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WO2018209167A1 - Compositions vaccinales contre le virus de la nécrose pancréatique infectieuse (ipnv) - Google Patents

Compositions vaccinales contre le virus de la nécrose pancréatique infectieuse (ipnv) Download PDF

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Publication number
WO2018209167A1
WO2018209167A1 PCT/US2018/032193 US2018032193W WO2018209167A1 WO 2018209167 A1 WO2018209167 A1 WO 2018209167A1 US 2018032193 W US2018032193 W US 2018032193W WO 2018209167 A1 WO2018209167 A1 WO 2018209167A1
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ibdv
vlp
monomers
protein
pvp2
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Daral John Jackwood
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Ohio State Innovation Foundation
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Ohio State Innovation Foundation
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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
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
    • 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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/10011—Birnaviridae
    • C12N2720/10022—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/10011—Birnaviridae
    • C12N2720/10023—Virus like particles [VLP]
    • 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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/10011—Birnaviridae
    • C12N2720/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Infectious bursal disease also known as IBD, Gumboro Disease, Infectious Bursitis and Infectious Avian Nephrosis
  • IBDV infectious bursal disease virus
  • IBDV infectious bursal disease virus
  • IBDV is a double stranded RNA virus that has a bi-segmented genome and belongs to the genus Avibirnavirus of family Bimaviridae. There are two distinct serotypes of the virus, but only serotype 1 viruses cause disease in poultry. At least six antigenic subtypes of IBDV serotype 1 have been identified by in vitro cross-neutralization assay. Viruses belonging to one of these antigenic subtypes are commonly known as variants, which were reported to break through high levels of maternal antibodies in commercial flocks, and cause immune suppression.
  • the IBDV genome consists of two segments, A and B, which are enclosed within a nonenveloped icosahedral capsid.
  • the genome segment B (2.9 kb) encodes VP1, the putative viral RNA polymerase.
  • the larger segment A (3.2 kb) encodes viral proteins VP2, VP3, VP4, and VP5.
  • VP2 protein contains important neutralizing antigenic sites and elicits a protective immune response and most of the amino acid (AA) changes between antigenically different IBDVs are clustered in the hypervariable region of VP2.
  • this hypervariable region of VP2 has been the target for the molecular techniques applied for IBDV detection and strain variation studies.
  • the virus-encoded RNA-dependent RNA polymerase, VP1 is incorporated into the capsid through its association with VP3.
  • VP3 also interacts extensively with the viral dsRNA genome.
  • the poultry vaccine industry currently makes inactivated IBDV vaccines for administration to breeder chickens.
  • Vaccinating parent breeder flocks produces maternal immunity in the chicks and protects them during the first few weeks of life from infectious bursal disease (IBD).
  • IBD infectious bursal disease
  • the vaccines for IBD are prepared in young chicks rather than eggs or cell culture because the quality and quantity of the antigen is considered to be superior. This is an expensive and time consuming process.
  • animal use issues have increased the risk of losing this source of high quality IBDV antigens. Using antigens produced in eggs or cell culture could reduce the potency and efficacy of these vaccines and thus increase IBD related morbidity, mortality and the cost of poultry meat and egg production.
  • Figure 1 shows IBDV pVP2 and VP3 clones excised from pGEM-T Easy vector using EcoRI.
  • Lane M contains a molecular DNA ladder
  • lanes 1-5 contain pVP2 clones
  • lanes 6-9 contain VP3 clones
  • lane 10 contains a negative (no insert) control
  • lane 11 contains an un-cut negative control.
  • Figure 2 shows purified pVP2 and VP3 inserts from the pGEM-T Easy vector.
  • Figure 3 A shows pVP2 clones excised from pVL1393 using EcoRI.
  • Figure 3B VP3 clones excised from pVL1393 using EcoRI.
  • Figure 4 shows virus-like particles (VLPs) prepared in Sf9 cell cultures infected with recombinant Baculoviruses.
  • the pVP2 from the variant USA08MD34p or classic Mol95 IBDV strains were co-expressed with USA08MD34p-VP3.
  • the mosaic VLPs contained pVP2 from the variant and classic viruses.
  • the horizontal bar on the bottom right of each electron micrograph represents 200nm.
  • FIG. 5 shows virus-like particles (VLPs) for infectious pancreatic necrosis virus (IPNV) prepared in Sf9 cell cultures infected with recombinant baculoviruses.
  • the pVP2 from Genogroup 1 strains (either IPNV2 or IPNV10) were co-expressed with IPNV10 VP3 to give the VLPs.
  • the horizontal bar on the bottom right of each electron micrograph represents 200nm.
  • composition can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
  • phrase "optionally the composition can comprise a combination" means that the composition may comprise a combination of different molecules or may not include a combination such that the description includes both the combination and the absence of the combination (i.e., individual members of the combination).
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • data is provided in a number of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15.
  • probe By “probe,” “primer,” or oligonucleotide is meant a single-stranded DNA or RNA molecule of defined sequence that can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the "target”).
  • target a complementary sequence
  • the stability of the resulting hybrid depends upon the extent of the base-pairing that occurs.
  • the extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the degree of stringency of the hybridization conditions.
  • the degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art.
  • Probes or primers specific for a nucleic acid have at least 80%-90% sequence complementarity, preferably at least 91%-95% sequence complementarity, more preferably at least 96%-99% sequence complementarity, and most preferably 100% sequence complementarity to the region of the nucleic acid to which they hybridize.
  • Probes, primers, and oligonucleotides may be detectably-labeled, either radioactively, or non-radioactively, by methods well-known to those skilled in the art. Probes, primers, and oligonucleotides are used for methods involving nucleic acid hybridization, such as: nucleic acid sequencing, reverse transcription and/or nucleic acid amplification by the polymerase chain reaction, single stranded
  • SSCP conformational polymorphism
  • RFLP restriction fragment polymorphism
  • Southern hybridization Southern hybridization
  • Northern hybridization in situ hybridization
  • electrophoretic mobility shift assay ESA
  • a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid (for example, a c-met nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.
  • high stringency conditions conditions that allow hybridization comparable with that resulting from the use of a DNA probe of at least 40 nucleotides in length, in a buffer containing 0.5 M NaHP04, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65°C, or a buffer containing 48% formamide, 4.8X SSC, 0.2 M Tris-Cl, pH 7.6, IX Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42°C.
  • compositions Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular VP2, VP3, or virus like particle (VLP) is disclosed and discussed and a number of modifications that can be made to a number of molecules including the VP2, VP3, or VLP are discussed, specifically contemplated is each and every combination and permutation of cancer gene or cooperation response gene and the modifications that are possible unless specifically indicated to the contrary.
  • VLP virus like particle
  • the genome of IBDV consists of two segments of double-stranded RNA (Dobos 1979).
  • the smaller genome segment encodes the RNA-dependent RNA polymerase, VP1 (von Amsterdamm 2004).
  • the larger genome segment encodes a polyprotein that is self-cleaved by the viral encoded protease VP4 (Birghan 2000) to yield pVP2, VP3 and VP4 (Kibenge 1988).
  • the pVP2 protein is further cleaved multiple times at the COOH terminus to yield the mature capsid protein VP2 (Galloux 2007). Trimers of the VP2 protein form a structure containing a base (B), shell (S) and projection (P) domain (Coulibaly 2010).
  • the surface of the IBDV capsid is a single protein layer made up of 260 VP2 trimers and beneath this layer are at least 200 trimers of the VP3 protein (Coulibaly 2005).
  • capsid formation may be initiated by a VP1- VP3 complex which interacts with VP2 trimers (Caston 2001; Moraver 2003; Lombardo 1999).
  • VP1- VP3 complex which interacts with VP2 trimers
  • the molecular co-expression of pVP2 and VP3 also produced the correct capsid structure (Martinez 2000).
  • the COOH terminal domain of pVP2 was essential for the assembly of the proteins into these virus-like particles (VLPs) (Ona 2004)
  • nucleotide sequences that encode the pVP2 proteins from a variant IBDV strain designated USA08MD34p and a classic IBDV strain designated Mol95 were produced using RT-PCR and cloned into a pGEM-T Easy vector.
  • a nucleotide sequence that encodes the VP3 protein was also produced from the USA08MD34p viral genome using RT-PCR and cloned into a pGEM-T Easy vector.
  • the VP3 and pVP2 clones were inserted into the pVL1393 Baculovirus transfer vector and sequenced to confirm their orientation to the promoter and to insure they contained uninterrupted open-reading-frames.
  • Recombinant Baculoviruses were constructed by transfection in Sf9 cells. Three recombinant Baculoviruses were produced and contained the USA08MD34p-VP3, USA08MD34p-pVP2 or Mol95-pVP2 genomic sequences.
  • VLPs Virus-like particles
  • the USA08MD34p, Mol95 and mosaic VLPs were used to vaccinate chickens. They induced an IBDV specific antibody response that was detected by ELISA and virus- neutralizing antibodies were detected in vitro.
  • VLPs Chickens vaccinated with the mosaic VLPs were protected from a virulent variant IBDV strain (VI) and a virulent classic IBDV strain (STC). The results indicate the mosaic VLPs maintained the antigenic integrity of the variant and classic viruses and have the potential to serve as a multivalent vaccine for use in breeder flocks.
  • VP2 of IBDV and “VP3 of IBDV'is meant the full-length or substantially full-length IBDV viral protein, a fragment thereof, a fusion, or a viral protein with internal deletions.
  • VP2 and VP3 can have the ability to form VLPs under conditions that favor VLP formation.
  • VP2 can include pVP2/VPX, and VP2 wherein a portion of the C-terminal domain sufficient to form VLPs exists.
  • the polyvalent VP2 can be a trimer comprised of three VP2 monomers, or trimer forming fragments thereof.
  • the term "polyvalent” refers to the VP2 trimer which is comprised of at least one VP2 monomer that is antigenically distinct from at least one other VP2 monomer in the VP2 trimer.
  • an "antigenically distinct” is meant that the monomers individually or as part of a trimer elicits a humoral/antibody response such that the monomers or trimers can be distinguished from other monomers or trimmers suing antibodies.
  • An "antigen” or “antigenic” refers to a molecule containing one or more epitopes (either linear, conformational or both) that will stimulate a host's immune-system to make a humoral and/or cellular antigen-specific response.
  • Immunogen refers to a molecule that is able to provoke a humoral and/or cell- mediated immune response.
  • a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids.
  • a T-cell epitope, such as a CTL epitope will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids.
  • an epitope will include between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids.
  • Immunogens include, but is not limited to, polypeptides which include
  • Such modifications may be deliberate, as through site- directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens.
  • An "immunological response” or “immune response” to an antigen, immunogen or composition is the development in a subject of a humoral and/or a cellular immune response to an antigen present in the composition of interest.
  • an "immune response” refers to any inflammatory, humoral, or cell-mediated response that occurs for the purpose of eliminating an antigen. Such responses can include, but are not limited to, antibody production, cytokine secretion,
  • the immune response is an antibody response.
  • a “humoral immune response” refers to an immune response mediated by antibody molecules, whereas a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells.
  • a “cellular immune response” can also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.
  • an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and/or the activation of T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest.
  • responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host.
  • ADCC antibody dependent cell cytotoxicity
  • Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.
  • an "immunogenic composition” refers to a composition that comprises an antigenic molecule where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigenic molecule of interest.
