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WO2010077712A1 - Particule de type viral du virus syncytial respiratoire bovin (vlps) - Google Patents

Particule de type viral du virus syncytial respiratoire bovin (vlps) Download PDF

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Publication number
WO2010077712A1
WO2010077712A1 PCT/US2009/067257 US2009067257W WO2010077712A1 WO 2010077712 A1 WO2010077712 A1 WO 2010077712A1 US 2009067257 W US2009067257 W US 2009067257W WO 2010077712 A1 WO2010077712 A1 WO 2010077712A1
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Prior art keywords
protein
rsv
vlp
brsv
pharmaceutically acceptable
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Michael J. Massare
Gale E. Smith
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Novavax Inc
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Novavax Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18523—Virus like particles [VLP]

Definitions

  • VLPS VLPS
  • Respiratory syncytial virus is a member of the genus Pneumovirus of the family Paramyxoviridae. This virus has a genome comprised of a single stranded negative- sense RNA, which is tightly associated with viral protein to form the nucleocapsid.
  • the viral envelope is composed of a plasma membrane derived lipid bilayer that contains virally encoded structural proteins.
  • a viral polymerase is packaged with the virion and transcribes genomic RNA into mRNA.
  • the RSV genome encodes three transmembrane structural proteins: F, G, and SH; two matrix proteins: M and M2; three nucleocapsid proteins: N, P and L; and two nonstructural proteins: NSl and NS2 (Collins et al, 1996, Respiratory syncytial virus, pp. 1313-1351, In B.N. Fields (ed.), Fields virology. Raven Press, New York, NY).
  • RSV Human respiratory syncytial virus
  • the RSV genome encodes 10 proteins: NSl , NS2, N, P, M, SH, G, F, M2, and L (Collins et al. (1994) J. Virol., 49, 572-578).
  • the M protein is expressed as a peripheral membrane protein
  • the F and G proteins are expressed as structural membrane proteins and are involved in virus attachment and viral entry into cells.
  • the F and G proteins are the major antigens that elicit neutralizing antibodies in vivo (as reviewed in Mclntosh and Chanock, 1990, Respiratory Syncytial Virus, In In Virology, 2nd ed, D. M. Knipe et al., (ed.). Raven Press New York, N.
  • FIG. 1 shows the amino acid sequence relatedness between proteins of HRSV subgroup A (HRSV-A) and HMPV (human metapneumovirus) subgroup A (HMPV).
  • Virus candidates were either under-attenuated or over-attenuated (Kim et al, 1973, Pediatrics, 52, 56-63; Wright et al, 1976, J. Pediatrics, 88, 931-936) and some of the vaccine candidates were genetically unstable which resulted in the loss of the attenuated phenotype (Hodes et al, 1974, Proc. Soc. Exp. Biol. Med., 145, 1158-1164).
  • VLPs Virus-like particles
  • VLPs closely resemble mature virions, but they do not contain viral genomic material ⁇ i.e., viral genomic RNA). Therefore, VLPs are non-replicative in nature, which make them safe for administration in the form of an immunogenic composition ⁇ e.g., a vaccine).
  • VLPs can express structural proteins on the surface of the VLP, which is the most physiological configuration.
  • VLPs since VLPs resemble intact virions and are multivalent particulate structures, VLPs may be more effective in inducing neutralizing antibodies to the structural protein than soluble envelope antigens.
  • VLPs can be administered repeatedly to vaccinated hosts, unlike many recombinant vaccine approaches.
  • novel VLP -based respiratory syncytial virus (RSV) vaccines comprising a bovine RSV ⁇ e.g. BRSV or bRSV) M protein and at least one immunogen capable of inducing an immune response for the prevention of diseases from RSV or other human or vertebrate diseases when administered to a subject in need thereof.
  • RSV respiratory syncytial virus
  • the present invention provides VLPs comprising at least one respiratory syncytial virus (RSV) protein and at least one immunogen.
  • the RSV protein of the VLP comprises a RSV matrix (M) protein, fragment, or variant thereof.
  • the RSV matrix (M) protein is a bovine respiratory syncytial virus (BRSV) M protein, fragment or variant thereof.
  • the VLP is chimeric VLP, comprising a BRSV M protein, fragment or variant thereof, and at least one non-RSV immunogen.
  • the VLPs of the present invention will generally comprise a bovine respiratory syncytial virus (BRSV) M protein, fragment, or variant thereof and at least one RSV and/or non-RSV immunogen.
  • the immunogen is a polypeptide.
  • the immunogen is a glycoprotein.
  • the immunogen is a lipoprotein.
  • the immunogen is co-expressed with the BRSV M protein, fragment, or variant thereof to form a VLP.
  • the immunogen is a chimeric protein, wherein the immunogen is operably linked to a signal sequence and a transmembrane protein from the same species or a different species from which the M protein is derived or a protein that is not an M protein.
  • the immunogen is a protein derived from one or more viruses, bacteria, parasite, protozoa, and/or diseased cells, such as, but not limited to cancer cells.
  • the immunogen may be a surface protein derived from one or more viruses, bacteria, parasites, protozoa and/or diseased cells such as cancer cells.
  • the immunogen may be from the same or different virus from which the matrix protein is obtained.
  • the VLPs comprise a BRSV M protein, fragment, or variant thereof and at least one RSV structural membrane protein.
  • the RSV structural protein may be an HRSV F protein and/or a G protein.
  • the VLPs comprise BRSV M protein, fragment, or variant thereof and at least one RSV structural membrane protein.
  • the VLPs are expressed in a eukaryotic cell under conditions that permit the formation of VLPs.
  • the present invention also provides methods of producing VLPs, comprising transfecting vectors encoding a BRSV M protein, fragment, or variant thereof and at least one immunogen into a suitable host cell and expressing the BRSV M protein, fragment, or variant thereof under conditions that allow VLP formation.
  • the method comprises producing chimeric VLPs which comprise a BRSV M protein, fragment, or variant thereof and at least one non-RSV immunogen.
  • the immunogen is co- expressed with the BRSV M protein, fragment, or variant thereof in suitable host cells, including insect cells.
  • the BRSV M protein, fragment, or variant thereof is operably linked to the immunogen.
  • the immunogen is a polypeptide.
  • the immunogen is a glycoprotein. In another embodiment, the immunogen is a lipoprotein. In a further embodiment, the immunogen is a chimeric protein operably linked to a signal sequence and a transmembrane and/or cytoplasmic domain sequence of a protein from the same species or different species from which the M protein is derived. In another embodiment, the immunogen is a protein derived from one or more viruses, bacteria, parasites, protozoa, and/or diseased cells, such as, but not limited to cancer cells. In a further embodiment, the immunogen may be a surface protein derived from one or more viruses, bacteria, parasites, protozoa and/or diseased cells.
  • the method comprises co-transfecting vectors encoding a BRSV M protein, fragment, or variant thereof and one or more HRSV structural membrane proteins. In another embodiment, the method comprises co-transfecting vectors encoding a BRSV M protein, fragment, or variant thereof and vectors encoding at least one chimeric RSV structural protein. In further embodiment, the co-transfecting vectors comprise a BRSV M protein and an RSV F protein and/or a G protein.
  • the present invention also provides pharmaceutically acceptable vaccine compositions for stimulating an immune response in a vertebrate, such as a human subject, against an infection or at least one disease symptom in a subject, comprising VLPs, which comprise a BRSV M protein, fragment, or variant thereof and at least one immunogen.
  • the pharmaceutically acceptable vaccine composition comprises chimeric VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one non-RSV immunogen.
  • the immunogen is co-expressed with a BRSV M protein, fragment, or variant thereof in a suitable host cell.
  • the BRSV M protein, fragment, or variant thereof is operably linked to the immunogen.
  • the immunogen is a polypeptide.
  • the immunogen is a glycoprotein. In another embodiment, the immunogen is a lipoprotein. In a further embodiment, the immunogen is a chimeric protein operably linked to a signal sequence and a transmembrane and/or cytoplasmic domain sequence of a protein from the same species or different species from which the M protein is derived or a protein that is not an M protein. In another embodiment, the immunogen is a protein derived from one or more viruses, bacteria, parasite, protozoa, and/or diseased cells, such as, but not limited to cancer cells. In a further embodiment, the immunogen may be a surface protein derived from one or more viruses, bacteria, parasites, protozoa and/or diseased cells.
  • the pharmaceutically acceptable vaccine composition comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one RSV structural membrane protein. In one embodiment, the pharmaceutically acceptable vaccine composition comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and a RSV structural membrane protein. In one embodiment, the pharmaceutically acceptable vaccine composition comprises chimeric VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one non-RSV structural membrane protein. In a further embodiment, the pharmaceutically acceptable vaccine composition comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and HRSV structural membrane protein such as a HRSV F protein and/or a G protein.
  • the pharmaceutically acceptable vaccine composition further comprises an adjuvant.
  • the adjuvant is a liposome.
  • the adjuvant is a non-phospholipid adjuvant.
  • the adjuvant is Novasomes ® .
  • kits for immunizing a vertebrate, such as a human subject, against an infection or at least one disease symptom in a subject comprising VLPs which comprise a BRSV M protein, fragment, or variant thereof and at least one immunogen.
  • the BRSV M protein, fragment, or variant thereof is operably linked to the immunogen.
  • the immunogen is a polypeptide.
  • the immunogen is a glycoprotein.
  • the immunogen is a lipoprotein.
  • the immunogen is a chimeric protein operably linked to a signal sequence and a transmembrane and/or cytoplasmic domain sequence of a protein from the same species or different species from which the M protein is derived.
  • the immunogen is a protein derived from one or more viruses, bacteria, parasite, protozoa, and/or diseased cells, such as but not limited to cancer cells.
  • the immunogen may be a surface protein derived from one or more viruses, bacteria, parasites, protozoa and/or diseased cells, including, but not limited to, cancer cells.
  • the kit comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one RSV structural membrane protein.
  • the kit comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one non-RSV protein. In one embodiment, the kit comprises chimeric VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one non-RSV immunogen. In further embodiment, the kit comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one RSV immunogen, including, but not limited to an RSV F and/or G protein.
  • the present invention also provides immunogenic formulations effective against an infection or disease or at least one symptom thereof in a subject, comprising administering to the mammal a protection-inducing amount of VLPs, wherein said VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one immunogen.
  • the formulation comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and an immunogen, wherein the BRSV M protein, fragment, or variant thereof is operably linked to the immunogen.
  • the immunogen is a polypeptide.
  • the immunogen is a glycoprotein.
  • the immunogen is a lipoprotein.
  • the immunogen is a chimeric protein operably linked to a signal sequence and a transmembrane and/or cytoplasmic domain sequence of a protein from the same species or different species from which the M protein is derived.
  • the immunogen is a protein derived from one or more viruses, bacteria, parasite, protozoa, and/or diseased cells, such as, but not limited to cancer cells.
  • the immunogen may be a surface protein derived from one or more viruses, bacteria, parasites, protozoa and/or diseased cells such as cancer cells.
  • the immunogenic formulation comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one RSV structural protein.
  • the immunogenic formulation comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one RSV structural proteins, such as an RSV F protein and/or RSV G protein.
  • the present invention also provides immunogenic formulations for inducing immunity to an infection or disease or at least one symptom thereof in a subject, comprising administering at least one effective dose of the VLPs of the present invention, wherein said VLPs comprise a BRSV M protein, fragment, or variant thereof and at least one immunogen.
  • the immunogenic formulation comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and an immunogen, wherein the BRSV M protein, fragment, or variant thereof is operably linked to the immunogen.
  • the immunogen is a polypeptide.
  • the immunogen is a glycoprotein.
  • the immunogen is a lipoprotein.
