-water emulsion.
  • the present invention includes the following method embodiments.
  • a method of vaccinating an avian comprising administering a composition comprising a Marek's Disease virus (MDV).
  • MDV Marek's Disease virus
  • a prime-boost regimen can be employed, which is comprised of at least one primary administration and at least one booster administration using at least one common polypeptide, antigen, epitope or immunogen.
  • the immunological composition or vaccine used in primary administration is different in nature from those used as a booster.
  • This administration protocol is called “prime-boost”.
  • a prime-boost regimen comprises at least one prime-administration and at least one boost administration using at least one common polypeptide and/or variants or fragments thereof.
  • the vaccine used in prime-administration may be different in nature from those used as a later booster vaccine.
  • the prime-administration may comprise one or more administrations.
  • the boost administration may comprise one or more administrations.
  • the dose volume of compositions is generally between about 0.1 to about 2.0 ml, between about 0.1 to about 1.0 ml, and between about 0.5 ml to about 1.0 ml.
  • the present invention contemplates at least one administration to an animal of an efficient amount of the therapeutic composition made according to the invention.
  • the animal may be male, female, pregnant female and newborn. This administration may be via various routes including, but not limited to, intramuscular (IM), intradermal (ID) or subcutaneous (SC) injection or via intranasal or oral administration.
  • IM intramuscular
  • ID intradermal
  • SC subcutaneous
  • the therapeutic composition according to the invention can also be administered by a needleless apparatus (as, for example with a Pigjet, Dermojet, Biojector, Avijet (Merial, GA, USA), Vetjet or Vitajet apparatus (Bioject, Oreg., USA)).
  • Another approach to administering plasmid compositions is to use electroporation (see, e.g.
  • the therapeutic composition is delivered to the animal by gene gun or gold particle bombardment.
  • the animal is a dog, ferret or seal.
  • kits for performing a method of eliciting or inducing an immunological or protective response against MDV in an animal comprising a recombinant MDV immunological composition or vaccine and instructions for performing the method of delivery in an effective amount for eliciting an immune response in the animal.
  • the subject matter disclosed herein is directed to a kit for performing a method of eliciting or inducing an immune response which may comprise any one of the recombinant MDV compositions or vaccines and instructions for performing the method.
  • G-CSF granulocyte colon
  • cytokines can be co-administered and/or sequentially administered with the immunological or vaccine composition of the present invention.
  • the vaccine of the instant invention can also contain an exogenous nucleic acid molecule that expresses in vivo a suitable cytokine, e.g., a cytokine matched to this host to be vaccinated or in which an immunological response is to be elicited (for instance, an avian cytokine for preparations to be administered to avians).
  • Marek's Disease Virus MDV
  • SR-1 Strains CVRM-2 RN1250 and RMI CN32399
  • a critical aspect of the instant invention is that after having completed the study outlined in this Example, inventors later determined the CVRM-2 MDV was not a clonal recombinant MDV, but in fact was a mixed population of RN1250 recombinant MDV and parental Rispens MDV. Thus, now that inventors have produced a stable, clonal RN1250 MDV, which virus' strong efficacy as a vaccine is demonstrated in later Examples, a skilled person will not be surprised by the broad (and unacceptable) range of protection (37-86%) apparently provided by the CVRM-2 MDV in this preliminary study.
  • One hundred fifty one-day-old SPF chicks were randomly assigned to five different groups of thirty birds each (Groups 1-5). Each treatment group was then randomly assigned to isolation units. The chicks in Group 1 used as the non-vaccinated, challenged controls were placed into negative pressure isolation units first, fifteen birds per unit. Each remaining group (Groups 2-5) were then subcutaneously (SQ) vaccinated with 0.2 ml per bird with either Intervet Rispens Rismavac®; MDV SR-1 RMI CN32399 P 4 ; MDV SR-1 CVRM-2 RN1250 P 4 ; or MDV Rispens CN32553 P 4 .
  • the SQ vaccinated birds in Groups 2-5 were also placed in negative pressure isolation units, 15 birds per unit. Four days later on Study Day 4, the birds in Groups 1-5 were challenged individually with MDV T. King, diluted 1:500 (protocol 02-085 02 January 3). Birds were challenged intraperitoneally (IP), with 0.2 ml per bird. Birds were observed for 49 days post-challenge. Any bird that died prior to Study Day 53 was necropsied and examined for MDV lesions. On Day 53, all remaining birds were terminated, necropsied and examined for MDV lesions.