  • the VP2 monomers, or trimer forming fragments thereof can be selected from known IBDV strains, for example as provided herein, including natural segment A and B reassortments, variant strains, classic strains, very virulent strains, or from future discovered strains of IBDV.
  • vvIBDV include but are not limited to UK661 (AJ878898), rA (GQ221682) and rB (GQ221683).
  • variant viruses include but are not limited to Del-E (AJ878905), Del- A (Y1459), GLS (AJ878906), VI (AF281235), GER (AF281228) and USA02CA viruses (HQ441143-HQ441158).
  • classic viruses include but are not limited to D78
  • reassorted viruses include but are not limited to IBDV strain 02015.1 (GenBank accession numbers AJ879932 and AJ88090), CA-D495 (HQ441142), CA- K785 (HQ441143), and GX (AJ878907).
  • strains that could be used as the VP2 of IBDV include, but are not limited to UPM97/61 (AF247006), UPM94/273 (AF527039), OKYM (D49706), UK661 (X92760), IBDKS (L42284), D6948 (AF240686), BD3/99
  • VP2s comprising variant and/or classic and/or very virulent monomers.
  • the VP2 monomer can comprise the identical amino acid sequences of a naturally occurring IBDV strain, modified amino acids not occurring naturally, fusions, or antigenic fragments thereof.
  • Amino acid and nucleic acid sequences of VP2 monomers can be one or more of the sequences identified herein as SEQ ID Nos. herein for VP2 and/or one or more of the sequences described in Genbank, such as Accession Number AAV68391.
  • the polyvalent VP2 can comprise a VP2 monomer of IBDV variant strain USA08MD34p, or a VP2 monomer of IBDV classic strain Mol95.
  • the polyvalent VP2 can comprise a VP2 monomer of IBDV variant strain USA08MD34p, or a VP2 monomer of IBDV classic strain Mol95 or a VP2 monomer from an IBDV strain which is not IBDV variant strain USA08MD34p and is not IBDV classic strain Mol95.
  • the polyvalent VP2 can also comprise two VP2 monomers from IBDV variant strain USA08MD34p and one VP2 monomer from IBDV classic strain Mol95.
  • the polyvalent VP2 can comprise one VP2 monomer from IBDV variant strain USA08MD34p and two VP2 monomers from IBDV classic strain Mol95.
  • the polyvalent VP2 can also comprise two VP2 monomers of one IBDV and one an antigenically distinct IBDV monomer.
  • the polyvalent VP2 of IBDV can have the following formula: R1-R2-R3, wherein R1-R2-R3 are each VP2 monomers selected from the group consisting of IBDV strain USA08MD34p, classic strain Mol95, UPM97/61 (AF247006), UPM94/273 (AF527039), OKYM (D49706), UK661 (X92760), IBDKS (L42284), D6948 (AF240686), BD3/99 (AF362776), Tasik94 (AF322444), Chinju (AF508176), HK46
  • AF092943 SH95 (AF13474), Gx (AY 444873), SDH1 (AY323952) and T09 (AY099456), D78 (AF499929), Cu-IM (AF362771), P2 (X84034), CT (AJ310185), CEF94 (AF194428), PBG-98 (D00868), JD1 (AF321055), HZ-2 (AF321054), TAD Gumboro, Delvax, Gumboro LZD, IBDVAC, 89163, Farager 52/70 and Edgar (AF462026); vvIBDV including but not limited to UK661 (AJ878898), rA (GQ221682) and rB (GQ221683); variant viruses including but not limited to Del-E (AJ878905), Del-A (Y1459), GLS (AJ878906), VI (AF281235), GER
  • Rl, R2 and R3 are antigenically distinct monomer from one or both of the other two monomers.
  • At least one of Rl, R2 and R3 is a VP2 monomer from a different strain of IBDV than the other monomers.
  • polyvalent VP2s comprising Rl, R2 and R3 comprised of variant and/or classic and/or very virulent strain monomers.
  • at least one of Rl, R2 and R3 is a VP2 monomer of variant strain USA08MD34p and is an antigenically distinct monomer from at least one other monomer.
  • at least one of Rl, R2 and R3 is a VP2 monomer of IBDV classic strain Mol95 and is an antigenically distinct monomer from at least one other monomer.
  • at least one monomer is antigenically distinct from both other monomers.
  • VLPs Virus-Like Particles
  • VLPs mosaic virus like particles
  • aVP2 as disclosed herein.
  • virus-like particle or “VLP” refer to a
  • VLPs are generally composed of one or more viral proteins, such as, but not limited to VP2s in the combinations disclosed herein, for example a combination of VP2 and VP3s. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. VLPs, when administered to an animal, can be immunogenic and thus can cause a protective or therapeutic immune response in the animal. Methods for producing VLPs are generally known in the art and discussed more fully below. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. See, e.g., Baker et al., Biophys. J.
  • VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density banding.
  • cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions.
  • mosaic virus like particles By “mosaic” is meant that the VLP comprises at least one VP2 trimer that is antigenicly distinct from at least one other VP2 trimer in the VLP. thus, for example disclosed herein are mosaic VLPs comprising a VP2 trimer from a different strain of IBDV than at least one VP2 trimers.
  • the mosaic VLP can generate a polyvalent immune response.
  • the VLP may can contain a multivalent VP2 and/or a monovalent VP2.
  • a “monovalent VP2” means a VP2 trimer comprised of the same or substantially the same VP2 monomer.
  • the mosaic VLP can comprise two or rmore monovalent VP2 trimers and/or one or more polyvalent VP2 trimers.
  • Mosaic VLPs comprising mixtures of monovalent and polyvalent VP2 trimers or exclusively monovalent or exclusively polyvalent VLP trimers are also disclosed herein.
  • antigenically distinct trimers can be derived from or represented by different strains of IBDV, for example as provided herein.
  • the VP2 trimers, or antigenic fragments thereof, whether polyvalent or monovalent can be selected from known IBDV strains, such as those disclosed herein, or from additional strains of IBDV.
  • the VP2 trimer can be selected for example from variant, classic, and very virulent strains.
  • mosaic VLPs comprised of VP2s from variant and/or classic and/or very virulent monomers.
  • the VLP VP2 trimer can be polyvalent comprising two or more antigenically distinct or variant monomers and/or monomers having the identical amino acid sequences of a naturally occurring IBDV strain (i.e., the VLP can simultaneously comprise polyvalent and monovalent trimmers), can comprise modified amino acids not occurring naturally, can comprise fusions, or can comprise antigenic fragments thereof.
  • the amino acid sequence of VP2 monomers making up the VP2 trimers in the mosaic VLP can be one or more of the sequences identified herein as SEQ ID Nos. and/or one or more of the sequences described, for example, in Genbank Accession No. CAI47764.
  • a polyvalent or monovalent VP2 trimer in the VLP can comprise a VP2 monomer of IBDV variant strain USA08MD34p, and/or a VP2 monomer of IBDV classic strain Mol95.
  • the polyvalent or monovalent VP2 can comprise a VP2 monomer of IBDV variant strain USA08MD34p, a VP2 monomer of IBDV classic strain Mol95 and a VP2 monomer from an IBDV strain which is not IBDV variant strain USA08MD34p and is not IBDV classic strain Mo 195.
  • Other strains that can be used as the VP2 of IBDV include, but are not limited to UPM97/61 (AF247006), UPM94/273 (AF527039), OKYM (D49706), UK661
  • the VP2 monomers that make up the monovalent or polyvalent VP2 trimers, or fragments thereof, can be selected from known IBDV strains, for example as provided herein, or from future discovered strains of IBDV. Any combination of monovalent or polyvalent VP2s can be utilized to form the VLP.
  • examples of the polyvalent VP2 trimers can, for example, comprise two VP2 monomers from IBDV variant strain USA08MD34p and one VP2 monomer from IBDV classic strain Mol95.
  • the polyvalent VP2 trimer can comprise one VP2 monomer IBDV variant strain USA08MD34p and two VP2 monomers from IBDV classic strain Mol95.
  • the polyvalent VP2 trimer can, for example, comprise two VP2 monomers of one IBDV monomer and one IBDV from an antigenically distinct monomer.
  • the VLP can further comprise a VP3 or a fragment thereof, or any other protein or polypeptide allowing the assembly of antigenic or immunogenic VP2 trimers as a VLP of the invention.
  • the VLP can also comprise other IBDV proteins such a VPl . Proteins from viruses having similar functionality to VP3 can also comprise the VLPs of this invention.
  • the antibodies provided herein are capable of neutralizing IBDV of other closely related species to IBDV.
  • the provided antibodies can be delivered directly, such as through needle injection, for example, to treat IBDV infections.
  • the provided antibodies can be delivered non-invasively, such as intranasally, for example.
  • the antibodies can also be encapsulated, for example into lipsomes, microspheres, or other transfection enhancement agents, for improved delivery into the cells to maximize the treatment efficiency.
  • the gene sequences encoding the provided antibodies, or their fragments such as Fab fragments can further be cloned into genetic vectors, such as plasmid or viral vectors, for example, and delivered into the hosts for
  • antibody encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class. Native antibodies are usually
  • heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains.
  • each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains.
  • Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • the light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (1), based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes.
  • variable is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR).
  • CDRs complementarity determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a b-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the b-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E. A. et al., "Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1987)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as Fv, sFv, F(ab')2, Fab', Fab and the like, including hybrid fragments.
  • fragments of the antibodies that retain the ability to bind their specific antigens are provided.
  • fragments of antibodies which maintain EFn binding activity are included within the meaning of the term "antibody or fragment thereof.”
  • Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity using general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
  • antibody or fragments thereof conjugates of antibody fragments and antigen binding proteins (single chain antibodies) as described, for example, in U.S. Pat. No. 4,704,692, the contents of which are hereby incorporated by reference.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity.
  • the disclosed monoclonal antibodies can be made using any procedure which produces monoclonal antibodies.
  • disclosed monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain.
  • Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.
  • the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen.
  • Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment.
  • antibody can also refer to a human antibody and/or a humanized antibody.
  • Many non-human antibodies e.g., those derived from mice, rats, or rabbits
  • are naturally antigenic in humans and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
  • human antibodies can be prepared using any technique. Examples of techniques for human monoclonal antibody production include those described by Cole et al. Human antibodies (and fragments thereof) can also be produced using phage display libraries.
  • the disclosed human antibodies can also be obtained from transgenic animals.
  • transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization.
  • the homozygous deletion of the antibody heavy chain joining region (J(H)) gene in these chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production, and the successful transfer of the human germ-line antibody gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge.
  • Antibodies having the desired activity are selected using Env- CD4-co-receptor complexes as described herein.
  • Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.
  • a humanized form of a non-human antibody is a chimeric antibody or antibody chain (or a fragment thereof, such as an Fv, Fab, Fab', or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.
  • CDRs complementarity determining regions
  • donor non-human antibody molecule
  • desired antigen binding characteristics e.g., a certain level of specificity and affinity for the target antigen.
  • Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues.
  • Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • humanized antibody has one or more amino acid residues introduced into it from a source which is non-human.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody
  • antigens expressed in baculovirus are disclosed herein.
  • the advantages to this system include ease of generation, high levels of expression, and post-translational modifications that are highly similar to those seen in mammalian systems.
  • the antigen is produced by inserting a gene fragment in-frame between the signal sequence and the mature protein domain of a nucleotide sequence.
  • nucleic acid refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing.
  • Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non- phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages).
  • nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof
  • nucleic acids such as SEQ ID NOS 1-24, as described herein, can be made using standard chemical synthesis methods or can be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see for example, Sambrook et al., Molecular Cloning: A
  • Protein nucleic acid molecules can be made using known methods such as those described by Nielsen et al., Bioconjug. Chem. 5:3-7 (1994).
  • compositions disclosed herein can be produced as described herein and can be prepared using standard recombinant techniques.
  • Polynucleotides encoding the VLP-forming protein(s) are introduced into a host cell and, when the proteins are expressed in the cell, they can assemble into the VP2s or VLPs.
  • incorporated into the VP2s and VLPs can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells expressing the gene, or by deriving the gene from a vector known to include the same.
  • plasmids which contain sequences that encode naturally occurring or altered cellular products may be obtained from a depository such as the A.T.C.C., or from commercial sources.
  • Plasmids containing the nucleotide sequences of interest can be digested with appropriate restriction enzymes, and DNA fragments containing the nucleotide sequences can be inserted into a gene transfer vector using standard molecular biology techniques.
  • cDNA sequences may be obtained from cells which express or contain the sequences, using standard techniques, such as phenol extraction and PCR of cDNA or genomic DNA. See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA. Briefly, mRNA from a cell which expresses the gene of interest can be reverse transcribed with reverse transcriptase using oligo-dT or random primers. The single stranded cDNA may then be amplified by PCR (see U.S. Pat. Nos.
  • the nucleotide sequence of interest can also be produced synthetically, rather than cloned, using a DNA synthesizer (e.g., an Applied Biosystems Model 392 DNA Synthesizer, available from ABI, Foster City, Calif).
  • the nucleotide sequence can be designed with the appropriate codons for the expression product desired.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223 : 1299; Jay et al. (1984) J. Biol. Chem. 259:6311.
  • any of the proteins used in the VP2s and VLPs described herein may be hybrid (or chimeric) proteins, referred to herein as mosaics. It will be apparent that all or parts of the polypeptides can be replaced with sequences from other viruses and/or sequences from other IBDV strains so long as the sequence does not prevent the formation of VP2 trimers and/or VLPs.
  • sequences employed to form the VP2s and VLPs disclosed herein can exhibit between about 60% to 80%> (or any value therebetween including 61%>, 62%>, 63%>, 64%>, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% and 79%) sequence identity to a naturally occurring IBDV polynucleotide sequence and more preferably the sequences exhibit between about 80%> and 100%> (or any value therebetween including 81%>, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%) sequence identity to a naturally occurring polynucleotide sequence.
  • any of the sequences described herein may further include additional sequences.
  • hybrid molecules are expressed and incorporated into the sub-viral structure. These hybrid molecules are generated by linking for example, at the DNA level, the sequences coding for the VP2s or VP3s with sequences coding for an adjuvant or immuno-regulatory moiety.
  • polypeptide immunomodulatory polypeptides e.g., adjuvants described herein
  • one or more additional molecules may be included in the VP2s and VLPs after production of the composition from the sequences described herein.
  • sequences may be expressed from the same promoter and/or from different promoters. As described below, sequences may be included on one or more vectors.
  • constructs comprising the sequences encoding the polypeptide(s) desired to be incorporated into the VP2s and VLPs have been synthesized, they can be cloned into any suitable vector or replicon for expression.
  • Numerous cloning vectors are known to those of skill in the art, and one having ordinary skill in the art can readily select appropriate vectors and control elements for any given host cell type in view of the teachings of the present specification and information known in the art about expression. See, generally, Ausubel et al, supra or Sambrook et al, supra. 70.
  • Non-limiting examples of vectors that can be used to express sequences that assembly into VP2s and VLPs as described herein include viral -based vectors (e.g., retrovirus, adenovirus, adeno-associated virus, lentivirus), baculovirus vectors (see Examples), plasmid vectors, non-viral vectors, mammalians vectors, mammalian artificial chromosomes (e.g., liposomes, particulate carriers, etc.) and combinations thereof.
  • viral -based vectors e.g., retrovirus, adenovirus, adeno-associated virus, lentivirus
  • baculovirus vectors see Examples
  • plasmid vectors e.g., non-viral vectors
  • mammalians vectors e.g., mammalians vectors
  • mammalian artificial chromosomes e.g., liposomes, particulate carriers, etc.
  • the expression vector(s) typically contain(s) coding sequences and expression control elements which allow expression of the coding regions in a suitable host.
  • the control elements generally include a promoter, translation initiation codon, and translation and transcription termination sequences, and an insertion site for introducing the insert into the vector.
  • one or more vectors may contain one or more sequences encoding proteins to be incorporated into the VP2s and VLPs.
  • a single vector may carry sequences encoding all the proteins found in the composition.
  • multiple vectors may be used (e.g., multiple constructs, each encoding a single polypeptide-encoding sequence or multiple constructs, each encoding one or more polypeptide-encoding sequences).
  • the sequences may be operably linked to the same or different transcriptional control elements (e.g., promoters) within the same vector.
  • vectors may contain additional gene expression controlling sequences including chromatin opening elements which prevent transgene silencing and confer consistent, stable and high level of gene expression, irrespective of the chromosomal integration site.
  • chromatin opening elements located in proximity of house-keeping genes, which in the vectors create a transcriptionally active open chromatin environment around the integrated transgene, maximizing transcription and protein expression, irrespective of the position of the transgene in the chromosome.
  • sequences encoding non-IBDV proteins may be expressed and incorporated into the VP2s and VLPs, including, but not limited to, sequences comprising and/or encoding immunomodulatory molecules (e.g., adjuvants described below), for example, immunomodulating oligonucleotides (e.g., CpGs), cytokines, detoxified bacterial toxins and the like.
  • immunomodulatory molecules e.g., adjuvants described below
  • immunomodulating oligonucleotides e.g., CpGs
  • cytokines detoxified bacterial toxins and the like.
  • particle-forming polypeptide derived from a particular viral protein is meant a full-length viral protein or a fragment thereof, a fusion of a viral protein, or a viral protein with internal deletions, which has the ability to form VLPs under conditions that favor VLP formation. Accordingly, the polypeptide may comprise the full-length sequence, fusions, fragments, truncated and partial sequences, as well as analogs and precursor forms of the reference molecule. Examples are disclosed herein, but can include VP2 proteins alone, or in combination with VP3 proteins, thereby forming VLPs.
  • a "particle-forming polypeptide” derived from VP2 of IBDV includes, but is notlimited to, full-length or near full-length viral protein, a fragment thereof, a fusion, or a viral protein with internal deletions, which has the ability to form VLPs under conditions that favor VLP formation.
  • “Particle-forming polypeptide” also includes, but is not limited to, deletions, additions and substitutions to the sequence, so long as the polypeptide retains the ability to form a VLP.
  • Particle-forming polypeptide also includes, but is not limited to, natural variations of the specified polypeptide since variations in coat proteins often occur between viral isolates as well as deletions, additions and substitutions that do not naturally occur in the reference protein, so long as the protein retains the ability to form a VLP.
  • substitutions are those which are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic— lysine, arginine, histidine; (3) non-polar— alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar— glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • polypeptide refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein.
  • a peptide can be a receptor.
  • a polypeptide is comprised of consecutive amino acids.
  • polypeptide encompasses naturally occurring or synthetic molecules.
  • peptide or “polypeptide” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications.
  • Modifications include, without limitation, acetylation, acylation, ADP- ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a
  • phosphytidylinositol disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer- RNA mediated addition of amino acids to protein such as arginylation.
  • amino acid sequence refers to a list of abbreviations, letters, characters or words representing amino acid residues.
  • Polyvalent compositions produced as described herein can be used to elicit an immune response when administered to a subject.
  • the compositions can comprise a variety of antigens (e.g., one or more modified IBVD antigens from one or more strains or isolates).
  • Purified polyvalent compositions can be administered to a vertebrate subject, usually in the form of vaccine compositions.
  • Combination vaccines may also be used, where such vaccines contain, for example, other subunit proteins derived from IBDV or other organisms and/or gene delivery vaccines encoding such antigens.
  • disclosed herein are vaccines comprising the VP2s and/or VLPs disclosed herein. It is understood that the disclosed vaccines can be therapeutic or prophylactic. Thus, for example, disclosed herein are vaccines comprising VP2s or VLps comprising a polyvalent or monovalent VP2 trimer in the VLP. For example, disclosed herein are vaccines wherein the VLP can comprise one or more VP2 monomers of IBDV variant strain
  • the vaccine can comprise VP2s or VLPs comprising polyvalent or monovalent VP2 trimers wherein the trimer comprises at least one VP2 monomer of IBDV selected from the group consisting of variant strain USA08MD34p, a VP2 monomer of IBDV classic strain Mol95 and a VP2 monomer from an IBDV strain which is not IBDV variant strain USA08MD34p and is not IBDV classic strain Mol95.
  • the vaccine can comprise VP2s or VLPs comprising polyvalent or monovalent VP2 trimers wherein the trimer comprises at least one or more VP2 monomer of IBDV selected from the group consisting of USA08MD34p, Mol95, UPM97/61 (AF247006), UPM94/273 (AF527039), OKYM (D49706), UK661 (X92760), IBDKS (L42284), D6948 (AF240686), BD3/99 (AF362776), Tasik94 (AF322444), Chinju (AF508176), HK46 (AF092943), SH95 (AF13474), Gx (AY 444873), SDH1 (AY323952) and T09
  • the trimer comprises at least one or more VP2 monomer of IBDV selected from the group consisting of USA08MD34p, Mol95, UPM97/61 (AF247006), UPM94/273 (AF527039), OKYM (D49706), UK661 (
  • compositions comprising the polyvalent VP2s and/or VLPs described herein, along with a pharmaceutically acceptable carrier.
  • VLPs and VP2s produced as described herein can be used to elicit an immune response when administered to a subject.
  • Purified VLPs or VP2s can be administered to a subject, usually in the form of vaccine compositions.
  • VP2/VLP immune-stimulating (or vaccine) compositions can include various excipients, adjuvants, carriers, auxiliary substances, modulating agents, and the like.
  • the immune stimulating compositions can include an amount of the VP2/antigen or VLP/antigen sufficient to mount an immunological response. An appropriate effective amount can be determined by one of skill in the art.
  • an effective amount of a compound as provided herein is meant a sufficient amount of the compound to provide the desired effect.
  • an effective amount of a compound can refer to a sufficient amount of the compound to generate an immune repsonse.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of disease (or underlying genetic defect) that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate "effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
  • an effective amount will fall in a relatively broad range that can be determined through routine trials and will generally be an amount on the order of about 0.1 ⁇ g to about 10 (or more) mg, more preferably about 1 ⁇ g to about 300 ⁇ g, even more preferably 25 ⁇ g to 50 ⁇ g of VP2/antigen or VLP/antigen.
  • Sub-viral structure vaccines are purified from the cell culture medium and formulated with the appropriate buffers and additives, such as a)
  • This vaccine can be prepared in a freeze-dried (lyophilized) form in order to provide for appropriate storage and maximize the shelf-life of the preparation. This will allow for stock piling of vaccine for prolonged periods of time maintaining immunogenicity, potency and efficacy.
  • compositions can also be administered in vivo in a
  • pharmaceutically acceptable carrier a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant.
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • the exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. 88.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced.
  • receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
  • compositions including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • compositions may also include one or more active ingredients such as
  • antimicrobial agents antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.. 97.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • adjuvants include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (International Publication No.