  • the immunogen is a chimeric protein operably linked to a signal sequence and a transmembrane and/or cytoplasmic domain sequence of a protein from the same species or different species from which the M protein is derived.
  • the immunogen is a protein derived from one or more viruses, bacteria, parasite, protozoa, and/or diseased cells, such as, but not limited to cancer.
  • the immunogen may be a surface protein derived from one or more viruses, bacteria, parasites, protozoa and/or diseased cells such as cancer cells.
  • the immunogenic formulation comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one RSV structural protein.
  • the immunogenic formulation comprises VLPs comprising a BRSV M protein, fragment, or variant thereof and an RSV structural proteins such as an RSV F protein and/or a G protein.
  • the immunogenic formulation comprises chimeric VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one non-RSV protein.
  • the present invention also provides methods of vaccinating a vertebrate, such as a human subject, against an infection or at least one disease symptom in a subject comprising providing at least one effective dose of the VLPs of the present invention, wherein the VLPs comprise a BRSV M protein, fragment, or variant thereof and at least one immunogen.
  • the methods comprise administering the VLPs to the vertebrate via any means, including orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.
  • the method comprises administering VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one immunogen.
  • the method comprises administering VLPs having a BRSV M protein, fragment, or variant thereof operably linked to the immunogen.
  • the immunogen is a polypeptide.
  • the immunogen is a glycoprotein.
  • the immunogen is a lipoprotein.
  • the immunogen is a chimeric protein operably linked to a signal sequence and a transmembrane domain and/or cytoplasmic domain of a protein from the same species or different species from which the M protein is derived.
  • the immunogen is a protein derived from one or more viruses, bacteria, parasite, protozoa, and/or diseased cells, such as, but not limited to cancer cells.
  • the immunogen may be surface protein derived from one or more viruses, bacteria, parasites, protozoa and/or diseased cells, including, but not limited to cancer cells.
  • the method of vaccinating comprises providing an effective dose of VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one RSV structural membrane protein, such as an RSV F protein and/or RSV G protein.
  • the vaccination method comprises providing VLPs comprising a BRSV M protein, fragment, or variant thereof and at least one chimeric RSV structural protein.
  • the method of vaccinating comprises providing chimeric VLPs comprising BRSV M protein and at least one non-RSV protein.
  • FIG. 1 compares the amino acid relatedness (% identity) between the BRSV-
  • FIG. 2 illustrates an SDS Coomassie-stained gel of total protein expression in the crude harvest (Panel A) and 100,000 x g harvest (Panel B) of the BRSV M VLP and HRSV-A M VLP with or without co-expression with HRSV-A F 0 protein.
  • FIG. 3 illustrates Western blot analyses for total intracellular protein (Panel
  • FIG. 4 illustrates an SDS Coomassie-stained gel of total protein expression in the crude harvest (Panel A, Intracellular) and 100,000 x g harvest (Panel B, Secreted Particles) of the influenza hemagglutinin (HA) and MMTV cytoplasmic domain associating with BRSV M protein.
  • FIG. 5 is a BRSV M protein versus RSV M protein sequence alignment. The alignment compares the cloned amino acid sequence for BRSV M with three published BRSV M sequences and three HRSV M protein sequences.
  • adjuvant refers to a compound that, when used in combination with a specific immunogen (e.g. a VLP) in a formulation, will augment or otherwise alter or modify the resultant immune response. Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses.
  • a specific immunogen e.g. a VLP
  • Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses.
  • an "effective dose” generally refers to that amount of VLPs of the invention sufficient to induce immunity, to prevent and/or ameliorate an infection or to reduce at least one symptom of an infection or disease, and/or to enhance the efficacy of another dose of a VLP.
  • An effective dose may refer to the amount of VLPs sufficient to delay or minimize the onset of an infection or disease.
  • An effective dose may also refer to the amount of VLPs that provides a therapeutic benefit in the treatment or management of an infection or disease. Further, an effective dose is the amount with respect to VLPs of the invention alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of an infection or disease.
  • An effective dose may also be the amount sufficient to enhance a subject's (e.g., a human's) own immune response against a subsequent exposure to an infectious agent or disease.
  • Levels of immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay, or by measuring cellular responses, such as, but not limited to cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and/or other cellular responses.
  • T cell response can be monitored, e.g., by measuring, for example, the amount of CD4 + and CD8 + cells present using specific markers by fluorescent flow cytometry or T cell assays, such as but not limited to T-cell proliferation assay, T-cell cytotoxic assay, TETRAMER assay, and/or ELISPOT assay.
  • an "effective dose" is one that prevents disease and/or reduces the severity of symptoms.
  • an effective amount refers to an amount of VLPs necessary or sufficient to realize a desired biologic effect.
  • An effective amount of the composition would be the amount that achieves a selected result, and such an amount could be determined as a matter of routine experimentation by a person skilled in the art.
  • an effective amount for preventing, treating and/or ameliorating an infection could be that amount necessary to cause activation of the immune system, resulting in the development of an antigen specific immune response upon exposure to VLPs of the invention.
  • the term is also synonymous with "sufficient amount.”
  • multivalent refers to VLPs which have one or more antigenic proteins/peptides or immunogens against multiple types or strains of infectious agents or diseases.
  • immunogen or "antigens” refers to a substance such as proteins, peptides, peptides, nucleic acids that are capable of eliciting an immune response. Both terms also encompass epitopes, and are used interchangeably.
  • immune stimulator refers to a compound that enhances an immune response via the body's own chemical messengers (cytokines). These molecules comprise various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interferons (IFN- ⁇ ), interleukins ⁇ e.g., IL-I, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors ⁇ e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1 ; B7.2, etc.
  • the immune stimulator molecules can be administered in the same formulation as VLPs of the invention, or can be administered separately. Either the protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect.
  • protection immune response refers to an immune response mediated by antibodies against an infectious agent or disease, which is exhibited by a vertebrate ⁇ e.g., a human), that prevents or ameliorates an infection or reduces at least one disease symptom thereof.
  • VLPs of the invention can stimulate the production of antibodies that, for example, neutralize infectious agents, blocks infectious agents from entering cells, blocks replication of the infectious agents, and/or protect host cells from infection and destruction.
  • the term can also refer to an immune response that is mediated by T-lymphocytes and/or other white blood cells against an infectious agent or disease, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates infection or disease, or reduces at least one symptom thereof.
  • a vertebrate e.g., a human
  • infectious agent refers to microorganisms that cause an infection in a vertebrate.
  • the organisms are viruses, bacteria, parasites, protozoa and/or fungi.
  • diseased cells refers to cells that are impaired in their normal cellular functions.
  • Non-limiting examples of diseased cells include cancer cells.
  • immunogenic formulation refers to a preparation which, when administered to a vertebrate, e.g. a mammal, will induce an immune response.
  • the term "pharmaceutically acceptable vaccine” refers to a formulation which contains VLPs of the present invention, which is in a form that is capable of being administered to a vertebrate and which induces a protective immune response sufficient to induce immunity to prevent and/or ameliorate an infection or disease, and/or to reduce at least one symptom of an infection or disease, and/or to enhance the efficacy of another dose of VLPs.
  • the vaccine comprises a conventional saline or buffered aqueous solution medium in which the composition of the present invention is suspended or dissolved.
  • the composition of the present invention can be used conveniently to prevent, ameliorate, or otherwise treat an infection.
  • the vaccine Upon introduction into a host, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies and/or cytokines and/or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and/or other cellular responses.
  • the term "vertebrate” or “subject” or “patient” refers to any member of the subphylum cordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species.
  • Farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats (including cotton rats) and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like are also non-limiting examples .
  • the terms “mammals” and “animals” are included in this definition. Both adult and newborn individuals are intended to be covered. In particular, infants and young children are appropriate subjects or patients for a RSV vaccine.
  • virus-like particle refers to a structure that in at least one attribute resembles a virus but which has not been demonstrated to be infectious.
  • Virus-like particles in accordance with the invention do not carry genetic information encoding for the proteins of the virus-like particles. In general, virus-like particles lack a viral genome and, therefore, are noninfectious. In addition, virus-like particles can often be produced in large quantities by heterologous expression and can be easily purified.
  • chimeric VLP refers to VLPs that contain proteins, or portions thereof, from at least two different infectious agents (heterologous proteins). Usually, one of the proteins is derived from a virus that can drive the formation of VLPs from host cells. Examples, for illustrative purposes, are BRSV M and/or HRSV G or F protein. The terms RSV VLPs and chimeric VLPs can be used interchangeably where appropriate.
  • RSV Matrix or "RSV M” protein refer to a RSV protein that, when expressed in a host cell, induces formation of VLPs.
  • RSV M protein An example of a RSV M protein is represented by SEQ ID NO: 1, which is a BRSV M protein.
  • SEQ ID NO: 1 is a BRSV M protein.
  • the term also comprises any variants, derivatives and/or fragments of RSV M that, when expressed in a host cell, induces formation of VLPs.
  • the term also encompasses nucleotide sequences which encode for RSV M and/or any variants, derivatives and/or fragments thereof that when transfected (or infected) into a host cell will express RSV M protein and induce formation of VLPs.
  • RSV M protein is a nonglycosylated internal virion protein that accumulates in the plasma membrane that interacts with RSV F protein and other factors during virus morphogenesis ⁇ Id., p. 1608).
  • SEQ ID NO: 1 depicts a representative amino acid sequence of BRSV M protein.
  • RSV M proteins that are about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 1, and all fragments and variants (including chimeric proteins) thereof.
  • F and G proteins Two structural membrane proteins, F and G proteins, are expressed on the surface of RSV, and have been shown to be targets of neutralizing antibodies (Sullender, W. (2000) Clinical Microbiology Review 13, 1-15). These two proteins are also primarily responsible for viral recognition and entry into target cells; G protein binds to a specific cellular receptor and the F protein promotes fusion of the virus with the cell. The F protein is also expressed on the surface of infected cells and is responsible for subsequent fusion with other cells leading to syncytia formation. Thus, antibodies to the F protein can neutralize virus or block entry of the virus into the cell or prevent syncytia formation.
  • the RSV F protein directs penetration of RSV by fusion between the virion's envelope protein and the host cell plasma membrane. Later in infection, the F protein expressed on the cell surface can mediate fusion with neighboring cells to form syncytia.
  • the F protein is a type I transmembrane surface protein that has a N-terminal cleaved signal peptide and a membrane anchor near the C-terminus.
  • RSV F is synthesized as an inactive Fo precursor that assembles into a homotrimer and is activated by cleavage in the trans-Golgi complex by a cellular endoprotease to yield two disulfide-linked subunits, Fj and F 2 subunits.
  • the N-terminus of the Fi subunit that is created by cleavage contains a hydrophobic domain (the fusion peptide) that inserts directly into the target membrane to initiate fusion.
  • the Fi subunit also contains heptad repeats that associate during fusion, driving a conformational shift that brings the viral and cellular membranes into close proximity (Collins and Crowe, 2007, Fields Virology, 5 th ed., D.M Kipe et al, Lipincott, Williams and Wilkons, p. 1604).
  • SEQ ID NO: 2 depicts a representative RSV F protein.
  • RSV F proteins that are about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 2, and all fragments and variants (including chimeric proteins) thereof.
  • RSV G protein is a type II transmembrane glycoprotein with a single hydrophobic region near the N-terminal end that serves as both an uncleaved signal peptide and a membrane anchor, leaving the C-terminal two-thirds of the molecule oriented externally.
  • RSV G is also expressed as a secreted protein that arises from translational initiation at the second AUG in the ORF (at about amino acid 48), which lies within the signal/anchor. Most of the ectodomain of RSV G is highly divergent between RSV strains ⁇ Id., p. 1607).