  • IP intraperitoneally
  • the experimental MDV SR-1 and Rispens strains provided protection rates of 37-86% against the early MDV T. King challenge.
  • Intervet's Rismavac® provided 83% protection.
  • the challenge was validated with the non-vaccinated, challenged control birds showing MD incidence of 97%.
  • the vaccinates were inoculated by the subcutaneous route (SQ) route (0.2 ml per bird) as follows: Group 1 was vaccinated with RN1250 pre MSV, P6 (isolate I); Group 2 was vaccinated with RN1250 pre MSV, P7 (isolate U); Group 3 was vaccinated with RN1250 pre MSV, P7 (isolate F); and Group 4 was vaccinated with Rismavac® serial 02760010, exp. 15 Apr. 2012. After the inoculations, the vaccinates were placed in the same colony houses where the non-vaccinated, contact control birds and shedders were placed previously.
  • SQL subcutaneous route
  • Clinical disease and mortality were monitored daily for 49 days post-vaccination.
  • the spleens from any contact control bird that died were collected for virus isolation.
  • the forty non-inoculated commercial broilers that were housed in isolation units, in order to serve as the second group of contact controls were placed in the colony houses, Groups 1-4, according to a randomization schedule, 10 birds per group. These birds were banded with numbered bands before placement for identification.
  • the spleens from these birds were also collected for virus isolation in the event that mortality occurred.
  • the remaining vaccinates and the initial non-vaccinated, contact control birds were terminated and necropsied at the of the 49 day observation period on study day 63.
  • Spleens and at least two feather follicles per bird were collected from 10 contact controls for virus isolation. The sampled birds were determined by a randomization schedule. On study day 83, the remaining contact control birds that were added to the groups on study day 53 (second group), were terminated. Spleens and at least two feather follicles per bird were collected from these contact control birds for virus isolation.
  • Marek's Disease Vaccine Serotype 1, RN 1250 Vaccine, pre MSV (I) and (F), were more efficacious than Intervet's Rismavac® in commercial broilers using MDV T. King in a shedder challenge model
  • Two hundred-fifty, one-day-old, SPF chickens were randomly assigned to five treatment groups, 50 chicks per group, as well as, randomly assigned to the negative pressure isolation units used for housing.
  • One hundred chicks were designated as vaccinates and were identified as Groups 1 and 2.
  • One hundred chicks were designated as the sham-vaccinated, contact controls of Groups 1 and 2 (Groups 4 and 5, respectively); the remaining sham-vaccinated chicks (50) were identified as Group 3 to serve as the negative controls.
  • the sham-vaccinated, contact controls and sham-vaccinated, negative controls were wing-banded with numbered bands for identification and were placed into their respective units (7-10 birds per unit).
  • the sham-vaccinated birds were inoculated with Marek's Disease diluent, 0.2 ml per bird.
  • the chicks designated as vaccinates were subcutaneously (SQ) inoculated with either: RN1250 (X+5) (Group 1) or Rismavac® (Group 2), 0.2 ml per bird ( ⁇ 5000-6500 plaque-forming units (pfu) per dose).
  • SQ subcutaneously
  • RN1250 X+5)
  • Rismavac® Group 2
  • 0.2 ml per bird ⁇ 5000-6500 plaque-forming units (pfu) per dose.
  • Organ samples (bursa, thymus and spleen) from five birds in each group (Groups 1-5) were harvested and fixed in 10% buffered formalin. Two to three birds per unit were used for the weighing and organ harvest. The birds were selected according to a randomization schedule. The procedure described on Study Day 14, was repeated on Day 28, and on Day 49 with the remaining birds.
  • the bursa ratios of birds vaccinated with MDVSR-1 Rismavac®-vaccine and the ratios of the sham-vaccinated, contacts controls were not significantly different from that of the sham-vaccinated, negative control birds (p ⁇ 0.0581).