  • WO 90/14837 containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RibiTM adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS),
  • cytokines such as interleukins (IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), beta chemokines (MIP, 1 -alpha, 1-beta Rantes, etc.
  • cytokines such as interleukins (IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), beta chemokines (MIP, 1 -alpha, 1-beta Rantes, etc.
  • cytokines such as interleukins (IL-1, IL-2, etc.
  • M-CSF macrophage colony stimulating factor
  • TNF tumor necrosis factor
  • MIP beta chemokines
  • coli heat-labile toxin particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63)
  • LT-R72 where arginine is substituted for the wild-type amino acid at position 72
  • CT-S109 where serine is substituted for the wild-type amino acid at position 109
  • PT- K9/G129 where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129)
  • Muramyl peptides include, but are not limited to, N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP), N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(l '-2'-dipalmitoyl-s- n- glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
  • thr-MDP N-acetyl- muramyl-L-threonyl-D-isoglutamine
  • nor-MDP N-acteyl-normuramyl-L-alanyl-D-isogluatme
  • MTP-PE N-acetylmuramyl
  • immunomodulatory molecules for use herein include adjuvants described above and the following: IL-1 and IL-2 (Karupiah et al. (1990) J. Immunology 144:290- 298, Weber et al. (1987) J. Exp. Med. 166: 1716-1733, Gansbacher et al. (1990) J. Exp. Med. 172: 1217-1224, and U.S. Pat. No. 4,738,927-); IL-3 and IL-4 (Tepper et al. (1989) Cell 57:503- 512, Golumbek et al. (1991) Science 254:713-716, and U.S. Pat. No. 5,017,691); IL-5 and IL-6 (Brakenhof et al. (1987) J. Immunol. 139:4116-4121, and International Publication No. WO
  • IL-7 U.S. Pat. No. 4,965,195
  • IL-8 IL-9, IL-10, IL-11, IL-12, and IL-13
  • IL-14 IL-15
  • alpha interferon Inter et al. (1991) Drugs 42:749- 765, U.S. Pat. Nos. 4,892,743 and 4,966,843, International Publication No. WO 85/02862, Nagata et al. (1980) Nature 284:316-320, Familletti et al. (1981) Methods in Enz. 78:387-394, Twu et al. (1989) Proc. Natl. Acad. Sci.
  • Immunomodulatory factors can also be agonists, antagonists, or ligands for these molecules.
  • soluble forms of receptors can often behave as antagonists for these types of factors, as can mutated forms of the factors themselves.
  • the VP2s, VLPs and compositions comprising them can be administered to a subject by any mode of delivery, including, for example, by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral (e.g. tablet, spray), vaginal, topical, transdermal (e.g. see W099/27961) or
  • transcutaneous e.g. see WO02/074244 and WO02/064162
  • intranasal e.g. see WO03/028760
  • ocular, aural, pulmonary or other mucosal administration e.g. see WO03/028760
  • ocular, aural, pulmonary or other mucosal administration e.g. see WO03/028760
  • ocular, aural pulmonary or other mucosal administration.
  • pulmonary or other mucosal administration e.g. see WO03/028760
  • ocular, aural e.g. see WO03/028760
  • ocular, aural, pulmonary or other mucosal administration e.g. see WO03/028760
  • ocular, aural, pulmonary or other mucosal administration e.g. see WO03/028760
  • the VLPs can be administered prior to, concurrent with, or subsequent to delivery of other vaccines.
  • the site of VLP administration may be the same or different as other vaccine compositions that are being administered.
  • Dosage treatment with the VLP composition can be a single dose schedule or a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals, chosen to maintain and/or reinforce the immune response.
  • the dosage regimen will also, at least in part, be determined by the potency of the modality, the vaccine delivery employed, the need of the subject and be dependent on the judgment of the practitioner.
  • two doses are given, and are administered 1, 2, 3 ,4 ,5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more days apart.
  • the first two doses are given 7-10 days apart.
  • the doses can be 3-4 weeks apart.
  • the vaccines disclosed herein can provide antibody titers of 100, 200, 300, 400, 500,
  • the dose of VLP or VP2 administered can vary, depending on the age or condition of the subject. Typically, for a VLP, the dose is between 15-75 ⁇ g, and more specifically between 25- 50 ⁇ ⁇ .
  • kits comprising the one or more of the compositions, including, but not limited to, the polyvalent VP2s, VLPs, nucleic acids, antibodies, or cells disclosed herein.
  • Infectious pancreatic necrosis virus is the causative agent of infectious pancreatic necrosis disease (TPN) that infects salmonids and remains a serious problem in the aquaculture industry.
  • IPN infectious pancreatic necrosis disease
  • IPN is especially contagious and destructive to juvenile trout and salmon. Highly virulent strains may cause greater than 70% mortality in hatchery stocks over a period of two months. This disease is especially destructive in salmonid eggs and fingerlings.
  • Survivors of infection can remain lifelong asymptomatic carriers and serve as reservoirs of infection, shedding vims in their feces and reproductive products. Losses due to IPNV on salmon smoltification have been estimated at 5%. Economic losses due to IPNV in aquaculture were estimated to be over 560 million in 1996. This has been reduced as vaccines for salmonids became available based on killed vims or recombinantly produced viral peptides. However, these vaccines are not completely effective and can only be used in fairly large fish due to the
  • Polyvalent VP2s and mosaic VLPs, and compositions of the invention can also be from members of the aquabirnavirus genus of birnavirus family.
  • the species infectious pancreatic necrosis vims (IPNV) can be used to form polyvalent VP2s and mosaic VLPs as described herein and exemplified for IBDV.
  • IPNV IP-binding protein
  • production of VP2s and VLPs for IPNV can be accomplished using the techniques described herein for IBDV utilizing sequences, for example, of pVP2 (ACY35990) and VP3 (AAM90322) from IPNV and other aquabirnavimses.
  • the IPNV sequences can be substuted for the IBDV sequences set forth in the examples.
  • the IPNV pVP2 gene can be amplified using RT-PCR and specific primers for that gene.
  • the IPNV VP3 gene can also be amplified using RT-PCR and specific primers for its sequence.
  • the amplified genes can then be ligated into a Baculovims expression vector (pVL1392) and then used to transfect Baculovimses Recombinant Baculovimses containing either the pVP2 or VP3 genes from IPNV can be used to express their respective proteins.
  • Co-expression of the pVP2 and VP3 proteins in insect cells can be conducted to produce VLPs.
  • the VLPs can be administered to fish for prevention of the diseases associated with IPNV in fish. Examples of such administration can be found, for example, in US 2010/0092521 Al, herein incorporated by reference in its entirety.
  • the VLPs can also be used to detect the presence of IPNV in a sample, as set forth for IBDV herein.
  • aquatic animal includes any multi-cellular organism that lives in water, typically fish.
  • said aquatic animal is an animal belonging to a fish species reared by aquaculture.
  • Illustrative examples of said aquatic animals include teleost fish, such as vertebrate fish, e.g. salmonids (e.g., rainbow trout, salmon, etc.), carp, turbot, gilthead sea bream, sea bass, etc.
  • IPNV polyvalent VP2 and/or mosaic VLP, composition, or nucleic acid encoding the VP2/VLP.
  • Another aspect of the invention also relates to a vaccine comprising IPNV polyvalent VP2 and/mosaic VLPs.
  • the vaccine can be used to protect aquatic animals that can be infected by aquatic animal pathogens.
  • said pathogens are pathogens of aquatic animals reared in aquaculture installations.
  • the vaccine of the invention can be administered by any appropriate route of administration that results in an immune response to protect against the pathogen in question, for which the vaccine will be formulated in a manner that is suitable for the chosen route of administration.
  • the vaccine of the invention can be administered orally, intramuscularly, by particle bombardment or by spraying using conventional methods (U.S. Pat. No. 5,780,448) for the simultaneous immunisation of a large number of aquatic animals, another method is to submerge said aquatic animals in a solution comprising the vaccine of the invention.
  • the vaccine of the invention can be prepared in the form of an aqueous solution or suspension, in a pharmaceutically acceptable vehicle, such as saline solution, phosphate buffered saline (PBS), or any other pharmaceutically acceptable vehicle.
  • a pharmaceutically acceptable vehicle such as saline solution, phosphate buffered saline (PBS), or any other pharmaceutically acceptable vehicle.
  • the vaccine of the invention can be prepared using conventional methods known by a person skilled in the art.
  • said vaccine can be prepared using the mixture, if applicable, of a vector of the invention, optionally having one or more adjuvants and/or pharmaceutically acceptable vehicles.
  • Another aspect of the invention relates to a method for simultaneously administering a vaccine to a plurality of aquatic animals (mass vaccination) that comprises the immersion of a plurality of aquatic animals in a bath containing the vaccine and the sonication of the bath containing said aquatic animals and said vaccine.
  • Other methods of IPNV vaccine administration can be found, for example, in US 2007/0248623 Al, herein incorporated by reference in its entirety for its teaching concerning IPNV.
  • IPNV infectious pancreatic necrosis virus
  • the polyvalent VP2 trimer comprises three VP2 monomers, and wherein at least one of the VP2 monomers is from a different strain of IPNV than the other monomers.
  • IPNV infectious pancreatic necrosis virus
  • the VP2 monomers are each independently from an IPNV strain selected from the group consisting of IPNV2, IPNV10, Te (AF342731; England), CI (AF342732; Canada), Ab (AF342729; Denmark), He (AF342730; Germany), C2 (AF342733; Canada), C3 (AF342734; Canada), SP (AF342728; Denmark), YTAV (AY283781; Japan), WB (AF342727;USA), and Jasper (AF342735; Canada).
  • IPNV2 IPNV10
  • Te AF342731; England
  • CI AF342732
  • Ab AF342729
  • He AF342730
  • C2 AF342733; Canada
  • C3 AF342734; Canada
  • SP AF342728; Denmark
  • YTAV AY283781; Japan
  • WB AF342727;USA
  • Jasper AF342735
  • the at least one of the VP2 monomers is from IPNV2 and at least one of the VP2 monomers is from IPNVIO. In some embodiments, the at least one of the VP2 monomers is SEQ ID NO:26 and at least one of the VP2 monomers is SEQ ID NO:28.
  • VLP virus like particle
  • IPNV infectious pancreatic necrosis virus
  • IPNV infectious pancreatic necrosis virus
  • IPNV infectious pancreatic necrosis virus
  • IPNV infectious pancreatic necrosis virus
  • IPNV infectious pancreatic necrosis virus
  • the polyvalent VP2 trimer comprises three VP2 monomers, and wherein at least one of the VP2 monomers is from a different strain of IPNV than the other monomers.
  • the at least one of the VP2 monomers is from an IPNV strain selected from the group consisting of IPNV2, IPNVIO, Te (AF342731; England), CI (AF342732; Canada), Ab (AF342729; Denmark), He (AF342730; Germany), C2 (AF342733; Canada), C3 (AF342734; Canada), SP (AF342728; Denmark), YTAV (AY283781; Japan), WB
  • the at least one of the VP2 monomers is from IPNV2. In some embodiments, the at least one of the VP2 monomers is SEQ ID NO:26. In some embodiments, the at least one of the VP2 monomers is from IPNVIO. In some embodiments, the at least one of the VP2 monomers is SEQ ID NO:28. In some embodiments, the at least one of the VP2 monomers is from IPNV2 and at least one of the VP2 monomers is from IPNVIO. In some embodiments, the at least one of the VP2 monomers is SEQ ID NO:26 and at least one of the VP2 monomers is SEQ ID NO:28.