  • SEQ ID NO: 3 depicts a representative RSV G protein.
  • RSV N proteins that are about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 3, and all fragments and variants (including chimeric proteins) thereof.
  • RSV N protein binds tightly to both genomic RNA and the replicative intermediate anti-genomic RNA to form RNAse resistant nucleocapsid.
  • SEQ ID NO: 4 depicts a representative RSV N protein. Encompassed in this invention are RSV N proteins that are about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 4, and all fragments and variants (including chimeric proteins) thereof.
  • the invention encompasses RSV VLPs that can be formulated into vaccines or antigenic formulations for protecting vertebrates ⁇ e.g. humans) against RSV infection or at least one disease symptom thereof.
  • the present invention also relates to RSV VLPs and vectors comprising wild-type and mutated RSV genes or a combination thereof derived from different strains of RSV virus, which when transfected into host cells, will produce virus like particles (VLPs) comprising RSV proteins.
  • RSV virus-like particles may include at least a viral matrix protein (e.g. RSV M and/or RSV N proteins) and at least one viral surface envelope protein (e.g. RSV F and/or G proteins).
  • RSV M protein e.g. RSV M and/or RSV N proteins
  • RSV F and/or G proteins viral surface envelope protein
  • the inventors have discovered that expressing RSV M protein in cells induces VLP formation.
  • the inventors discovered that the VLPs produced using BRSV M protein are more efficiently produced as compared to VLPs using the HRSV M protein and that the BRSV M is expressed at higher levels than HRSV M protein in various cells, including insect cells.
  • one embodiment of the invention comprises RSV VLPs wherein the VLPs are formed from the expression of BRSV M protein.
  • VLPs of the invention further comprise at least one immunogen, such as but is not limited to a viral surface envelope RSV protein, such as HRSV surface protein.
  • the envelope RSV protein comprises RSV F protein.
  • the RSV F protein can be from the same or different RSV strain than that of the BRSV M protein.
  • the VLPs further comprise RSV G protein.
  • the G protein may be from HRSV group A.
  • the G protein may be from HRSV group B.
  • the RSV G may be derived from HRSV group B and/or group A.
  • VLPs of the invention comprise RSV N and/or P protein.
  • VLPs of the invention comprise heterologous immunogens, such as influenza hemagglutinin (HA) and/or neuraminidase (NA).
  • the invention also comprises combinations of different RSV F, N and/or G proteins from same and/or different strains in one or more VLPs.
  • the invention comprises co-expression of BRSV M protein and at least one immunogen such as but not limited to RSV F, N and/or G protein.
  • the invention comprises co-expression of BRSV M protein and at least one immunogen such as HRSV F, N and/or G protein.
  • the VLPs can include one or more additional molecules for the enhancement of an immune response.
  • the RSV VLPs can carry agents such as nucleic acids, siRNA, microRNA, chemotherapeutic agents, imaging agents, and/or other agents that need to be delivered to a patient.
  • Chimeric VLPs are VLPs having at least two proteins in the VLPs, wherein one protein can drive VLP formation (e.g. BRSV M) and the other protein is from a heterologous infectious agent or from more than one strain, group, subtype etc. of the same or unrelated agent.
  • the infectious agent may be a virus, a bacterium, a parasite, a fungi or a protozoan. Proteins from the infectious agent may have antigenic variations of the same protein or can be a protein from an unrelated agent.
  • chimeric VLPs of the invention comprise BRSV M protein and RSV F proteins from the same or different RSV strains.
  • chimeric VLPs can comprise F protein from HRSV group A and HRSV group B.
  • chimeric VLPs can comprise BRSV M protein and G protein from HRSV group A and HRSV group B.
  • chimeric VLPs can comprise HRSV F protein from group A and HRSV G protein from group B, or vice a versa.
  • chimeric VLPs of the invention further comprise HA and/or NA from influenza virus, or other known viral proteins, and F and/or G proteins from RSV, including but not limited to HRSV F and/or G protein.
  • the chimeric VLPs of the present invention comprising at least two proteins may have at least a heterologous protein from a disease cell other than an infectious agent.
  • a diseased cell such as a cancer cell
  • proteins from a diseased cell include but not limited to melanoma antigens, e.g., MART-I, gp 100, MAGE-3, MAGE-6, MAGE-D; breast cancer antigens, e.g., CA-153, HER2-Neu; NY-ESO-I and other cancer antigens known to a person skill in the art.
  • VLPs of the invention are useful for preparing vaccines and immunogenic compositions.
  • One important feature of VLPs is the ability to express surface proteins of interest so that the immune system of a vertebrate induces an immune response against the protein of interest.
  • not all proteins can be expressed on the surface of VLPs.
  • certain proteins are not expressed, or be poorly expressed, on the surface of VLPs.
  • One reason is that the protein is not directed to the membrane of a host cell or that the protein does not have a transmembrane domain.
  • sequences near the carboxyl terminus of influenza hemagglutinin may be important for incorporation of HA into the lipid bilayer of the mature influenza enveloped nucleocapsids and for the assembly of HA trimer interaction with the influenza matrix protein Ml (AIi, et al, (2000) J. Virol. 74, 8709-19).
  • one embodiment of the invention comprises chimeric VLPs comprising a M protein from BRSV and at least one immunogen which is not normally efficiently expressed on the cell surface or is not a normal RSV protein.
  • the BRSV M protein is fused to the immunogen of interest.
  • the BRSV M protein associates with the immunogen via the transmembrane domain and cytoplasmic tail of a heterologous viral surface membrane protein, e.g., MMTV envelope protein.
  • the chimeric VLPs comprise a M protein from BRSV, a portion of an RSV structural protein such as F or G protein and at least one immunogen or heterologous immunogen is fused to the transmembrane domain and/or cytoplasmic tail of the RSV F or G, including but not limited to human, bovine or avian RSV F or G proteins.
  • the chimeric RSV structural protein is fused to the immunogen via the transmembrane domain and cytoplasmic tail of F and/or G protein.
  • the transmembrane domain and cytoplasmic tail of influenza HA protein are fused to the F and/or G protein by removing and replacing the transmembrane domain and cytoplasmic tail of the F and/or G protein with influenza HA protein transmembrane domain and cytoplasmic tail.
  • the transmembrane domain and cytoplasmic tail of influenza HA protein are fused to the F and/or G protein by removing and replacing the transmembrane domain and cytoplasmic tail of the influenza HA protein with RSV F and/or G protein transmembrane domain and cytoplasmic tail.
  • influenza HA is from A/Indonesia (HlNl or H5N1) or A/Brisbane (H3N2).
  • VLPs comprising chimeric BRSV M protein, a RSV F and/or G protein from HRSV group A and/or group B.
  • the G protein (group A and/or B) is fused to the transmembrane domain and cytoplasmic tail of RSV F protein.
  • the HA protein is fused to the transmembrane domain and cytoplasmic tail of the RSV F and/or G protein.
  • the chimeric VLPs comprise a chimeric F protein, NA and/or HA from influenza virus and M protein from BRSV.
  • VLP constructs as disclosed by the inventors in the present invention take advantage of the capability and efficiency of the BRSV M protein to make virus particles.
  • the BRSV M-proteinVLPs co- expressed with RSV F protein have been found by the inventors to express unexpected high levels of BRSV M protein as well as RSV F protein and are highly efficient in VLP formation.
  • VLPs of the invention comprise VLPs comprising a BRSV M protein and at least one protein from a heterologous infectious agent.
  • heterologous infectious agent include but are not limited to a virus, a bacterium, a protozoan, a fungi and/or a parasite.
  • the immunogen from another infectious agent is a heterologous viral protein.
  • the protein from a heterologous infectious agent is an envelope-associated protein.
  • the protein from another heterologous infectious agent is expressed on the surface of VLPs.
  • viral surface envelope proteins include a precursor fusion protein (F 0 ), a fusion protein (Fi and F 2 ), and/or a glycoprotein (G).
  • the protein from an infectious agent comprises an epitope that will generate a protective immune response in a vertebrate.
  • the protein from another infectious agent is co-expressed with a RSV M protein.
  • the protein from another infections agent is co-expressed with a BRSV M protein.
  • the protein from another infectious agent is fused to a RSV protein.
  • only a portion of a protein from another infectious agent is fused to a RSV protein.
  • only a portion of a protein from another infectious agent is fused to a portion of a RSV protein.
  • the portion of the protein from another infectious agent fused to RSV protein is expressed on the surface of VLPs.
  • the RSV protein, or portion thereof is derived from BRSV M protein, RSV F, G, N and/or P proteins.
  • heterologous proteins may associate with RSV naturally.
  • some heterologous proteins and even some native RSV proteins may need to be engineered to make them associate with RSV or BRSV M protein (the term associate with BRSV M implies both direct association and indirect association).
  • the protein from another infectious agent can associated directly with BRSV M protein.
  • the protein from another agent, or portion thereof is fused to a protein, including but not limited to a BRSV, HRSV or avian RSV protein that associates with the BRSV M protein.
  • an approach to make chimeric VLPs comprising heterologous proteins and BRSV M is to engineer chimeric molecules with RSV proteins that can associate with BRSV M by fusing heterologous proteins (or native RSV proteins) to the BRSV M protein or RSV proteins.
  • heterologous proteins or native RSV proteins
  • the external domains of proteins from infective agents such as VZV, metapneumovirus, dengue, influenza, HIV, MMTV and/or other proteins can be used to generate chimeric molecules by fusing the proteins to RSV proteins, or portion thereof, that associates with BRSV M.
  • proteins include but are not limited RSV F and/or G protein from human, bovine, avian or other species.
  • the invention comprises a VLP comprising a chimeric molecule comprising the transmembrane domain and/or cytoplasmic tail of RSV F or G fused to heterologous proteins.
  • the transmembrane domain and/or cytoplasmic tail of the G protein extends from the N-terminus to the approximately 0, 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 to about 50 amino acids past the transmembrane domain and is fused to a protein from another infectious agent.
  • the transmembrane domain and/or cytoplasmic tail of the F protein extends from the N-terminus to the approximately 0, 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 to about 50 amino acids past the transmembrane domain and is fused to a protein from another infectious agent.
  • the portion of the F or G protein that comprises the cytoplasmic and the transmembrane domain is fused to a portion of the protein from another infectious agent.
  • the portion of the protein from another infectious agent is HA and/or NA from influenza virus (all serotypes, including avian and human strains).
  • the HA and/or NA do not comprise their natural cytoplasmic and/or transmembrane domain.
  • the cytoplasmic and/or transmembrane domain of HA and/or NA is replaced with the transmembrane and/or cytoplasmic domains of RSV G and/or F proteins.
  • the VLP comprises RSV N and/or P protein.
  • the protein from an infectious agent is fused to the RSV N protein.
  • the protein from an infectious agent is HA and/or NA from influenza virus (all serotypes, including avian and human strains).
  • the protein from an infectious agent is fused to the RSV M protein.
  • the protein from an infectious agent is fused to the BRSV M protein.
  • the chimeric genes which may be codon optimized, are synthesized and cloned through a series of steps into a bacmid construct followed by rescue of recombinant baculovirus by plaque isolation and expression analyses.
  • the VLPs for each of these targets can then be rescued by co-infection with the use of two recombinant baculoviruses (1) expressing the RSV M, and (2) expressing the chimeric protein from an infectious agent (e.g.
  • VZV, metapneumovirus, HIV, MMTV, RSV, dengue, influenza with cytoplasmic and transmembrane domain of RSV F or G protein from, e.g., human, bovine or avian RSV.
  • the VLPs for each of these targets can then be rescued by infection with a recombinant baculovirus expressing the BRSV M, and the chimeric protein from an infectious agent (e.g. VZV, metapneumovirus, HIV, MMTV, RSV, dengue, influenza) with cytoplasmic and transmembrane domain of RSV F or G from, e.g., human, bovine or avian RSV.