  • the thymus ratios of birds vaccinated with MDVSR-1 Rismavac®-vaccine and the ratios of the sham-vaccinated, contacts controls were not significantly different from that of the sham-vaccinated, negative control birds (p ⁇ 0.4004).
  • the ratios were not significantly different on any other days (p ⁇ 0.5558) Histological Examination Results from the histological examination did not shown evidence of atrophy in the bursa, thymus or spleen of the birds inoculated with the Marek's Disease SR-1 RN1250 or Rismavac®.
  • MDV SR-1, RN1250, X+5 was safe when administered SQ, as evaluated by thymic, bursa and/or spleen atrophy.
  • One hundred twenty (120) one-day-old SPF chickens were randomized into three different treatment groups and six units with each unit containing 15 vaccinates and five contacts.
  • the birds randomized as contacts were banded in the nape of the neck with colored numbered bands per the randomization schedule and then placed into their respective units.
  • the birds randomized as vaccinates were vaccinated with either MDV RN1250 X+5 or Rispens.
  • the experimental and commercial vaccines were diluted in Marek's diluent to yield approximately 100,000 pfu per dose (approximately 17 ⁇ the expected field dose) administered by the SQ route. After vaccination, the birds were placed into their respective units along with the contacts that were previously placed.
  • the vaccinated birds were banded at eight days of age in the nape of the neck with colored numbered bands according to a randomization schedule. Personnel involved with clinical assessments during the study were not present for the banding of the contacts or vaccinates nor did they perform any clinical observations during this time period in order to maintain blinding.
  • tracheal and cloacal swabs were taken for virus recovery from all vaccinated birds.
  • the swabs were pooled by group, with five tracheal or cloacal swabs per swab tube containing 5 ml of SPGA stabilizer.
  • Primary feather follicles were collected for virus recovery from two vaccinated birds in each group, collecting two to three feather samples per bird. The two birds per group for feather follicle sampling were selected according to a randomization schedule and were kept alive after sampling. All samples were taken to Merial Select's analytical department for processing. The same sampling procedure was repeated two times at seven day intervals.
  • Group one MDV SR-1 RN1250 X+5 arithmetic mean titer (AMT) was 94,160 pfu/0.2 ml dose.
  • Group two MDV SR-1 Rispens was 51,800 pfu/0.2 ml dose. All tracheal and cloacal swabs were negative for virus recovery.
  • Group 1 MDV SR-1 RN1250 All of the samples tested for this group were negative for virus recovery from feather follicles.
  • Two hundred eighty (280) one-day-old SPF chicks were randomized into eight different groups and 24 different isolation units according to a randomization schedule (11-12 birds per unit; 35 birds per treatment). After the randomization, the birds in Groups 6 and 7, the sham-vaccinated, challenged and sham-vaccinated/sham challenged negative controls, were sham vaccinated with Marek's vaccine diluent and were placed into their designated units according to the randomization schedule.
  • the remaining birds were then vaccinated with MDV SR-1 RN1250, at either 287, 578, 736, 1085, or 1392 plaque forming units (pfu's) per bird dose, or with MDV SR-3 HVT, 1728 pfu's per bird dose.
  • the birds were vaccinated subcutaneously (SQ) with 0.2 ml per chick. After the vaccinations, each vaccinated group was placed into its designated unit according to the randomization schedule.
  • the birds in Group 7 were sham challenged with Marek's vaccine diluent and Groups 1-6 and 8 were challenged with vvMDV RB1B, by the intraperitoneal (IP) route, 0.2 ml per bird.
  • IP intraperitoneal
  • the birds were observed daily for 45 days post-challenge for any unfavorable reactions to the challenge, particularly death or depression.
  • the birds were terminated and necropsied to examine for gross lesions associated with Marek's disease.
  • the prevented fraction rates against the vvMDV RB1B challenge in Groups 1-5 had a range of 0.84 to 0.94.
  • the prevented fraction in Group 8 the HVT vaccinated group, was 0.73.
  • the incidence of MDV in the sham vaccinated, challenge control Group 6 was 91.2%.
  • the MDV vaccine, Serotype-1, Live Virus, RN1250 Experimental Vaccine (X+5) administered subcutaneously (SQ) to day-old SPF chickens was efficacious at 287 plaque forming units per dose using the RB1B virus as the challenge.

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