  • two VP2 monomers from IPNV2 (SEQ ID NO:26) and one VP2 monomer from IPNVIO (SEQ ID NO:28). In some embodiments, one VP2 monomer from IPNV2 (SEQ ID NO:26) and two VP2 monomers from IPNVIO (SEQ ID NO:28).
  • the IPNV2 and IPNVIO virus sequences are both in Genogroup 1, but their sequences are not identical.
  • Genogroup 1 contains both serotype Al and A9 strains.
  • the IPNV2 and IPNVIO sequences are most similar to the WB and Jasper strains.
  • the VP3 proteins are from IPNVIO. In some embodiments, the VP3 proteins are SEQ ID NO:30. In some embodiments, the VP3 proteins are from IPNV2. In some embodiments, the VP3 proteins are from more than one IPNV strain.
  • the VP3 proteins are from an IPNV strain selected from the group consisting of IPNV2, IPNVIO, Te (AF342731; England), CI (AF342732; Canada), Ab (AF342729; Denmark), He (AF342730; Germany), C2 (AF342733; Canada), C3 (AF342734; Canada), SP (AF342728; Denmark), YTAV (AY283781; Japan), WB (AF342727;USA), and Jasper (AF342735; Canada).
  • composition comprising a virus like particle (VLP) comprising VP3 proteins from at least one strain of infectious pancreatic necrosis virus (IPNV) and a polyvalent VP2 trimer of infectious pancreatic necrosis virus (IPNV), wherein the polyvalent VP2 trimer comprises three VP2 monomers, and wherein at least one of the VP2 monomers is from a different strain of IPNV than the other monomers, and a pharmaceutically acceptable carrier.
  • VLP virus like particle
  • IPNV infectious pancreatic necrosis virus
  • IPNV infectious pancreatic necrosis virus
  • IPNV infectious pancreatic necrosis virus
  • VLP virus like particle
  • IPNV infectious pancreatic necrosis virus
  • IPNV infectious pancreatic necrosis virus
  • IPNV infectious pancreatic necrosis virus
  • IPNV infectious pancreatic necrosis virus
  • the cell is an insect cell. In some embodiments, the insect cell is a Sf9 cell.
  • Disclosed herein is a method of eliciting an immune response against IPNV in a subject comprising administering to the subject a composition comprising a virus like particle
  • VLP comprising VP3 proteins from at least one strain of infectious pancreatic necrosis virus (IPNV) and a polyvalent VP2 trimer of infectious pancreatic necrosis virus (IPNV), wherein the polyvalent VP2 trimer comprises three VP2 monomers, and wherein at least one of the VP2 monomers is from a different strain of IPNV than the other monomers.
  • the subject is a fish.
  • the fish is a salmonid.
  • the fish is a salmon, trout (for example, rainbow trout), char, or other freshwater whitefish.
  • the fish is a carp, turbot, gilthead sea bream, sea bass, etc.
  • the method further comprising administering to the subject a virulent IPNV to monitor the immune response.
  • the composition is administered in a single dose. In some embodiments, the composition is administered in two doses.
  • Disclosed herein are methods of eliciting an immune response against IBDV in a subject comprising administering to the animal one or more VP2s or VLPs, or a composition or nucleic acid thereof.
  • the immune response can be considered to be efficacious if the desired result is obtained.
  • an "efficacious” immune response is a response that is able to afford the subject an acceptable degree of immune protection from the immunizing antigen.
  • the present methods disclose methods of assessing the ability of an immune response to provide immune protection against future antigenic encounter. Traditionally, such methods involve antigenic challenge. It is understood that the present methods provide an alternative means to achieve the goal of antigenic challenge and can be used separately or in conjunction with a challenge to determine efficacy or sufficiency.
  • a "sufficient or effective immune response” is used to describe an immune response of a large enough magnitude to provide an acceptable immune protection to the subject against future antigen encounter. It is understood that immune protection does not necessarily mean prevention of future antigenic encounter (e.g., infection), nor is it limited to a lack of any pathogenic symptoms. "Immune protection” means a prevention of the full onset of a pathogenic condition.
  • a "sufficient immune response” is a response that reduces the symptoms, magnitude, or duration of an infection or other disease condition when compared with an appropriate control.
  • the control can be a subject that is exposed to an antigen before or without a sufficient immune response.
  • protective against can mean that the subject is prevented from acquiring one or more symptoms associated with IBD virus infection, or from having any negative response when exposed to the virus, or is prevented from dying from the disease, or dying from the disease within a given time period, such as 1, 2, 3 ,4 ,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks, or 5, 6, 7, 8, 9, 10, 11, or 12 months, or any amount of time in between.
  • the survival rate of a group of subjects to which the molecules are administered can have a 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% improvement in mortality rate.
  • mortality rate is meant that the subject has an increased lifespan. For example, if 100 broilers are given a vaccine (as disclosed herein) which immunizes them against IBDV, and 95% of them survive past a given time, such as 3-4 weeks, then the vaccine is considered to impart a 95% survival rate.
  • the subject (such as a parental line, broiler or layer) can be administered a virulent IBDV to monitor the immune response.
  • a virulent IBDV can increase the lifespan of the subject by a given time period.
  • the virulent IBDV can also be administered to a subject that has not received the vaccine (a control), and both can be monitored to determine the effectiveness of the vaccine.
  • the immune response for both can be measured either by observation of the onset of symptoms of IBDV, or by monitoring the immune response of the subjects.
  • strains of IBDV there are multiple strains of IBDV, and any of these strains can be given to a subject who has been vaccinated, and/or a control, in order to monitor the effectiveness of the vaccine.
  • examples of such strains include, but are not limited to, the VI variant virus and STC classic virus strain.
  • compositions as disclosed herein such as one comprising a VP2s or
  • the subject can be an avian, such as a chicken.
  • reducing immunosuppression is meant that the amount of immunosuppression in a given subject after vaccination is less than that in an unvaccinated subject.
  • immunosuppression is meant suppression of the immune systems by IBDV.
  • the amount that immunosuppression is reduced in a given individual subject can be 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% when compared to a control.
  • reducing death in a subject comprising the steps of: providing a composition comprising a VP2s or VLPs, as described herein, and administering said composition to the subject.
  • reducing death is meant increasing survival rate of the subject as compared to a control.
  • treatment refers to the medical management of a subject with the intent to produce a therapy, cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • the treatment can be any reduction from native levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition.
  • the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • a subject such as an animal (for example, a chicken), that has a condition or disease, such as IBDV, an increased susceptibility for developing such a disease, in order to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects of the disease.
  • treat can also refer to non-pharmacological methods of preventing or delaying a worsening of the effects of the disease or condition, or to partially or fully reversing the effects of the disease.
  • “treat” is meant to mean a course of action to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects of the disease other than by administering a compound.
  • prevent is meant to minimize the chance that a subject who has a susceptibility for developing disease, such as IBDV induced disease, will develop a such a disease, or one or more symptoms associated with the disease.
  • subject is meant any member of the subphylum chordata, and in particular the aves class, although the methods and compositions disclosed herein are relevant to any animal that can for example contract IBDV (infectious bursal disease virus) or IPNV (infectious pancreatic necrosis virus).
  • members of the aves class include, but are not limited to, those found in the superorder palaeognathae:struthioniformes (ostriches, emus, kiwis, and allies), and tinamiformes (tinamous).
  • neognathae which includes anseriformes (waterfowl), galliformes (fowl, including chickens, ducks, geese, guinea, quail, grouse, pheasant and turkeys), charadriiformes (gulls, button-quails, plovers and allies), gaviiformes (loons), podicipediformes (grebes), procellariiformes (albatrosses, petrels, and allies), sphenisciformes (penguins), pelecaniformes (pelicans and allies), phaethontiformes (tropicbirds), ciconiiformes (storks and allies), cathartiformes (New World vultures), phoenicopteriformes (flamingos), falconiformes (falcons, eagles, hawks and allies
  • VLPs disclosed herein can be used as a diagnostic tool to detect, for example IBDV or IPNV antibodies in a sample.
  • diagnosis means having been subjected to an examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example,
  • diagnosis with IBDV means having been subjected to an examination by a person of skill, for example, a verterinarian, and found to have a condition that can be diagnosed or treated by one or more of the compositions described herein.
  • diagnostic tools include but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight
  • MALDI-TOF mass spectrometry
  • microcytometry microarray
  • microscopy microscopy
  • fluorescence activated cell sorting FACS
  • flow cytometry as well as assays based on a property of the protein including but not limited to enzymatic activity or interaction with other protein partners.
  • VLP antigens can react with serum antibodies to IBDV and produce a positive result in the ELISA.
  • Chicken serum samples from birds not exposed to IBDV were negative in the assay. The results indicate that VLP antigens can be used in the ELISA to detect antibodies to IBDV strains.
  • this invention provides a method of detecting or determining the presence of IBDV or IBDV in a sample comprising contacting a VLP of this invention with an antibody containing sample from a patient and detecting the precence and/or absence of binding between the VLP and the antibodies in the sample.
  • the immune response can be measured in a subject to assess the viability or usefulness of the composition, or to determine if multiple doses need to be administered.
  • Immunoassays that involve the detection of as substance, such as a protein or an antibody to a specific protein, include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection.
  • Label-free assays are generally diagnostic means of determining the presence or absence of a specific protein, or an antibody to a specific protein, in a sample.
  • Protein separation methods are additionally useful for evaluating physical properties of the protein, such as size or net charge.
  • Capture assays are generally more useful for quantitatively evaluating the concentration of a specific protein, or antibody to a specific protein, in a sample.
  • in vivo detection is useful for evaluating the spatial expression patterns of the substance, i.e., where the substance can be found in a subject, tissue or cell.
  • the molecular complexes ([Ab-Ag]n) generated by antibody-antigen interaction are visible to the naked eye, but smaller amounts may also be detected and measured due to their ability to scatter a beam of light.
  • the formation of complexes indicates that both reactants are present, and in immunoprecipitation assays a constant concentration of a reagent antibody is used to measure specific antigen ([Ab-Ag]n), and reagent antigens are used to detect specific antibody ([Ab-Ag]n). If the reagent species is previously coated onto cells (as in hemagglutination assay) or very small particles (as in latex agglutination assay), "clumping" of the coated particles is visible at much lower concentrations.
  • Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving.
  • electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique.
  • the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel.
  • the matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage.
  • the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels.
  • a porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than
  • polyacrylamide of the same concentration agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes.
  • Polyacrylamide which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation.
  • proteins are fractionated first on the basis of one physical property, and, in a second step, on the basis of another.
  • isoelectric focusing can be used for the first dimension, conveniently carried out in a tube gel, and SDS
  • electrophoresis in a slab gel can be used for the second dimension.
  • a procedure is that of O'Farrell, P.H., High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem. 250:4007-4021 (1975), herein incorporated by reference in its entirety for its teaching regarding two-dimensional electrophoresis methods.
  • Other examples include but are not limited to, those found in Anderson, L and Anderson, NG, High resolution two-dimensional electrophoresis of human plasma proteins, Proc. Natl. Acad. Sci. 74:5421-5425 (1977), Ornstein, L., Disc electrophoresis, L. Ann. N.Y. Acad. Sci. 121 :321349 (1964), each of which is herein incorporated by reference in its entirety for teachings regarding electrophoresis methods.