  • an infectious agent e.g. VZV, metapneumovirus, HIV, MMTV, RSV, dengue, influenza
  • the VLPs for each of these targets can then be rescued by infection with a recombinant baculovirus expressing the BRSV M, and the chimeric protein from an infectious agent (e.g. VZV, metapneumovirus, HIV, RSV, dengue, influenza) with cytoplasmic and transmembrane domain of MMTV env protein.
  • infectious agent e.g. VZV, metapneumovirus, HIV, RSV, dengue, influenza
  • Infectious agents can be viruses, bacteria, protozoa and/or parasites.
  • a protein that may be expressed on the surface of RSV VLPs can be derived from viruses, bacteria, fungi, protozoa, and/or parasites.
  • the proteins derived from viruses, bacteria, fungi, protozoa and/or parasites can induce an immune response (cellular and/or humoral) in a vertebrate that will prevent, treat, manage and/or ameliorate an infectious disease in the vertebrate.
  • Non-limiting examples of virus families from which the infectious agent proteins can be derived are: Paramyxoviridae; Orthomyxoviridae; Hepdnaviridae; Adenoviridae; Poxviridae; Herpesviridae; Papillomaviridae; Polyomaviridae; Parvoviridae; Rhabdoviridae; Filoviridae; Bunyaviridae; Arenaviridae; Coronaviridae; Flaviviradae; Togaviridae; Picornaviridae; Caliciviridae; Astroviridae; Retroviridae and Reoviridae.
  • influenza A and B, e.g. HA and/or NA
  • coronavirus e.g. SARS
  • hepatitis viruses A, B, C, D & E3, human immunodeficiency virus (HIV)
  • HAV human immunodeficiency virus
  • herpes viruses 1, 2, 6 & 7, cytomegalovirus varicella zoster
  • papilloma virus Epstein Barr virus
  • parainfluenza viruses adenoviruses
  • bunya viruses e.g.
  • hanta virus coxsackie viruses, picona viruses, rotaviruses, rhinoviruses, rubella virus, mumps virus, measles virus, Rubella virus, polio virus (multiple types), adeno virus (multiple types), parainfluenza virus (multiple types), avian influenza (various types), shipping fever virus, Western and Eastern equine encephalomyelitis, Japanese encephalomyelitis, fowl pox, rabies virus, slow brain viruses, Rous sarcoma virus, Papovaviridae, Parvoviridae, Piconaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), Togaviridae (e.g., Rubivirus), Newcastle disease virus, West Nile fever virus, Tick borne encephalitis, yellow fever
  • the specific proteins from viruses may comprise: HA and/or NA from influenza virus (including avian); S protein from coronavirus; gpl60, gpl40 and/or gp41 from HIV; gp I to IV and Vp from varicella zoster; E and preM/M from yellow fever virus, Dengue (all serotypes) or any flavivirus. Also included are any proteins from a virus that can induce an immune response (cellular and/or humoral) in a vertebrate that can prevent, treat, manage and/or ameliorate an infectious disease in the vertebrate.
  • Non-limiting examples of bacteria from which the infectious agent proteins can be derived are the following: B. pertussis; Leptospira Pomona; S. paratyphi A and B; C. diphtheriae, C. tetani, C. botulinum, C. perfringens, C. feseri and other gas gangrene bacteria; B. anthracis; P. pestis; P. multocida; Neisseria meningitides; N.
  • gonorrheae Hemophilus influenzae; Actinomyces (e.g., Norcardia); Acinetobacter; Bacillaceae (e.g., Bacillus anthrasis); Bacteroides (e.g., Bacteroides fragilis); Blastomycosis; Bordetella (Bordetella pertusi); Borrelia (e.g., Borrelia burgdorferi); Brucella; Campylobacter; Chlamydia; Cocci dioides; Corynebacterium (e.g., Corynebacterium diptheriae); E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E.
  • Bacillaceae e.g., Bacillus anthrasis
  • Bacteroides e.g., Bacteroides fragilis
  • Blastomycosis e.g., Bordetella pertusi
  • Borrelia
  • Enterobacter e.g. Enterobacter aerogenes
  • Enterobacteriaceae Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Serratia, Yersinia, Shigella); Erysipelothrix; Haemophilus (e.g., Haemophilus influenza type B); Helicobacter; Legionella (e.g., Legionella pneumophila); Leptospira; Listeria (e.g., Listeria monocytogenes); Mycoplasma; Mycobacterium (e.g., Mycobacterium leprae, Mycobacterium tuberculosis and Mycobacterium bovis); Vibrio (e.g., Vibrio cholerae); Pasteurellacea ⁇ Pasteurella haemolytica); Proteus; Pseudomonas ⁇ e.g., Pseudomonas aeruginosa);
  • Non-limiting examples of parasites or protozoa from which the infectious agent proteins can be derived from are the following: leishmaniasis ⁇ Leishmania tropica mexicana, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania chagasi); trypanosomiasis ⁇ Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense); toxoplasmosis ⁇ Toxoplasma gondii); schistosomiasis ⁇ Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Schistosoma mekongi, Schistosoma intercalatum); malaria ⁇ Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium oval
  • the invention also encompasses variants of the proteins expressed on or in the
  • the variants may contain alterations in the amino acid sequences of the constituent proteins.
  • the term "variant" with respect to a protein refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence.
  • the variant can have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine.
  • a variant can have "nonconservative” changes, e.g., replacement of a glycine with a tryptophan.
  • Analogous minor variations can also include amino acid deletion or insertion, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software.
  • Natural variants can occur due to mutations in the proteins. These mutations may lead to antigenic variability within individual groups of infectious agents, for example influenza. Thus, a person infected with, for example, an influenza strain develops antibody against that virus, as newer virus strains appear, the antibodies against the older strains no longer recognize the newer virus and re-infection can occur.
  • the invention encompasses all antigenic and genetic variability of proteins from infectious agents for making VLPs.
  • the invention also encompasses using known methods of protein engineering and recombinant DNA technology to improve or alter the characteristics of the proteins expressed on or in the VLPs of the invention.
  • Various types of mutagenesis can be used to produce and/or isolate variant nucleic acids that encode for protein molecules and/or to further modify/mutate the proteins in or on the VLPs of the invention.
  • mutagenesis include but are not limited to site-directed, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like. Mutagenesis, e.g., involving chimeric constructs, is also included in the present invention. In one embodiment, mutagenesis can be guided by known information of the naturally occurring molecule or altered or mutated naturally occurring molecule, e.g., sequence, sequence comparisons, physical properties, crystal structure or the like.
  • the invention further comprises protein variants which show substantial biological activity, e.g., able to elicit an effective antibody response when expressed on or in VLPs of the invention.
  • Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
  • An example of a mutation is to remove the cleavage site in the RSV F protein and/or remove or add a glycosylation site.
  • the gene encoding a specific RSV protein can be isolated by RT-PCR from polyadenylated mRNA extracted from cells which had been infected with a RSV virus.
  • the resulting product gene can be cloned as a DNA insert into a vector.
  • vector refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components.
  • Vectors include plasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell.
  • a vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating.
  • the vectors of the present invention are plasmids or bacmids.
  • the invention comprises nucleotides that encode proteins, including chimeric molecules, cloned into an expression vector that can be expressed in a cell that induces the formation of VLPs of the invention.
  • An "expression vector” is a vector, such as a plasmid that is capable of promoting expression, as well as replication of a nucleic acid incorporated therein.
  • the nucleic acid to be expressed is “operably linked" to a promoter and/or enhancer, and is subject to transcription regulatory control by the promoter and/or enhancer.
  • the nucleotides encode for a chimeric BRSV M protein (as discussed above).
  • the vector further comprises nucleotides that encode the F and/or G RSV proteins.
  • the vector comprises nucleotides that encode the M, F, G and/or N RSV proteins.
  • the vector comprises nucleotides that encode the chimeric BRSV M protein, HRSV F and/or G protein, or influenza HA and/or NA protein.
  • the nucleotides encode a chimeric RSV G and/or F protein with an influenza HA and/or NA protein.
  • the expression vector is a baculovirus vector.
  • the BRSV M amino acid sequence or nucleic acid sequence is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 1 or SEQ ID NO: 7, respectively.
  • the invention also utilizes nucleic acids and polypeptides which encode a
  • the RSV F amino acid sequence or nucleic acid sequence is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 2 or SEQ ID NO: 8, respectively.
  • the invention also utilizes nucleic acids and polypeptides which encode a
  • the RSV G amino acid sequence or nucleic acid sequence is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3 or SEQ ID NO: 9, respectively.
  • the invention also utilizes nucleic acids and polypeptides which encode a
  • RSV N protein In one embodiment, the RSV N amino acid sequence or nucleic acid sequence is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4 or SEQ ID NO: 10, respectively.
  • the invention also utilizes nucleic acids and polypeptides which encode a
  • the RSV M amino acid sequence or nucleic acid sequence is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5 or SEQ ID NO: 11 , respectively.
  • the invention also provides chimeric molecules made from HRSV F and/or G proteins with a BRSV F 0 cytoplasmic domain replacement.
  • the chimeric molecule's amino acid or nucleic acid sequence is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 6 or SEQ ID NO: 12, respectively.
  • the proteins may comprise mutations containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded protein or how the proteins are made.
  • Nucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by insect cells such as Sf9 cells. See U.S. patent publication 2005/0118191, herein incorporated by reference in its entirety for all purposes.
  • nucleotides can be sequenced to ensure that the correct coding regions were cloned and do not contain any unwanted mutations.
  • the nucleotides can be subcloned into an expression vector (e.g. baculovirus) for expression in any cell.
  • an expression vector e.g. baculovirus
  • the above is only one example of how the RSV viral proteins can be cloned. A person with skill in the art understands that additional methods are available and are possible.
  • the invention also provides for constructs and/or vectors that comprise RSV nucleotides that encode for RSV structural genes, including M, F, G, N or portions thereof, and/or any chimeric molecule described above.
  • the vector may be, for example, a phage, plasmid, viral, or retroviral vector.
  • the constructs and/or vectors that comprise RSV structural genes, including M, F, G, N or portions thereof, and/or any chimeric molecule described above, should be operatively linked to an appropriate promoter, such as the AcMNPV polyhedrin promoter (or other baculovirus), phage lambda PL promoter, the E.
  • the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome-binding site for translation.
  • the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
  • Expression vectors will preferably include at least one selectable marker.
  • virus vectors such as baculovirus, poxvirus (e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoonpox virus, swinepox virus, etc.), adenovirus (e.g., canine adenovirus), herpesvirus, and retrovirus.
  • poxvirus e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoonpox virus, swinepox virus, etc.
  • adenovirus e.g., canine adenovirus
  • herpesvirus e.g., canine adenovirus
  • retrovirus e.g., canine adenovirus
  • vectors for use in bacteria comprise vectors for use in bacteria, which comprise pQE70, pQE60 and pQE-9, pBluescript vectors, Phagescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5.
  • preferred eukaryotic vectors are pFastBacl pWINEO, ⁇ SV2CAT, pOG44, pXTl and pSG, pSVK3, pBPV, pMSG, and pSVL.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • the vector that comprises nucleotides encoding for RSV genes is pFastBac.
  • the vector that comprises an insert that consists of nucleotides encoding for RSV genes, comprising BRSV M and RSV F is pFastBac.
  • the vector that comprises an insert that consists of nucleotides encoding for RSV genes, comprising BRSV M, and RSV G protein is pFastBac.
  • the vector that comprises an insert that consists of nucleotides encoding for RSV genes, comprising BRSV M, and RSV F and G is pFastBac.
  • the vector that comprises an insert that consists of nucleotides encoding for RSV genes, comprising BRSV M and N is pFastBac.
  • the vector that comprises an insert that consists of nucleotides encoding for RSV genes, comprising BRSV M, and RSV F and N is pFastBac.
  • the vector that comprises an insert that consists of nucleotides encoding for RSV genes comprising chimeric BRSV M, and RSV G and N.
  • the vector that comprises an insert that comprises nucleotides encoding for BRSV M protein and at least one protein or immunogen from another infectious agent is pFastBac.
  • the vector that comprises an insert that consists of nucleotides encoding for BRSV M protein and at least one protein from another infectious agent is pFastBac.
  • the vector comprises one or more of SEQ ID NOs: 7-12.
  • the recombinant constructs mentioned above could be used to transfect, infect, or transform and can express chimeric RSV proteins, including BRSV M protein and at least one immunogen.
  • the recombinant construct comprises BRSV M protein; RSV F, G, N, or portions thereof, and/or any chimeric molecule described above, into eukaryotic cells and/or prokaryotic cells.
  • the invention provides for host cells which comprise a vector (or vectors) that contain nucleic acids which code for RSV structural genes, including BRSV M; and at least one immunogen such as but not limited to RSV F, G, N, or portions thereof, and/or any chimeric molecule described above, and permit the expression of chimeric genes, including BRSV M; RSV F, G, N, or portions thereof, and/or any chimeric molecule described above in the host cell under conditions which allow the formation of VLPs.
  • eukaryotic host cells are yeast, insect, avian, plant, C. elegans (or nematode) and mammalian host cells.
  • Non limiting examples of insect cells are, Spodoptera frugiperda (Sf) cells, e.g. Sf9, Sf21, Trichoplusia ni cells, e.g. High Five cells, and Drosophila S2 cells.
  • fungi including yeast host cells are S. cerevisiae, Kluyveromyces lactis (K. lactis), species of Candida including C. albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica.
  • mammalian cells examples include COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, and African green monkey cells, CVl cells, HeLa cells, MDCK cells, Vero and Hep-2 cells. Xenopus laevis oocytes, or other cells of amphibian origin, may also be used.
  • prokaryotic host cells include bacterial cells, for example, E. coli, B. subtilis, Salmonella typhi and mycobacteria.
  • Vectors e.g., vectors comprising polynucleotides of BRSV M protein; and at least one immunogen including but not limited to RSV F, G, N, or portions thereof, and/or any chimeric molecule described above, can be transfected into host cells according to methods well known in the art.
  • introducing nucleic acids into eukaryotic cells can be by calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection employing polyamine transfection reagents.
  • the vector is a recombinant baculovirus.
  • the recombinant baculovirus is transfected into a eukaryotic cell.
  • the cell is an insect cell.
  • the insect cell is a Sf9 cell.
  • the vector and/or host cell comprise nucleotides that encode chimeric RSV genes, BRSV M protein and at least one immunogen including but not limited to RSV F, G, or portions thereof, and/or any chimeric molecule described above.
  • the vector and/or host cell consists essentially of BRSV M, RSV F, G, N, or portions thereof, and/or any chimeric or heterologous molecule described above.
  • the vector and/or host cell consists of RSV protein comprising BRSV M, RSV F, G, N, or portions thereof, and/or any chimeric or heterologous molecule described above.
  • vectors and/or host cells contain BRSV M, RSV F, G, N, or portions thereof, and/or any chimeric or heterologous molecule described above, and may contain additional cellular constituents such as cellular proteins, baculovirus proteins, lipids, carbohydrates etc.
  • This invention also provides for constructs and methods that will increase the efficiency of VLPs production. For example, the addition of leader sequences to the BRSV M protein, RSV F, G, N, or portions thereof, and/or any chimeric or heterologous molecules described above, can improve the efficiency of protein transporting within the cell.
  • a heterologous signal sequence can be fused to the BRSV M, RSV F, G, N, or portions thereof, and/or any chimeric or heterologous molecule described above.
  • the signal sequence can be derived from the gene of an insect cell and fused to BRSV M, RSV F, G, N, or portions thereof, and/or any chimeric or heterologous molecules described above.
  • the signal peptide is the chitinase signal sequence, which works efficiently in baculovirus expression systems.
  • Another method to increase efficiency of VLP production is to codon optimize the nucleotides that encode RSV including BRSV M protein, RSV F, G, N, or portions thereof, and/or any chimeric or heterologous molecules described above for a specific cell type.
  • codon optimizing nucleic acids for expression in Sf9 cell see SEQ ID NOs: 7-12.
  • the invention also provides for methods of producing VLPs, the methods comprising expressing RSV genes include BRSV M, and at least an immunogen including but not limited to RSV F, G, N, or portions thereof, and/or any chimeric or heterologous molecules described above under conditions that allow VLP formation.
  • the VLPs are produced by growing host cells transformed by an expression vector under conditions whereby the recombinant proteins are expressed and VLPs are formed.
  • the invention comprises a method of producing a VLP, comprising transfecting vectors encoding at least one BRSV M protein into a suitable host cell and expressing the immunogen such as a RSV virus protein under conditions that allow VLP formation.
  • the eukaryotic cell is selected from the group consisting of, yeast, insect, amphibian, avian or mammalian cells.
  • the selection of the appropriate growth conditions is within the skill or a person with skill of one of ordinary skill in the art.
  • Methods to grow cells engineered to produce VLPs of the invention include, but are not limited to, batch, batch- fed, continuous and perfusion cell culture techniques.
  • Cell culture means the growth and propagation of cells in a bioreactor (a fermentation chamber) where cells propagate and express protein (e.g. recombinant proteins) for purification and isolation.
  • protein e.g. recombinant proteins
  • cell culture is performed under sterile, controlled temperature and atmospheric conditions in a bioreactor.
  • a bioreactor is a chamber used to culture cells in which environmental conditions such as temperature, atmosphere, agitation and/or pH can be monitored.
  • the bioreactor is a stainless steel chamber.
  • the bioreactor is a pre-sterilized plastic bag (e.g. Cellbag ® , Wave Biotech, Bridgewater, NJ).
  • the pre-sterilized plastic bags are about 50 L to 1000 L bags.
  • VLPs are then isolated using methods that preserve the integrity thereof, such as by gradient centrifugation, e.g., cesium chloride, sucrose and iodixanol, as well as standard purification techniques including, e.g., ion exchange and gel filtration chromatography.
  • gradient centrifugation e.g., cesium chloride, sucrose and iodixanol
  • standard purification techniques including, e.g., ion exchange and gel filtration chromatography.
  • VLPs of the invention can be made, isolated and purified.
  • VLPs are produced from recombinant cell lines engineered to create VLPs when the cells are grown in cell culture (see above).
  • a person of skill in the art would understand that there are additional methods that can be utilized to make and purify VLPs of the invention, thus the invention is not limited to the method described.
  • Production of VLPs of the invention can start by seeding Sf9 cells (non- infected) into shaker flasks, allowing the cells to expand and scaling up as the cells grow and multiply (for example from a 125-ml flask to a 50 L Wave bag).
  • the medium used to grow the cell is formulated for the appropriate cell line (preferably serum free media, e.g. insect medium ExCell-420, JRH).
  • the cells are infected with recombinant baculovirus at the most efficient multiplicity of infection (e.g. from about 1 to about 3 plaque forming units per cell).
  • the BRSV M, RSV F, G, N, or portions thereof, and/or any chimeric or heterologous molecule described above are expressed from the virus genome, self assemble into VLPs and are secreted from the cells approximately 24 to 72 hours post infection. Usually, infection is most efficient when the cells are in mid-log phase of growth (4-8 x 10 6 cells/ml) and are at least about 90% viable.
  • VLPs of the invention can be harvested approximately 48 to 96 hours post infection, when the levels of VLPs in the cell culture medium are near the maximum but before extensive cell lysis.
  • the Sf9 cell density and viability at the time of harvest can be about 0.5 x 10 6 cells/ml to about 1.5 x 10 6 cells/ml with at least 20% viability, as shown by dye exclusion assay.
  • the medium is removed and clarified. NaCl can be added to the medium to a concentration of about 0.4 to about 1.0 M, preferably to about 0.5 M, to avoid VLP aggregation.
  • the removal of cell and cellular debris from the cell culture medium containing VLPs of the invention can be accomplished by tangential flow filtration (TFF) with a single use, pre-sterilized hollow fiber 0.5 or 1.00 ⁇ m filter cartridge or a similar device.
  • TMF tangential flow filtration
  • VLPs in the clarified culture medium can be concentrated by ultrafiltration using a disposable, pre-sterilized 500,000 molecular weight cut off hollow fiber cartridge.
  • the concentrated VLPs can be diafiltrated against 10 volumes pH 7.0 to 8.0 phosphate-buffered saline (PBS) containing 0.5 M NaCl to remove residual medium components.
  • PBS phosphate-buffered saline
  • the concentrated, diafiltered VLPs can be furthered purified on a 20% to 60% discontinuous sucrose gradient in pH 7.2 PBS buffer with 0.5 M NaCl by centrifugation at 6,500 x g for 18 hours at about 4° C to about 10° C.
  • VLPs will form a distinctive visible band between about 30% to about 40% sucrose or at the interface (in a 20% and 60% step gradient) that can be collected from the gradient and stored.
  • This product can be diluted to comprise 200 mM of NaCl in preparation for the next step in the purification process.
  • This product contains VLPs and may contain intact baculovirus particles.
  • VLPs Further purification of VLPs can be achieved by anion exchange chromatography, or 44% isopycnic sucrose cushion centrifugation.
  • anion exchange chromatography the sample from the sucrose gradient (see above) is loaded into column containing a medium with an anion (e.g. Matrix Fractogel EMD TMAE) and eluded via a salt gradient (from about 0.2 M to about 1.0 M of NaCl) that can separate the VLP from other contaminates (e.g. baculovirus and DNA/RNA).
  • a medium with an anion e.g. Matrix Fractogel EMD TMAE
  • a salt gradient from about 0.2 M to about 1.0 M of NaCl
  • the sucrose cushion method the sample comprising the VLPs is added to a 44% sucrose cushion and centrifuged for about 18 hours at 30,000 g.
  • VLPs form a band at the top of 44% sucrose, while baculovirus precipitates at the bottom and other contaminating proteins stay in the 0% sucrose layer at the top.
  • the VLP peak or band is collected.
  • the intact baculovirus can be inactivated, if desired. Inactivation can be accomplished by chemical methods, for example, formalin or ⁇ -propiolactone (BPL). Removal and/or inactivation of intact baculovirus can also be largely accomplished by using selective precipitation and chromatographic methods known in the art, as exemplified above. Methods of inactivation comprise incubating the sample containing the VLPs in 0.2% of BPL for 3 hours at about 25 °C to about 27 0 C. The baculovirus can also be inactivated by incubating the sample containing the VLPs at 0.05% BPL at 4 °C for 3 days, then at 37 0 C for one hour.
  • the product comprising VLPs can be run through another diafiltration step to remove any reagent from the inactivation step and/or any residual sucrose, and to place the VLPs into the desired buffer (e.g. PBS).
  • the solution comprising VLPs can be sterilized by methods known in the art (e.g. sterile filtration) and stored in the refrigerator or freezer.
  • the above techniques can be practiced across a variety of scales. For example, T-flasks, shake-flasks, spinner bottles, up to industrial sized bioreactors.
  • the bioreactors can comprise either a stainless steel tank or a pre-sterilized plastic bag (for example, the system sold by Wave Biotech, Bridgewater, NJ). A person with skill in the art will know what is most desirable for their purposes.