  • Laemmli U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227:680 (1970), which is herein incorporated by reference in its entirety for teachings regarding electrophoresis methods, discloses a discontinuous system for resolving proteins denatured with SDS.
  • the leading ion in the Laemmli buffer system is chloride, and the trailing ion is glycine.
  • the resolving gel and the stacking gel are made up in Tris-HCl buffers (of different concentration and pH), while the tank buffer is Tris-glycine. All buffers contain 0.1% SDS.
  • Western blot analysis allows the determination of the molecular mass of a protein and the measurement of relative amounts of the protein present in different samples. Detection methods include chemiluminescence and chromagenic detection. Standard methods for Western blot analysis can be found in, for example, D.M. Bollag et al., Protein Methods (2d edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Patent 4,452,901, each of which is herein incorporated by reference in their entirety for teachings regarding Western blot methods.
  • proteins are separated by gel electrophoresis, usually SDS-PAGE.
  • the proteins are transferred to a sheet of special blotting paper, e.g., nitrocellulose, though other types of paper, or membranes, can be used.
  • the proteins retain the same pattern of separation they had on the gel.
  • the blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose.
  • An antibody is then added to the solution which is able to bind to its specific protein.
  • Probes for the detection of antibody binding can be conjugated anti-immunoglobulins, conjugated staphylococcal Protein A (binds IgG), or probes to biotinylated primary antibodies (e.g., conjugated avidin/ streptavidin).
  • the power of the technique lies in the simultaneous detection of a specific protein by means of its antigenicity, and its molecular mass. Proteins are first separated by mass in the SDS- PAGE, then specifically detected in the immunoassay step. Thus, protein standards (ladders) can be run simultaneously in order to approximate molecular mass of the protein of interest in a heterogeneous sample.
  • the gel shift assay or electrophoretic mobility shift assay can be used to detect the interactions between DNA binding proteins and their cognate DNA recognition sequences, in both a qualitative and quantitative manner. Exemplary techniques are described in Ornstein L., Disc electrophoresis - 1: Background and theory, Ann. NY Acad. Sci. 121 :321-349 (1964), and Matsudiara, PT and DR Burgess, SDS microslab linear gradient polyacrylamide gel electrophoresis, Anal. Biochem. 87:386-396 (1987), each of which is herein incorporated by reference in its entirety for teachings regarding gel-shift assays.
  • purified proteins or crude cell extracts can be incubated with a labeled (e.g., 32P-radiolabeled) DNA or RNA probe, followed by separation of the complexes from the free probe through a nondenaturing polyacrylamide gel. The complexes migrate more slowly through the gel than unbound probe.
  • a labeled probe can be either double-stranded or single-stranded.
  • DNA binding proteins such as transcription factors
  • nuclear cell extracts can be used.
  • RNA binding proteins either purified or partially purified proteins, or nuclear or cytoplasmic cell extracts can be used.
  • the specificity of the DNA or RNA binding protein for the putative binding site is established by competition experiments using DNA or RNA fragments or oligonucleotides containing a binding site for the protein of interest, or other unrelated sequence. The differences in the nature and intensity of the complex formed in the presence of specific and nonspecific competitor allows identification of specific interactions.
  • Gel shift methods can include using, for example, colloidal forms of COOMASSIE (Imperial Chemicals Industries, Ltd) blue stain to detect proteins in gels such as polyacrylamide electrophoresis gels.
  • COOMASSIE International Chemicals Industries, Ltd
  • Such methods are described, for example, in Neuhoff et al., Electrophoresis 6:427-448 (1985), and Neuhoff et al., Electrophoresis 9:255-262 (1988), each of which is herein incorporated by reference in its entirety for teachings regarding gel shift methods.
  • a combination cleaning and protein staining composition is described in U.S. Patent 5,424,000, herein incorporated by reference in its entirety for its teaching regarding gel shift methods.
  • the solutions can include phosphoric, sulfuric, and nitric acids, and Acid Violet dye.
  • Protein arrays are solid-phase ligand binding assay systems using immobilized proteins on surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles.
  • the assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). Their advantages include being rapid and automatable, capable of high sensitivity, economical on reagents, and giving an abundance of data for a single experiment. Bioinformatics support is important; the data handling demands sophisticated software and data comparison analysis. However, the software can be adapted from that used for DNA arrays, as can much of the hardware and detection systems.
  • capture array in which ligand-binding reagents, which are usually antibodies but can also be alternative protein scaffolds, peptides or nucleic acid aptamers, are used to detect target molecules in mixtures such as plasma or tissue extracts.
  • ligand-binding reagents which are usually antibodies but can also be alternative protein scaffolds, peptides or nucleic acid aptamers, are used to detect target molecules in mixtures such as plasma or tissue extracts.
  • capture arrays can be used to carry out multiple immunoassays in parallel, both testing for several analytes in individual sera for example and testing many serum samples simultaneously.
  • proteomics capture arrays are used to quantitate and compare the levels of proteins in different samples in health and disease, i.e. protein expression profiling.
  • Proteins other than specific ligand binders are used in the array format for in vitro functional interaction screens such as protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc.
  • the capture reagents themselves are selected and screened against many proteins, which can also be done in a multiplex array format against multiple protein targets.
  • sources of proteins include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for peptides. Many of these methods can be automated for high throughput production.
  • proteins For capture arrays and protein function analysis, it is important that proteins should be correctly folded and functional; this is not always the case, e.g. where recombinant proteins are extracted from bacteria under denaturing conditions. Nevertheless, arrays of denatured proteins are useful in screening antibodies for cross-reactivity, identifying autoantibodies and selecting ligand binding proteins.
  • Protein arrays have been designed as a miniaturization of familiar immunoassay methods such as ELISA and dot blotting, often utilizing fluorescent readout, and facilitated by robotics and high throughput detection systems to enable multiple assays to be carried out in parallel.
  • Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads. While microdrops of protein delivered onto planar surfaces are the most familiar format, alternative architectures include CD
  • centrifugation devices based on developments in microfluidics (Gyros, Monmouth Junction, NJ) and specialized chip designs, such as engineered microchannels in a plate (e.g., The Living ChipTM, Biotrove, Woburn, MA) and tiny 3D posts on a silicon surface (Zyomyx, Hayward CA).
  • Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include color coding for microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDotsTM, Quantum Dot, Hayward, CA), and barcoding for beads (UltraPlexTM, SmartBead Technologies Ltd, Babraham, Cambridge, UK) and multimetal microrods (e.g., NanobarcodesTM particles, Nanoplex Technologies, Mountain View, CA). Beads can also be assembled into planar arrays on semiconductor chips (LEAPS technology, BioArray Solutions, Warren, NJ).
  • Immobilization of proteins involves both the coupling reagent and the nature of the surface being coupled to.
  • a good protein array support surface is chemically stable before and after the coupling procedures, allows good spot morphology, displays minimal nonspecific binding, does not contribute a background in detection systems, and is compatible with different detection systems.
  • the immobilization method used are reproducible, applicable to proteins of different properties (size, hydrophilic, hydrophobic), amenable to high throughput and
  • Orientation of the surface-bound protein is recognized as an important factor in presenting it to ligand or substrate in an active state; for capture arrays the most efficient binding results are obtained with orientated capture reagents, which generally require site-specific labeling of the protein.
  • Noncovalent binding of unmodified protein occurs within porous structures such as HydroGelTM (PerkinElmer, Wellesley, MA), based on a 3-dimensional polyacrylamide gel; this substrate is reported to give a particularly low background on glass microarrays, with a high capacity and retention of protein function.
  • Widely used biological coupling methods are through biotin/streptavidin or hexahistidine/Ni interactions, having modified the protein appropriately.
  • Biotin may be conjugated to a poly-lysine backbone immobilised on a surface such as titanium dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).
  • Array fabrication methods include robotic contact printing, ink-jetting, piezoelectric spotting and photolithography.
  • a number of commercial arrayers are available [e.g. Packard Biosciences] as well as manual equipment [V & P Scientific].
  • Bacterial colonies can be robotically gridded onto PVDF membranes for induction of protein expression in situ.
  • Fluorescence labeling and detection methods are widely used. The same
  • instrumentation as used for reading DNA microarrays is applicable to protein arrays.
  • capture e.g., antibody
  • fluorescently labeled proteins from two different cell states, in which cell lysates are directly conjugated with different fluorophores (e.g. Cy-3, Cy-5) and mixed, such that the color acts as a readout for changes in target abundance.
  • Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (PerkinElmer Lifesciences).
  • TSA tyramide signal amplification
  • Planar waveguide technology Zeptosens
  • High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (Luminex) or the properties of semiconductor nanocrystals (Quantum Dot).
  • Luminex phycoerythrin as label
  • Quantum Dot semiconductor nanocrystals
  • a number of novel alternative readouts have been developed, especially in the commercial biotech arena. These include adaptations of surface plasmon resonance (HTS Biosystems,
  • Intrinsic Bioprobes Tempe, AZ
  • rolling circle DNA amplification Molecular Staging, New Haven CT
  • mass spectrometry Intrinsic Bioprobes; Ciphergen, Fremont, CA
  • resonance light scattering Genicon Sciences, San Diego, CA
  • atomic force microscopy [BioForce
  • Capture arrays form the basis of diagnostic chips and arrays for expression profiling.
  • high affinity capture reagents such as conventional antibodies, single domains, engineered scaffolds, peptides or nucleic acid aptamers, to bind and detect specific target ligands in high throughput manner.
  • Antibody arrays have the required properties of specificity and acceptable background, and some are available commercially (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays are made either by
  • the term "scaffold” refers to ligand-binding domains of proteins, which are engineered into multiple variants capable of binding diverse target molecules with antibody-like properties of specificity and affinity.
  • the variants can be produced in a genetic library format and selected against individual targets by phage, bacterial or ribosome display.
  • Such ligand-binding scaffolds or frameworks include 'Affibodies' based on Staph, aureus protein A (Affibody, Bromma, Sweden), 'Trinectins' based on fibronectins (Phylos, Lexington, MA) and 'Anticalins' based on the lipocalin structure (Pieris Proteolab, Freising-Weihenstephan, Germany). These can be used on capture arrays in a similar fashion to antibodies and may have advantages of robustness and ease of production.
  • An alternative to an array of capture molecules is one made through 'molecular imprinting' technology, in which peptides (e.g., from the C-terminal regions of proteins) are used as templates to generate structurally complementary, sequence-specific cavities in a
  • the cavities can then specifically capture (denatured) proteins that have the appropriate primary amino acid sequence (ProteinPrintTM, Aspira Biosystems, Burlingame, CA).
  • PROTEINCHIP® array (Ciphergen, Fremont, CA), in which solid phase chromatographic surfaces bind proteins with similar characteristics of charge or hydrophobicity from mixtures such as plasma or tumour extracts, and SELDI-TOF mass spectrometry is used to detection the retained proteins.
  • protein arrays can be in vitro alternatives to the cell-based yeast two-hybrid system and may be useful where the latter is deficient, such as interactions involving secreted proteins or proteins with disulphide bridges.
  • High-throughput analysis of biochemical activities on arrays has been described for yeast protein kinases and for various functions (protein-protein and protein-lipid interactions) of the yeast proteome, where a large proportion of all yeast open-reading frames was expressed and immobilised on a microarray. Large-scale 'proteome chips' promise to be very useful in identification of functional interactions, drug screening, etc. (Proteometrix, Branford, CT).