  • Expansion and production of baculovirus expression vectors and infection of cells with recombinant baculovirus to produce recombinant RSV VLPs can be accomplished in insect cells, for example Sf9 insect cells as previously described.
  • the cells are SF9 infected with recombinant baculovirus engineered to produce RSV VLPs.
  • compositions useful herein contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to the vertebrate receiving the composition, and which may be administered without undue toxicity and a VLP of the invention.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopia, European Pharmacopia or other generally recognized pharmacopia for use in mammals, and more particularly in humans.
  • These compositions can be useful as a vaccine and/or antigenic compositions for inducing a protective immune response in a vertebrate.
  • the invention encompasses a pharmaceutically acceptable vaccine composition comprising VLPs which comprises at least one RSV protein.
  • the pharmaceutically acceptable vaccine composition comprises VLPs comprising RSV M protein and at least one immunogen.
  • the pharmaceutically acceptable vaccine composition comprises VLPs comprising BRSV M protein and at least one immunogen.
  • the pharmaceutically acceptable vaccine composition comprises VLPs further comprising RSV F protein, such as but not limited to a HRSV F protein.
  • the pharmaceutically acceptable vaccine composition comprises VLPs further comprising RSV G protein, including but not limited to a HRSV G protein.
  • the pharmaceutically acceptable vaccine composition comprises VLPs further comprising RSV N protein, including but not limited to a HRSV, BRSV or avian RSV.
  • the pharmaceutically acceptable vaccine composition comprises VLPs comprising BRSV M protein and F and/or G protein from HRSV group A.
  • the pharmaceutically acceptable vaccine composition comprises VLPs comprising BRSV M protein and F and/or G protein from HRSV group B.
  • the invention encompasses a pharmaceutically acceptable vaccine composition comprising chimeric VLPs such as VLPs comprising chimeric M protein from a BRSV and optionally HA protein derived from an influenza virus, wherein the M protein is fused to the influenza HA protein.
  • the invention encompasses a pharmaceutically acceptable vaccine composition
  • a pharmaceutically acceptable vaccine composition comprising chimeric VLPs such as VLPs comprising BRSV M, and a chimeric F and/or G protein from a RSV and optionally HA protein derived from an influenza virus, wherein the chimeric influenza HA protein is fused to the transmembrane domain and cytoplasmic tail of RSV F and/or G protein.
  • the invention encompasses a pharmaceutically acceptable vaccine composition
  • chimeric VLPs such as VLPs comprising BRSV M and a chimeric F and/or G protein from a RSV and optionally HA or NA protein derived from an influenza virus, wherein the HA or NA protein is a fused to the transmembrane domain and cytoplasmic tail of RSV F and/or G protein.
  • the invention also encompasses a pharmaceutically acceptable vaccine composition comprising a chimeric VLP that comprises at least one RSV protein.
  • the pharmaceutically acceptable vaccine composition comprises VLPs comprising a BRSV M protein and at least one immunogen from a heterologous infectious agent or diseased cell.
  • the immunogen from a heterologous infectious agent is a viral protein.
  • the viral protein from a heterologous infectious agent is an envelope associated protein.
  • the viral protein from a heterologous infectious agent is expressed on the surface of VLPs.
  • the protein from an infectious agent comprises an epitope that will generate a protective immune response in a vertebrate.
  • the protein from another infectious agent can associate with BRSV M protein.
  • the protein from a heterologous infectious agent is fused to the BRSV M protein.
  • only a portion of a protein from a heterologous infectious agent is fused to the BRSV M protein.
  • only a portion of a protein from a heterologous infectious agent is fused to a portion of the BRSV M protein.
  • the portion of the protein from a heterologous infectious agent fused to the BRSV M protein is expressed on the surface of VLPs.
  • a second RSV protein, or portion thereof, fused to the protein from another infectious agent associates with the BRSV M protein.
  • the RSV protein, or portion thereof is derived from RSV F, G, N and/or P.
  • the chimeric VLPs further comprise N and/or P protein from RSV.
  • the chimeric VLPs comprise more than one protein from an infectious agent.
  • the chimeric VLPs comprise more than one infectious agent proteins, thus creating a multivalent VLP.
  • the invention also encompasses a kit for immunizing a vertebrate, such as a human subject, comprising VLPs that comprise at least one RSV protein.
  • the kit comprises VLPs comprising a BRSV M protein.
  • the kit further comprises a RSV F protein.
  • the kit further comprises a RSV G protein.
  • the kit comprises VLPs comprising a RSV F and/or G protein from HRSV group A.
  • the kit comprises VLPs comprising a RSV F and/or G protein from HRSV group B.
  • the invention encompasses a kit comprising VLPs which comprises a chimeric M protein from a BRSV and optionally HA protein derived from an influenza virus, wherein the M protein is fused to the BRSV M.
  • the invention encompasses a kit comprising VLPs which comprises a chimeric M protein from a BRSV, a RSV F and/or G protein and an immunogen from a heterologous infectious agent.
  • the invention encompasses a kit comprising VLPs which comprises a M protein from a BRSV, a chimeric RSV F and/or G protein and optionally HA protein derived from an influenza virus, wherein the HA protein is fused to the transmembrane domain and cytoplasmic tail of RSV F or G protein.
  • the invention encompasses a kit comprising VLPs which comprises M protein from a BRSV, a chimeric RSV F and/or G protein and optionally HA or NA protein derived from an influenza virus, wherein the HA protein is fused to the transmembrane domain and cytoplasmic tail of RSV F and/or G protein.
  • the invention also encompasses a kit comprising chimeric VLPs that comprise at least one RSV protein.
  • the kit comprises VLPs comprising a BRSV M protein and at least one immunogen from a heterologous infectious agent or diseased cell.
  • the protein from heterologous infectious agent is a viral protein.
  • the protein from heterologous infectious agent is an envelope associated protein.
  • the protein from heterologous infectious agent is expressed on the surface of VLPs.
  • the protein from heterologous infectious agent comprises an epitope that will generate a protective immune response in a vertebrate.
  • the protein from heterologous infectious agent can associate with RSV M protein.
  • the protein from heterologous infectious agent is fused to a second RSV protein other than the BRSV M protein.
  • only a portion of a protein from heterologous infectious agent is fused to a second RSV protein.
  • only a portion of a protein from another infectious agent is fused to a portion of the second RSV protein.
  • the portion of the protein from heterologous infectious agent fused to the second RSV protein is expressed on the surface of VLPs.
  • the RSV protein, or portion thereof, fused to the protein from heterologous infectious agent associates with the BRSV M protein.
  • the RSV protein, or portion thereof is derived from RSV F, G, N and/or P.
  • the chimeric VLPs further comprise N and/or P protein from RSV. In another embodiment, the chimeric VLPs comprise more than one protein from an infectious agent. In another embodiment, the chimeric VLPs comprise more one infectious agent proteins, thus creating a multivalent VLP.
  • the invention encompasses an immunogenic formulation comprising VLPs which comprises at least one RSV protein.
  • the immunogenic formulation comprises VLPs comprising RSV M protein and at least one immunogen.
  • the immunogenic formulation comprises VLPs comprising BRSV M protein and at least one immunogen.
  • the immunogenic formulation comprises VLPs further comprising RSV F protein.
  • the immunogenic formulation composition comprises VLPs further comprising RSV G protein.
  • the immunogenic formulation comprises VLPs further comprising RSV N protein.
  • the immunogenic formulation comprises VLPs comprising F and/or G protein from HRSV group A.
  • the immunogenic formulation comprises VLPs comprising F and/or G protein from HRSV group B.
  • the invention encompasses an immunogenic formulation comprising chimeric VLPs such as VLPs comprising chimeric M protein from BRSV and optionally HA protein derived from an influenza virus, wherein the M protein is fused to the HA protein.
  • the invention encompasses an immunogenic formulation comprising VLPs which comprises M protein from a BRSV, a chimeric RSV F and/or G protein and an immunogen from a heterologous infectious agent.
  • the invention encompasses an immunogenic formulation comprising VLPs which comprises a M protein from a BRSV, a chimeric RSV F and/or G protein and optionally HA protein derived from an influenza virus, wherein the chimeric influenza HA protein is fused to the transmembrane domain and cytoplasmic tail of RSV F and/or G protein.
  • the invention encompasses an immunogenic formulation comprising VLPs which comprises M protein from a BRSV, a chimeric RSV F and/or G protein and optionally HA and/or NA protein derived from an influenza virus, wherein the HA and/or NA protein is fused to the transmembrane domain and cytoplasmic tail of RSV F and/or G protein.
  • the invention also encompasses an immunogenic formulation comprising a chimeric VLP that comprises at least one RSV protein.
  • the immunogenic formulation comprises VLPs comprising a BRSV M protein and at least one immunogen from a heterologous infectious agent or diseased cell.
  • the immunogen from a heterologous infectious agent is a viral protein.
  • the viral protein from a heterologous infectious agent is an envelope associated protein.
  • the viral protein from a heterologous infectious agent is expressed on the surface of VLPs.
  • the protein from an infectious agent comprises an epitope that will generate a protective immune response in a vertebrate.
  • the protein from another infectious agent can associate with BRSV M protein.
  • the protein from a heterologous infectious agent is fused to the BRSV M protein. In another embodiment, only a portion of a protein from a heterologous infectious agent is fused to the BRSV M protein. In another embodiment, only a portion of a protein from a heterologous infectious agent is fused to a portion of the BRSV M protein. In another embodiment, the portion of the protein from a heterologous infectious agent fused to the BRSV M protein is expressed on the surface of VLPs. In other embodiment, a second RSV protein, or portion thereof, fused to the protein from another infectious agent associates with the BRSV M protein. In other embodiment, the second RSV protein, or portion thereof, is derived from RSV F, G, N and/or P.
  • the chimeric VLPs further comprise N and/or P protein from RSV. In another embodiment, the chimeric VLPs comprise more than one protein from an infectious agent. In another embodiment, the chimeric VLPs comprise more than one infectious agent proteins, thus creating a multivalent VLP.
  • the immunogenic formulation of the invention comprises VLPs comprising BRSV M, and RSV F, G, N, or portions thereof, and/or any chimeric or heterologous molecule described above and a pharmaceutically acceptable carrier or excipient.
  • Pharmaceutically acceptable carriers include but are not limited to saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof.
  • saline buffered saline
  • dextrose dextrose
  • water glycerol
  • sterile isotonic aqueous buffer and combinations thereof.
  • the formulation should suit the mode of administration.
  • the formulation is suitable for administration to humans, preferably is sterile, non-particulate and/or non-pyrogenic.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a solid form, such as a lyophilized powder suitable for reconstitution, a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the invention also provides for a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the vaccine formulations of the invention.
  • the kit comprises two containers, one containing VLPs and the other containing an adjuvant.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the invention also provides that the VLP formulation be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of composition.
  • the VLP composition is supplied as a liquid, in another embodiment, as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject.
  • the VLP composition is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the VLP composition.
  • the liquid form of the VLP composition is supplied in a hermetically sealed container at least about 50 ⁇ g/ml, more preferably at least about 100 ⁇ g/ml, at least about 200 ⁇ g/ml, at least 500 ⁇ g/ml, or at least 1 mg/ml.
  • chimeric BRSV VLPs comprising HRSV F and/or G protein of the invention are administered in an effective amount or quantity (as defined above) sufficient to stimulate an immune response, each a response against one or more strains of RSV.
  • Administration of the VLPs of the invention elicits immunity against RSV.
  • the dose can be adjusted within this range based on, e.g., age, physical condition, body weight, sex, diet, time of administration, and other clinical factors.
  • the prophylactic vaccine formulation is systemically administered, e.g., by subcutaneous or intramuscular injection using a needle and syringe, or a needle-less injection device.
  • the vaccine formulation is administered intranasally, either by drops, large particle aerosol (greater than about 10 microns), or spray into the upper respiratory tract. While any of the above routes of delivery results in an immune response, intranasal administration confers the added benefit of eliciting mucosal immunity at the site of entry of many viruses, including RSV and influenza.
  • the invention also comprises a method of formulating a vaccine or antigenic composition that induces immunity to an infection or at least one disease symptom thereof to a mammal, comprising adding to the formulation an effective dose of RSV VLPs.
  • the infection is an RSV infection.
  • While stimulation of immunity with a single dose is possible, additional dosages can be administered, by the same or different route, to achieve the desired effect.
  • multiple administrations may be required to elicit sufficient levels of immunity.
  • Administration can continue at intervals throughout childhood, as necessary to maintain sufficient levels of protection against infections, e.g. RSV infection.
  • adults who are particularly susceptible to repeated or serious infections such as, for example, health care workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary function may require multiple immunizations to establish and/or maintain protective immune responses.
  • Levels of induced immunity can be monitored, for example, by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to elicit and maintain desired levels of protection.
  • compositions comprising VLPs include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral or pulmonary routes or by suppositories).
  • parenteral administration e.g., intradermal, intramuscular, intravenous and subcutaneous
  • epidural e.g., epidural and mucosal
  • mucosal e.g., intranasal and oral or pulmonary routes or by suppositories.
  • compositions of the present invention are administered intramuscularly, intravenously, subcutaneously, transdermally or intradermally.
  • compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucous, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, urinary bladder and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • intranasal or other mucosal routes of administration of a composition comprising VLPs of the invention may induce an antibody or other immune response that is substantially higher than other routes of administration.
  • intranasal or other mucosal routes of administration of a composition comprising VLPs of the invention may induce an antibody or other immune response that will induce cross protection against other strains of RSV. Administration can be systemic or local.
  • the vaccine and/or immunogenic formulation is administered in such a manner as to target mucosal tissues in order to elicit an immune response at the site of immunization.
  • mucosal tissues such as gut associated lymphoid tissue (GALT) can be targeted for immunization by using oral administration of compositions which contain adjuvants with particular mucosal targeting properties.
  • Additional mucosal tissues can also be targeted, such as nasopharyngeal lymphoid tissue (NALT) and bronchial-associated lymphoid tissue (BALT).
  • Vaccines and/or immunogenic formulations of the invention may also be administered on a dosage schedule, for example, an initial administration of the vaccine composition with subsequent booster administrations.
  • a second dose of the composition is administered anywhere from two weeks to one year, preferably from about 1, about 2, about 3, about 4, about 5 to about 6 months, after the initial administration.
  • a third dose may be administered after the second dose and from about three months to about two years, or even longer, preferably about 4, about 5, or about 6 months, or about 7 months to about one year after the initial administration.
  • the third dose may be optionally administered when no or low levels of specific immunoglobulins are detected in the serum and/or urine or mucosal secretions of the subject after the second dose.
  • a second dose is administered about one month after the first administration and a third dose is administered about six months after the first administration.
  • the second dose is administered about six months after the first administration.
  • the VLPs of the invention can be administered as part of a combination therapy.
  • VLPs of the invention can be formulated with other immunogenic compositions, antivirals and/or antibiotics.
  • the dosage of the pharmaceutical composition can be determined readily by the skilled artisan, for example, by first identifying doses effective to elicit a prophylactic or therapeutic immune response, e.g., by measuring the serum titer of virus specific immunoglobulins or by measuring the inhibitory ratio of antibodies in serum samples, or urine samples, or mucosal secretions.
  • the dosages can be determined from animal studies.
  • a non-limiting list of animals used to study the efficacy of vaccines include the guinea pig, hamster, ferrets, chinchilla, mouse and cotton rat. Most animals are not natural hosts to infectious agents but can still serve in studies of various aspects of the disease.
  • any of the above animals can be dosed with a vaccine candidate, e.g. VLPs of the invention, to partially characterize the immune response induced, and/or to determine if any neutralizing antibodies have been produced.
  • a vaccine candidate e.g. VLPs of the invention
  • many studies have been conducted in the mouse model because mice are small size and their low cost allows researchers to conduct studies on a larger scale.
  • the immunogenicity of a particular composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants have been used experimentally to promote a generalized increase in immunity against unknown antigens (e.g., U.S. Pat. No. 4,877,611). Immunization protocols have used adjuvants to stimulate responses for many years, and as such, adjuvants are well known to one of ordinary skill in the art. Some adjuvants affect the way in which antigens are presented. For example, the immune response is increased when protein antigens are precipitated by alum. Emulsif ⁇ cation of antigens also prolongs the duration of antigen presentation.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • Other adjuvants comprise GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • RIBI which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion also is contemplated.
  • MF-59, Novasomes ® , MHC antigens may also be used.
  • the adjuvant is a paucilamellar lipid vesicle having about two to ten bilayers arranged in the form of substantially spherical shells separated by aqueous layers surrounding a large amorphous central cavity free of lipid bilayers.
  • Paucilamellar lipid vesicles may act to stimulate the immune response several ways, as non-specific stimulators, as carriers for the antigen, as carriers of additional adjuvants, and combinations thereof.
  • Paucilamellar lipid vesicles act as non-specific immune stimulators when, for example, a vaccine is prepared by intermixing the antigen with the preformed vesicles such that the antigen remains extracellular to the vesicles.
  • the vesicle acts both as an immune stimulator and a carrier for the antigen.
  • the vesicles are primarily made of nonphospholipid vesicles.
  • the vesicles are Novasomes ® .
  • Novasomes ® are paucilamellar nonphospholipid vesicles ranging from about 100 nm to about 500 nm. They comprise Brij 72, cholesterol, oleic acid and squalene. Novasomes have been shown to be an effective adjuvant for influenza antigens (see, U.S. Patents 5,629,021, 6,387,373, and 4,911,928, herein incorporated by reference in their entireties for all purposes).
  • the VLPs of the invention can also be formulated with "immune stimulators.” These are the body's own chemical messengers (cytokines) to increase the immune system's response. Immune stimulators include, but not limited to, various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-I, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc.
  • interleukins e.g., IL-I, IL-2, IL-3, IL-4, IL-12, IL-13
  • growth factors e.g., granulocyte-macrophage (GM)-colony stimulating factor (
  • the immunostimulatory molecules can be administered in the same formulation as the chimeric BRSV VLPs, or can be administered separately. Either the protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect.
  • the invention comprises antigentic and vaccine formulations comprising an adjuvant and/or an immune stimulator.
  • the VLPs of the invention are useful for preparing compositions that stimulate an immune response that confers immunity or substantial immunity to infectious agents. Both mucosal and cellular immunity may contribute to immunity to infectious agents and disease. Antibodies secreted locally in the upper respiratory tract are a major factor in resistance to natural infection. Secretory immunoglobulin A (slgA) is involved in the protection of the upper respiratory tract and serum IgG in protection of the lower respiratory tract.
  • the immune response induced by an infection protects against reinfection with the same virus or an antigenically similar viral strain. For example, RSV undergoes frequent and unpredictable changes; therefore, after natural infection, the effective period of protection provided by the host's immunity may only be effective for a few years against the new strains of virus circulating in the community.
  • the invention encompasses a method of inducing immunity to infections or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of BRSV M VLPs.
  • the method comprises administering VLPs comprising at least one RSV protein.
  • the method comprises administering VLPs comprising RSV M protein and at least one immunogen.
  • the method comprises administering VLPs comprising BRSV M protein and at least one immunogen.
  • the method comprises administering VLPs comprising RSV F protein, for example, HRSV F protein.
  • the method comprises administering VLPs further comprising RSV N protein.
  • the method comprises administering VLPs further comprising F and/or G protein from HRSV group A and/or group B.
  • the method comprises administering VLPs comprising M protein from BRSV and a chimeric RSV F and/or G protein or MMTV envelope protein, for example, HA or NA protein derived from an influenza virus, wherein the HA and/or NA protein is fused to the transmembrane domain and cytoplasmic tail of the RSV F and/or G protein or MMTV envelope protein.
  • the method comprises administering VLPs comprising M protein from BRSV and a chimeric RSV F and/or G protein and optionally HA or NA protein derived from an influenza virus, wherein the HA or NA protein is fused to the transmembrane domain and cytoplasmic tail of RSV F and/or G protein.
  • the subject is a mammal.
  • the mammal is a human.
  • RSV VLPs are formulated with an adjuvant or immune stimulator.
  • Another embodiment of the invention comprises a method to induce immunity to RSV infection or at least one disease symptom thereof in a subject, comprises administering at least one effective dose of a RSV VLPs, wherein the VLPs comprise RSV M (including chimeric M), F, G, and/or N proteins.
  • a method of inducing immunity to RSV infection or at least one symptom thereof in a subject comprises administering at least one effective dose of a RSV VLPs, wherein the VLPs consists essentially of BRSV M (including chimeric M), and RSV F, G, and/or N proteins.
  • the VLPs may comprise additional RSV proteins and/or protein contaminates in negligible concentrations.
  • a method of inducing immunity to RSV infection or at least one symptom thereof in a subject comprises administering at least one effective dose of a RSV VLPs, wherein the VLPs consists of BRSV M (including chimeric M), RSV G and/or F.
  • a method of inducing immunity to RSV infection or at least one disease symptom in a subject comprises administering at least one effective dose of a RSV VLPs comprising RSV proteins, wherein the RSV proteins consist of BRSV M (including chimeric M), F, G, and/or N proteins, including chimeric F, G, and/or N proteins.
  • VLPs contain BRSV M (including chimeric M), RSV F, G, and/or N proteins and may contain additional cellular constituents such as cellular proteins, baculovirus proteins, lipids, carbohydrates etc., but do not contain additional RSV proteins (other than fragments of BRSV M (including chimeric M), BRSV/RSV F, G, and/or N proteins.
  • the subject is a vertebrate.
  • the vertebrate is a mammal.
  • the mammal is a human.
  • the method comprises inducing immunity to RSV infection or at least one disease symptom by administering the formulation in one dose.
  • the method comprises inducing immunity to RSV infection or at least one disease symptom by administering the formulation in multiple doses.
  • the invention also encompasses inducing immunity to an infection, or at least one symptom thereof, in a subject caused by an infectious agent, comprising administering at least one effective dose of VLPs of the invention.
  • the method comprises administering VLPs comprising a RSV M protein and at least one protein from a heterologous infectious agent.
  • the method comprises administering VLPs comprising a BRSV M protein and at least one protein from the same or a heterologous infectious agent.
  • the protein from the heterologous infectious agent is a viral protein.
  • the protein from the infectious agent is an envelope associated protein.
  • the protein from the infectious agent is expressed on the surface of VLPs.
  • the protein from the infectious agent comprises an epitope that will generate a protective immune response in a vertebrate.
  • the protein from the infectious agent can associate with RSV M protein such as BRSV M protein, RSV F, G and/or N protein.
  • the protein from the infectious agent is fused to a RSV protein such as a BRSV M protein, RSV F, G and/or N . protein.
  • only a portion of a protein from the infectious agent is fused to a RSV protein such as a BRSV M protein, RSV F, G and/or N protein.
  • a portion of a protein from the infectious agent is fused to a portion of a RSV protein such as a BRSV M protein, RSV F, G and/or N protein.
  • the portion of the protein from the infectious agent fused to the RSV protein is expressed on the surface of VLPs.
  • the RSV protein, or portion thereof, fused to the protein from the infectious agent associates with the RSV M protein.
  • the RSV protein, or portion thereof is derived from RSV F, G, N and/or P.
  • the chimeric VLPs further comprise N and/or P protein from RSV.