  • a protein array can be used to screen phage or ribosome display libraries, in order to select specific binding partners, including antibodies, synthetic scaffolds, peptides and aptamers. In this way, 'library against library' screening can be carried out. Screening of drug candidates in combinatorial chemical libraries against an array of protein targets identified from genome projects is another application of the approach.
  • a multiplexed bead assay such as, for example, the BDTM Cytometric Bead Array, is a series of spectrally discrete particles that can be used to capture and quantitate soluble analytes. The analyte is then measured by detection of a fluorescence-based emission and flow cytometric analysis. Multiplexed bead assay generates data that is comparable to ELISA based assays, but in a "multiplexed" or simultaneous fashion. Concentration of unknowns is calculated for the cytometric bead array as with any sandwich format assay, i.e. through the use of known standards and plotting unknowns against a standard curve.
  • multiplexed bead assay allows quantification of soluble analytes in samples never previously considered due to sample volume limitations.
  • powerful visual images can be generated revealing unique profiles or signatures that provide the user with additional information at a glance.
  • the methods disclosed herein comprise assessing/measuring the efficacy or sufficiency of an immune response to a selected antigen in a subject.
  • the disclosed methods utilize tissue samples from the subject to provide the basis for assessment.
  • tissue samples can include, but are not limited to, blood (including peripheral blood and peripheral blood mononuclear cells), tissue biopsy samples (e.g., spleen, liver, bone marrow, thymus, lung, kidney, brain, salivary glands, skin, lymph nodes, and intestinal tract), and specimens acquired by pulmonary lavage (e.g., bronchoalveolar lavage (BAL)).
  • BAL bronchoalveolar lavage
  • non-lymphoid tissue examples include but are not limited to lung, liver, kidney, and gut. Lymphoid tissue includes both primary and secondary lymphoid organs such as the spleen, bone marrow, thymus, and lymph nodes.
  • reaction conditions e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.
  • Example 1 Mosaic Virus-Like-Particle Vaccine Protects Against Classic and Variant Infectious Bursal Disease Viruses
  • Nucleotide sequences that encode the pVP2 proteins from a variant IBDV strain designated USA08MD34p and a classic IBDV strain designated Mol95 were produced using RT- PCR and cloned into a pGEM-T Easy vector.
  • a nucleotide sequence that encodes the VP3 protein was also produced from the USA08MD34p viral genome using RT-PCR and cloned into a pGEM-T Easy vector.
  • the VP3 and pVP2 clones were inserted into the pVL1393 Baculovirus transfer vector and sequenced to confirm their orientation to the promoter and to insure they contained uninterrupted open-reading-frames.
  • Recombinant Baculoviruses were constructed by transfection in Sf9 cells. Three recombinant Baculoviruses were produced and contained the USA08MD34p-VP3, USA08MD34p-pVP2 or Mol95-pVP2 genomic sequences. Virus-like particles (VLPs) were observed using transmission electron microscopy when the USA08MD34p- VP3 Baculovirus was co-inoculated into Sf9 cells with either of the pVP2 constructs. VLPs were also observed when the USA08MD34p-pVP2 and Mol95-pVP2 were co-expressed with VLPs.
  • VLPs Virus-like particles
  • USA08MD34p-VP3 These mosaic VLPs contained both classic and variant pVP2s.
  • the USA08MD34p, Mol95 and mosaic VLPs were used to vaccinate chickens. They induced an IBDV specific antibody response that was detected by ELISA and virus-neutralizing antibodies were detected in vitro.
  • Chickens vaccinated with the mosaic VLPs were protected from a virulent variant IBDV strain (VI) and a virulent classic IBDV strain (STC). The results indicate the mosaic VLPs maintained the antigenic integrity of the variant and classic viruses and have the potential to serve as a multivalent vaccine for use in breeder flock.
  • VI virulent variant IBDV strain
  • STC virulent classic IBDV strain
  • the IBDV variant strain US A08MD34p (GenBank Accession #GQ856676) was used to obtain pVP2 and VP3 sequences.
  • the IBDV variant strain USA08MD34p was isolated from a Maryland broiler flock in 2008 (Jackwood 2010).
  • the IBDV used to obtain the classic pVP2 sequences was Mol95 (GenBank Accession #AY780324). It was isolated from a Missouri broiler flock in 2004 (Jackwood 2005).
  • the STC and VI IBDV strains were used to challenge vaccinated specific-pathogen-free (SPF) chickens (Charles River Laboratories, North Franklin, CT).
  • the VI variant virus (GenBank Accession #AF281235) is pathogenic in SPF chickens and its amino acid sequence across the four P domains of VP2 is identical to Del-E (Jackwood 2001).
  • the STC strain (GenBank Accession #D00499) is the classic standard challenge virus from the U.S. Animal and Plant Health Inspection Service (APHIS), National Veterinary Services
  • the VP3 from IBDV strain USA08MD34p (variant strain) was amplified using a reverse transcriptase - polymerase chain reaction (RT-PCR) kit (Superscript III One-step RT- PCR, Invitrogen, Life Technologies, Grand Island, NY).
  • RT-PCR reverse transcriptase - polymerase chain reaction
  • the primers VP3F 5'- GTACCTGATCACC4 JGGCTGC ATC AGAGTTC-3 ' (SEQ ID NO: 1) and
  • VP3R 5 ' -C AGGATGATCACTC AAGGTCCTC ATC AGAG-3 ' (SEQ ID NO: 2) amplified the entire VP3 sequence from base 2,323 to base 3,123 of genome segment A.
  • the start codon (ATG) in the forward primer is listed in italics and the underlined sequences are a genetic marker (not IBDV sequence) designed to identify the cloned gene.
  • the pVP2 portion of genome segment A was amplified from both the USA08MD34p and Mo 195 viruses using a RT-PCR kit (Superscript III One-step RT-PCR, Invitrogen) and the following primers: VP2F: 5 ' -TTCGATGATC ACG4 JGAC AAACCTGTC AGATC-3 ' (SEQ ID NO: 3) and VP2R: 5 ' -ACTACTGATC ACCCCTTGTCGGCGGCGAGAG-3 ' (SEQ ID NO: 4).
  • the start codon (ATG) in the forward primer is listed in italics and the underlined sequences are a genetic marker (not IBDV sequence) designed to identify the cloned gene.
  • These primers amplify the entire pVP2 sequence from base 64 to base 1,635 of genome segment A.
  • the 801bp VP3 RT-PCR product and the l,572bp pVP2 RT-PCR products were ligated into the pGEM-T Easy vector (Promega Corp., Madison WI) using a Rapid Ligation Kit (Promega Corp.). Incubation for the ligation reaction was overnight at 4°C.
  • the plasmids were then used to transform the E. coli strain HB-101 (Promega Corp.). The transformed bacteria were grown on L-agar containing lOC ⁇ g/ml ampicillin, lOC ⁇ g/ml X-gal and 0.1 ⁇ IPTG at 37°C overnight.
  • the pVL1393 baculovirus transfection vector (BD Biosciences, San Jose, CA) was used.
  • the vector was linearized using EcoKI and then treated with 0.05U Calf Intestinal Alkaline Phosphatase (Promega Corp.) at 37°C for lhr.
  • the reaction was stopped by adding 300 ⁇ 1 of a 20% SDS solution and the vector was phenol/chloroform extracted and ethanol precipitated with a 0.5 volume of 7.5M Ammonium Acetate (Sigma Chemical Co.).
  • the VP3 and pVP2 inserts that were excised from the pGEM-T Easy vectors were ligated into the linear pVL1393 vector using a Rapid Ligation Kit (Promega Corp.). Incubation was for 12 hours at 4°C and the plasmids were then used to transform the E. coli strain HB-101 (Promega Corp.). The bacteria were placed onto L-agar containing 100 ⁇ g/ml ampicillin and incubated at 37°C overnight. Bacterial colonies that grew on the agar plates were selected and grown in 4.0ml L-Broth containing 100 ⁇ g/ml ampicillin at 37°C with shaking overnight. The plasmids were extracted from a 1.0ml volume of the bacterial cultures using a Wizard Plus SV Minipreps DNA Purification System (Promega Corp.).
  • the constructs were cut with the restriction enzyme Pstl and the fragments were visualized on a 0.8% agarose gel.
  • the pVL1393 constructs containing VP3s and pVP2s in the correct orientation were used to transfect Sf9 insect cells.
  • the BD BaculoGold Transfection Kit (BD Biosciences) was used. Briefly, the cell culture medium was removed from Sf9 cells growing in 6-well culture plates (Becton Dickinson Labware, Franklin Lakes, NJ) and 1.0ml of Transfection Buffer A was added to each of the 6 wells. Approximately 2 ⁇ g of each pVL1393 construct was added to 0 ⁇ g of the linearized Baculovirus DNA supplied in the BaculoGold kit. After a 5 min incubation at room temperature, 1.0ml of Transfection Buffer B was added and the solution was gently agitated.
  • This DNA mixture was then added slowly to the Sf9 cells and incubated at 27°C for 4hrs. Following this incubation, the transfection solution was removed from the cells and 3.0ml of T M-FH medium (BD-Biosciences) was gently added. Incubation continued at 27°C for 4 days. The supernatants were then removed and half was stored at -70°C until they could be examined for VP3 and pVP2 expression products while the other half was stored at 4°C until they were used to inoculate new Sf9 cell cultures.
  • T M-FH medium BD-Biosciences
  • a 743 -bp segment of the hypervariable region of VP2 (hvVP2) from nucleotides 737 to 1479 was amplified using primers 743-1 (5'- GCCCAGAGTCTACACCAT-3 ') (SEQ ID NO: 5) and 743-2 (5'-CCCGGATTATGTCTTTGA- 3') (SEQ ID NO: 6) (Jackwood 2005).
  • the pVP2 protein was generated by infecting Sf9 cells with either the USA08MD34p - pVP2 baculovirus construct or the Mol95-pVP2 construct.
  • the VLPs were prepared by inoculating Sf9 cells with the pVP2 constructs plus the USA08MD34p -VP3 baculovirus construct. Expression of the pVP2 proteins and VLPs was examined using an antigen-capture (AC)-ELISA (Synbiotics Corp., Kansas City, MO).
  • the AC-ELISA plate contained the IBD screening monoclonal antibodies B69, R63 and #10. These monoclonal antibodies were reported to bind classic and variant VP2 proteins (Vakharia 1994).
  • Supernatants from recombinant Baculovirus infected Sf9 cells were tested undiluted and at the following dilutions: 1 :2, 1 :4, 1 :8, 1 : 16 and 1 :64.
  • VLPs A 25 ⁇ 1 volume of each VLP was place in an eppendorf tube and particulates were pelleted at 16,000 x g for 2 min. The supernatants were then placed on formvar coated grids and stained with uranyl acetate for 2 min in the dark. Samples were examined using a transmission electron microscope (Hitachi H-7500) for VLPs.
  • VLPs were used to vaccinate 3 week old SPF chickens. The birds were bled prior to being vaccinated and were negative for IBDV antibodies. They were vaccinated with a 0.1ml dose of the Sf9 cell cultures containing the USA08MD34p, Mol95 or mosaic VLPs. Inoculations were via the intramuscular route. Two weeks later, they were vaccinated with a second 0.1ml dose of the same VLP samples via the subcutaneous route. The birds were then bled two weeks following the booster vaccination and the sera were examined for IBDV specific antibodies using an IBDxr ELISA kit (TDEXX, Corp.). The sera were also examined for virus-neutralizing (VN) antibody titers using a standard protocol and classic (S706) and variant (Del-E) antigens (Dybing 1998).