  • the chimeric VLPs comprise more than one protein from the same and/or a heterologous infectious agent.
  • the chimeric VLPs comprise more than one infectious agent protein, thus creating a multivalent VLP.
  • VLPs of the invention can induce substantial immunity in a vertebrate (e.g. a human) when administered to the vertebrate.
  • the substantial immunity results from an immune response against VLPs of the invention that protects or ameliorates infection or at least reduces a symptom of infection in the vertebrate.
  • the infection will be asymptomatic.
  • the response may not be a fully protective response.
  • the vertebrate is infected with an infectious agent, the vertebrate will experience reduced symptoms or a shorter duration of symptoms compared to a non- immunized vertebrate.
  • the invention comprises a method of inducing substantial immunity to RSV virus infection or at least one disease symptom in a subject, comprising administering at least one effective dose of RSV VLPs.
  • the invention comprises a method of vaccinating a mammal against RSV comprising administering to the mammal a protection-inducing amount of VLPs comprising at least one RSV protein, hi one embodiment, the method comprises administering VLPs comprising BRSV M protein.
  • the method further comprises administering VLPs comprising RSV F protein, for example a HRSV F protein.
  • the method further comprises administering VLPs comprising the G protein from HRSV group A.
  • the method further comprises administering VLPs comprising the G protein from HRSV group B.
  • the method comprises administering VLPs comprising chimeric M protein from BRSV and F and/or G protein derived from RSV wherein the F and/or G protein is fused to the transmembrane and cytoplasmic tail of the M protein.
  • the method comprises administering VLPs comprising M protein from BRSV and chimeric RSV F and/or G protein wherein the F and/or G protein is a fused to the transmembrane domain and cytoplasmic tail of influenza HA and/or NA protein.
  • the method comprises administering VLPs comprising M protein from BRSV and chimeric RSV F and/or G protein and optionally an influenza HA and/or NA protein wherein the F and/or G protein is a fused to the transmembrane domain and cytoplasmic tail of the HA protein.
  • the method comprises administering VLPs comprising M protein from BRSV and chimeric RSV F and/or G protein, and optionally an influenza HA and/or NA protein wherein the HA and/or NA protein is fused to the transmembrane domain and cytoplasmic tail of RSV F and/or G protein.
  • the invention also encompasses a method of inducing substantial immunity to an infection, or at least one disease symptom in a subject caused by an infectious agent, comprising administering at least one effective dose of VLPs of the invention.
  • the method comprises administering VLPs comprising a RSV M protein and at least one protein from another infectious agent.
  • the method comprises administering VLPs comprising a BRSV M protein and at least one protein from the same or a heterologous infectious agent.
  • the protein from the infectious agent is a viral protein.
  • the protein from the infectious agent is an envelope associated protein.
  • the protein from the infectious agent is expressed on the surface of VLPs.
  • the protein from the infectious agent comprises an epitope that will generate a protective immune response in a vertebrate.
  • the protein from the infectious agent can associate with RSV M protein.
  • the protein from the infectious agent can associate with BRSV M protein.
  • the protein from the infectious agent is fused to a RSV protein.
  • only a portion of a protein from the infectious agent is fused to a RSV protein.
  • only a portion of a protein from the infectious agent is fused to a portion of a RSV protein.
  • the portion of the protein from the infectious agent fused to the RSV protein is expressed on the surface of VLPs.
  • the RSV protein, or portion thereof, fused to the protein from the infectious agent associates with the RSV M protein.
  • the RSV protein, or portion thereof, fused to the protein from the infectious agent associates with the BRSV M protein.
  • the RSV protein, or portion thereof is derived from RSV F, G, N and/or P.
  • the VLPs further comprise N and/or P protein from RSV.
  • the VLPs comprise more than one protein from the infectious agent.
  • the VLPs comprise more than one infectious agent protein, thus creating a multivalent VLP.
  • the invention comprises a method of inducing a protective antibody response to an infection or at least one symptom thereof in a subject, comprising administering at least one effective dose of BRSV VLPs, wherein the VLPs comprises RSV proteins including BRSV M, RSV F, G, N, or portions thereof, and/or any chimeric molecule described above.
  • an "antibody” is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy" chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • the invention comprises a method of inducing a protective cellular response to RSV infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a BRSV VLP, wherein the VLP comprises including BRSV M and RSV F, G, N or portions thereof, and/or any chimeric molecule described above.
  • Cell-mediated immunity also plays a role in recovery from RSV infection and may prevent RSV-associated complications.
  • RSV-specific cellular lymphocytes have been detected in the blood and the lower respiratory tract secretions of infected subjects. Cytolysis of RS V-infected cells is mediated by CTLs in concert with RSV- specific antibodies and complement.
  • the primary cytotoxic response is detectable in blood after 6-14 days and disappears by day 21 in infected or vaccinated individuals (Ennis et ah, 1981).
  • Cell-mediated immunity also plays a role in recovery from RSV infection and may prevent RSV-associated complications.
  • RSV-specific cellular lymphocytes have been detected in the blood and the lower respiratory tract secretions of infected subjects.
  • the VLPs of the invention prevent or reduce at least one symptom of RSV infection in a subject.
  • Symptoms of RSV are well known in the art. They include rhinorrhea, sore throat, headache, hoarseness, cough, sputum, fever, rales, wheezing, and dyspnea.
  • the method of the invention comprises the prevention or reduction of at least one symptom associated with RSV infection.
  • a reduction in a symptom may be determined subjectively or objectively, e.g., self assessment by a subject, by a clinician's assessment or by conducting an appropriate assay or measurement (e.g.
  • the objective assessment comprises both animal and human assessments.
  • RSV-VLPs were generated with the M protein of BRSV alone, the M protein of type A HRSV (HRS V-A) alone, and in combination with HRSV-A precursor fusion (F 0 ) protein. Additional constructs were created comprising the HRSV-A G protein in combination with the BRSV M protein.
  • SEQ ID NOs: 1-6 The protein sequences disclosed in SEQ ID NOs: 1-6 were used to synthesize genes in which the nucleotides were codon optimized for expression in Sf9 insect cells. The resultant codon optimized genes were cloned into bacmids.
  • Sf9 insect cells were then transfected to make recombinant virus stocks and plaque purification was performed. To accomplish this transfection, different bacmid DNAs from above were made for each construct and were isolated. These DNAs were precipitated and added to Sf9 cells for 5 hours.
  • Equal volumes of cell samples from crude harvest and 2x sample buffer containing ⁇ ME (beta-mercaptoehtanol) were loaded, approximately 15 to 20 ⁇ l (about to 7.5 to 10 ⁇ l of the culture)/lane, onto SDS Laemmli gel.
  • Figure 2(A) shows the total protein expression from crude harvest containing cells (intracellular) from each of culture described above by Coomassie staining.
  • Figure 2(B) showed the total protein expression from resuspended 100,000 x g VLPs-containing pellet from each of the culture described above by Coomassie staining.
  • the experiment illustrated in Figure 2(A) shows unexpectedly high intracellular levels of BRSV M (lane 2, M-B) compared to HRSV-A M (lane 1, M-R) when expressed individually.
  • BRSV M protein remains high when co-expressed with HRSV-A F 0 clone 541 (lane 4, M-B F0541) or with HRSV-A F 0 clone 576 (lane 6, M-B F0576) whereas HRSV-A M protein cannot be detected when co- expressed with either of these two HRSV-A F 0 clones (lane 3, M-R F0541 and lane 5, M-R F0576 respectively).
  • Figure 2(B) shows high levels of BRSV M expression in the 100,000 x g VLP s- containing pellets when expressed alone or when co-expressed with HRSV-A F 0 .
  • no visible HRSV M protein was observed in the 100,000 x g pellets of HRSV-A M alone or co-expressed with either clones for HRSV-A F 0.
  • Figure 3(A) shows the total F protein expression from crude harvest containing cells (intracellular) from each of culture described above by Western blot analysis.
  • Figure 3(B) shows the total F protein expression from resuspended 100,000 x g VLPs- containing pellets from each of the culture described above by Western blot analysis.
  • the amount of HRSV F protein detected in the 100,000 x g VLP pellet shows an increased level in the presence of BRSV M with HRSV-A FO clone 541 (lane 4, M-B F0541) and equal amounts with HRSV-A FO clone 576 (lane 6, M-B F0576) compared to when HRSV-A M protein was present (lane 3, M-R F0541; and lane 5, M-R F0576 respectively).
  • Influenza hemagglutinin/neuraminidase (HANA) and HIV chimeric gpl40- MMTV Env constructs were made.
  • the transmembrane (TM) and the cytoplasmic domain (CT) of the influenza HANA was replaced with MMTV Env proteins to make chimeric construct which was co-expressed with BRSV M.
  • VLPs For total protein expression analysis in the VLPs, a culture of insect cells was infected at ⁇ 3 MOI with baculovirus carrying the various constructs. The culture and supernatant were harvested 68 h post-infection and the samples were processed for analysis by SDS-PAGE set forth in Example 1. Protein expression of the resuspended 100,000 x g VLPs-containing pellets (secreted particles) and crude cell harvests containing VLPs (intracellular) were analyzed by SDS-PAGE and stained for total proteins by Coomassie stain.
  • FIG. 4(A) shows intracellular levels of BRSV M only (B-M, lane 1) or with the co-expressed proteins: influenza hemagglutinin/neuraminidase (B-M HANA, lane 2); chimeric HIV gpl40-MMTV Env (B-M HIV Env, lane 3); and A/Indonesia/5/05 (avian) Ml co-expressed with A/Brisbane/ 10/07 HANA (M avian HANA, lane 4).
  • Figure 4(B) shows high levels of BRSV M expression in the 100,000 x g VLPs-containing pellets when expressed alone or when co-expressed with influenza hemagglutinin/neuraminidase (B-M HANA, lane 6); and chimeric HIV gpl40-MMTV Env (B-M HIV Env, lane 7).
  • influenza hemagglutinin/neuraminidase level is much less when co-expressed with A/Indonesia/5/05 (avian) Ml compared to expression with BRSV M (compare lane 8 to lane 6).
  • HIV env was expressed at high level when BRSV M was present (Iane7).
  • Figure 5 shows a protein alignment using the CLC Protein Workbench version 3.3.2 software package (CLC bio, Finlandsgade 10-12, DK 8200 Aarhus N, Denmark) between the amino acid sequence of BRSV M construct used (top line) with three published BRSV M sequences (lines 2, 3, and 4) and three published HRSV-A M protein sequences (lines 5, 6, and 7). The consensus is show in line 8.
  • All publications, patents and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

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Abstract

La présente invention porte sur des particules de type viral (VLP) comprenant des protéines matricielles de RSV bovin (BRSV). L'invention comprend des produits de construction de vecteur contenant des protéines matricielles de RSV bovin (BRSV), des cellules contenant les produits de construction, des formulations et des vaccins contenant les VLP de l'invention. L'invention porte également sur des procédés de fabrication et des méthodes d'administration des VLP à des vertébrés, comprenant des méthodes d'induction d'une immunité vis-à-vis des infections, dont le RSV.
PCT/US2009/067257 2008-12-09 2009-12-09 Particule de type viral du virus syncytial respiratoire bovin (vlps) Ceased WO2010077712A1 (fr)

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US9815873B2 (en) 2011-03-23 2017-11-14 Medicago Inc. Method for recovering plant-derived proteins
US12139512B2 (en) 2011-05-13 2024-11-12 Glaxosmithkline Biologicals Sa Pre-fusion RSV F antigens
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CN108348593A (zh) * 2015-08-31 2018-07-31 泰克诺瓦克斯股份有限公司 基于人呼吸道合胞病毒(hrsv)病毒样颗粒(vlps)的疫苗

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