  • VN virus-neutralizing
  • S706 standard protocol and classic
  • Del-E variant antigens
  • bursa/body weight ratio B/BW
  • Bursa/body weight (B/BW) ratios were calculated as the bursa weight (g)/body weight (g) x 1000. These B/BW ratios were compared for statistical differences among the groups using the SAS: Proc GLM program.
  • the pVL1393 Baculovirus transfer vector was used to insert the pVP2 and VP3 clones into the Baculovirus genome.
  • the pVP2 and VP3 inserts from the pGEM-T Easy vectors were excised using EcoKI and purified on an agarose gel ( Figure 2).
  • the inserts were then ligated into the pVL1393 plasmid that was cut with EcoKI and dephosphorlated.
  • the resulting plasmids were used to transform E. coli HB-101 cells and then examined for IBDV sequences using the EcoKI enzyme and agarose gel electrophoresis ( Figure 3).
  • Baculovirus polyhedron promoter and in the correct direction Because the pVL1393 vector was cut with one restriction enzyme, the pVP2 and VP3 clones could have been ligated into this vector in either direction.
  • the orientation of the pVP2 inserts in the pVL1393 vector was determined using the enzyme Pstl. This enzyme was selected because it cuts once in the plasmid and has multiple cut sites in the inserts.
  • the pVP2 clones in the correct direction produce bands at 1,01 lbp, 332bp and 74bp after digestion with Pstl.
  • the VP3 clones were cut with BgHI to determine if they were in the correct orientation.
  • This enzyme produces a band at 155bp if the VP3 insert was ligated into the plasmid in the correct direction.
  • the pVL1393 pVP2 and VP3 constructs in the correct direction were selected for Transfection into the Baculovirus genome. These constructs were also RT-PCR amplified and sequenced to insure they were in the correct orientation and contained uninterrupted open reading frames.
  • the Sf9 cells and cell culture media were harvested 4 days following transfection of the pVP2 and VP3 sequences into the baculovirus genome. The samples were stored at 4°C and then used to inoculate new cultures of Sf9 cells. Cytopathic effects (CPE) that consisted of floating cells and holes in the monolayer were observed at 3 and 4 days post-inoculation.
  • CPE Cytopathic effects
  • Samples for protein expression were collected at 4 days post-inoculation and frozen at -70°C.
  • VLPs virus-like particles
  • VLPs The production of VLPs was initiated by inoculating monolayers of Sf9 cells with the USA08MD34p-VP3 Baculovims. These cell cultures were then inoculated with either the USA08MD34p-pVP2 or Mol95-pVP2 Baculovimses. After 4 days of incubation at 27°C the cultures were observed to have CPE and they were frozen at -70°C. The USA08MD34p- pVP2/VP3 and Mol95-pVP2/VP3 cultures were tested in the AC-ELISA for protein expression (Table 2). The optical density readings for the Mo 195 VLPs were strongly positive.
  • the USA08MD34p VLPs were also positive in the AC-ELISA, showing the monoclonal antibodies do not bind the USA08MD34p-pVP2 but they do bind the USA08MD34p proteins when the pVP2 is combined with VP3 into a VLP.
  • VLPs IBDV like particles
  • Figure 4 the un-inoculated control Sf9 cultures were negative for VLPs
  • the VLPs varied in size from 40nm to 80nm but most were approximately 60nm. Table 2. AC -ELISA and assays for antibody titers to VLPs.
  • VN titers were only determined for VLP samples.
  • the cell culture adapted viruses used were Del-E for the variant antigen and S706 for the classic antigen.
  • the mosaic VLPs contained VP2 from both the USA08MD34p and Mol95 strains. 209.
  • the Mol95-VP2, USA08MD34p-VP2 and USA08MD34p-VP3 were inoculated into Sf9 cells to produce VLPs containing both classic and variant pVP2 antigens.
  • the resulting mosaic VLPs resembled the capsid structure of IBDV ( Figure 4).
  • the Mol95, USA08MD34p and mosaic VLPs were used to vaccinate SPF chickens.
  • the three-week-old SPF birds were negative for IBDV antibodies prior to being vaccinated.
  • Two weeks following booster vaccination using either USA08MD34p, Mol95 or mosaic VLPs, the sera from vaccinated birds were positive in the ELISA and VN assays.
  • the ELISA antibody titers to IBDV and the virus-neutralization (VN) antibody titers are reported in Table 2.
  • USA08MD34p and Mol95 VLPs were immunogenic in chickens and produced IBDV specific antibodies detected in the ELISA.
  • the VN data indicate the antibodies produced were capable of neutralizing IBDV strains in cell culture.
  • the mosaic VLPs containing VP2 from both USA08MD34p and Mol95 produced ELISA titers and neutralizing antibodies to both the classic (S706) and variant (Del-E) IBDV strains (Table 2).
  • the challenge viruses were given at 10 2 0 CIDso/bird. The birds were challenged 7 days following the last booster vaccination.
  • the mosaic VLPs contained VP2 from both the USA08MD34p and Mol95 strains.
  • Baculovirus expression system was used to create an alternative to bursa derived IBDV antigens that can be used in breeder flock vaccines.
  • USA08MD34p and Mol95 represent variant and classic viruses respectively.
  • the AC-ELISA screening plate Synbiotics,
  • Detection of the USA08MD34p VLP in the AC-ELISA indicates one or more of the monoclonal antibodies bind a tertiary epitope formed by the pVP2 trimer or an epitope was hidden when the VP2 was singularly expressed but exposed when it was co- expressed with VP3.
  • the result shows that VLP capsid structures are higher quality antigens than VP2 tubules and polyprotein derived mixed structures (Martinez 2003).
  • Antigen for coating ELIS A plates was prepared by infecting SF9 insect cells with a combination of recombinant Baculoviruses expressing pVP2 and VP3. The infected cells produced VLPs and were harvested 4 days following infection. The VLPs were diluted 1 :5 in PBS (1.9 mM NaH2P04, 8.1 mM Na2HP04, 154 mM NaCl (pH7.2]) containing 0.05% (wt/vol) sodium azide and used to coat 96-well flat-bottom plates (Falcon; Becton Dickinson, Lincoln Park, N. J.). This dilution of antigen was determined to be optimal using standard procedures.
  • a 50ul volume of the diluted VLP antigen was used to coat each well of a 96-well plate for 24hrs at room temperature.
  • the antigen-coated 96-well plates were washed three times in water and then incubated at room temperature for 30 min in blocking buffer (170 mM"H3B04 [pH 8.5], 120 mM NaCI, 1 mM EDTA, 0.05% [wt/vol] sodium azide, 0.25% [wt/vol] bovine serum albumin, 0.05% [vol/vol] Tween 20). After three washes in water, a 50ul volume of serum diluted in blocking buffer was added to each well. Incubation continued at room temperature for 30 min.
  • the ELISA plates were then washed three times in water, once for 10 min in blocking buffer, and then three times in water.
  • a horseradish peroxidase-labeled goat anti -chicken immunoglobulin G was used according to the manufacturer's directions.
  • a 50 ul volume was added to each well. Plates were incubated at room temperature for 30 min and then washed in water, in blocking buffer, and again in water as described above.
  • a 75 ul volume of the substrate was added, and after 15 min the color development was stopped with 5% (wt/vol) SDS in water. Test wells were read on an ELISA reader at a wavelength of 620 nm.
  • VLP antigens reacted with the serum antibodies to IBDV and produced a positive result in the ELISA.
  • Chicken serum samples from birds not exposed to IBDV were negative in the assay. The results indicate that VLP antigens can be used in the ELISA to detect antibodies to IBDV strains.
  • the VLP vaccine for vvIBDV can be used to produce maternal immunity in chickens.
  • the VLP vvIBDV vaccine can be added to an existing commercial product designed for breeder bird vaccination or can be used alone. To test the efficacy of such a vaccine, breeder birds are vaccinated and their progeny are tested for immunity to a vvIBDV challenge strain.
  • Vaccines Commercial Killed Breeder vaccine
  • VP2 variable domain are associated with typical and atypical antigenicity in very virulent infectious bursal disease viruses. Arch Virol 1998; 143 : 1627-36.
  • Kibenge FSB Jackwood DJ, Mercado CC. Nucleotide sequence analysis of genome segment A of infectious bursal disease virus. J Gen Virol 1990;71 :569-77.

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Abstract

L'invention concerne des méthodes et des compositions en rapport avec le virus de la bursite infectieuse (IBDV) et le virus de la nécrose pancréatique infectieuse (IPNV), et des vaccins pour le traitement et la prévention de celles-ci. L'invention porte, de manière spécifique, sur une particule de type viral (VLP) comprenant une proétine de capside IPNV formée par des trimères de VP2 et VP3. L'invention concerne en outre des monomères provenant de souches du génogroupe 1 (soit IPNV2 soit IPNV10) co-exprimées avec IPNV10 pour produire les VLP.
PCT/US2018/032193 2017-05-12 2018-05-11 Compositions vaccinales contre le virus de la nécrose pancréatique infectieuse (ipnv) Ceased WO2018209167A1 (fr)

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US15/593,940 US10086062B2 (en) 2012-07-05 2017-05-12 Infectious pancreatic necrosis virus (IPNV) vaccine compositions
US15/593,940 2017-05-12

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100092521A1 (en) * 2006-12-20 2010-04-15 Advanced Bionutrition Corporation Antigenicity of infectious pancreatic necrosis virus vp2 sub-viral particles expressed in yeast
US20150104475A1 (en) * 2012-07-05 2015-04-16 The Ohio State University Compositions and methods related to viral vaccines
US20170348409A1 (en) * 2012-07-05 2017-12-07 Ohio State Innovation Foundation Compositions and methods related to viral vaccines

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100092521A1 (en) * 2006-12-20 2010-04-15 Advanced Bionutrition Corporation Antigenicity of infectious pancreatic necrosis virus vp2 sub-viral particles expressed in yeast
US20150104475A1 (en) * 2012-07-05 2015-04-16 The Ohio State University Compositions and methods related to viral vaccines
US20170348409A1 (en) * 2012-07-05 2017-12-07 Ohio State Innovation Foundation Compositions and methods related to viral vaccines

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DA COSTA ET AL.: "Blotched Snakehead Virus Is a New Aquatic Birnavirus That Is Slightly More Related to Avibirnavirus Than to Aquabirnavirus", JOURNAL OF VIROLOGY, vol. 77, no. 1, January 2003 (2003-01-01), pages 719 - 725, XP055550561 *
DATABASE Protein [O] 14 October 2015 (2015-10-14), "Structural polyprotein", XP055550457, Database accession no. P05844 *
DATABASE UniProtKB 1 November 1996 (1996-11-01), "Structural polyprotein", XP055550445, Database accession no. Q82731 *
DATABASE UniProtKB 3 April 2013 (2013-04-03), "Structural polyprotein", XP055550464, Database accession no. L7RDD8 *
LAUKSUND ET AL.: "Infectious pancreatic necrosis virus proteins VP2, VP3, VP4 and VP5 antagonize IFNa1 promoter activation while VP1 induces IFNa1", VIRUS RESEARCH, vol. 196, 22 January 2015 (2015-01-22), pages 113 - 121, XP029127397 *

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