WO2009062532A1 - Souches de métapneumovirus atténués vivants et leur utilisation dans des formulations de vaccin et souches de métapneumovirus chimériques - Google Patents

Souches de métapneumovirus atténués vivants et leur utilisation dans des formulations de vaccin et souches de métapneumovirus chimériques Download PDF

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WO2009062532A1
WO2009062532A1 PCT/EP2007/009942 EP2007009942W WO2009062532A1 WO 2009062532 A1 WO2009062532 A1 WO 2009062532A1 EP 2007009942 W EP2007009942 W EP 2007009942W WO 2009062532 A1 WO2009062532 A1 WO 2009062532A1
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virus
protein
metapneumovirus
gene
hmpv
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PCT/EP2007/009942
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Ron A.M. Fouchier
Sander Herfst
Miranda De Graaf
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Vironovative Bv
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • C12N2760/18311—Metapneumovirus, e.g. avian pneumovirus
    • C12N2760/18322—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
    • C12N2760/18311—Metapneumovirus, e.g. avian pneumovirus
    • C12N2760/18334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2760/18311—Metapneumovirus, e.g. avian pneumovirus
    • C12N2760/18361—Methods of inactivation or attenuation
    • C12N2760/18364—Methods of inactivation or attenuation by serial passage

Definitions

  • the invention relates to an isolated mammalian negative strand RNA virus, metapneumovirus (MPV), within the sub-family Pneumoviridae, of the family Paramyxoviridae with one or more genetic modifications.
  • MPV metapneumovirus
  • the present invention also relates to the mutant components, i.e., nucleic acids and proteins, of these mutant mammalian MPVs. These mutant mMPV can be attenuated. These mutant mMPVs can encode non- native sequences.
  • the invention further relates to vaccine formulations comprising the mMPV, including recombinant and chimeric forms of said viruses.
  • the vaccine preparations of the invention encompass multivalent vaccines, including bivalent and trivalent vaccine preparations.
  • the invention relates to chimeric viral RNA polymerase complex and assays using these chimeric RNA polymerase complexes.
  • the chim eric RNA polymerase complexes of the invention are composed of different RNA polymerase components from different viruses of the family of paramyxoviridae.
  • hMPV human metapneumovirus
  • Human metapneumovirus is an enveloped, non-segmented, negative- strand RNA virus that causes respiratory tract illnesses primarily in infants, young children, frail elderly and immunocompromised individuals (Crowe, 2004, Pediatr. Infect. Dis. 23, S215-221 ; Falsey et al., 2003, J. Infect. Dis. 187, 785-790; Kahn, 2006, Clin. Microbiol. Rev. 19, 546-557; Pelletier et al., 2002, Emerg. Infect. Dis. 8, 976-978; van den Hoogen et al., 2001, Nat. Med. 7, 719-724; van den Hoogen et al., 2003, J. Infect.
  • HMPV is a member of the Paramyxoviridae family, subfamily Pneumovirinae, genus Metapneumovirus, and can be divided into two main genetic lineages (A and B) each consisting of two sublineages (Al, A2, Bl and B2) (van den Hoogen et al., 2004, Emerg. Infect. Dis. 10, 658-666).
  • a and B main genetic lineages
  • the only other identified member of the Metapneumovirus genus is the avian metapneumovirus (aMPV).
  • AMPV has been found to infect domestic poultry worldwide, causing acute respiratory infections (Cook, 2000, Rev. Sci. Tech. 19, 602-613).
  • AMPVs have been classified into four subgroups, A through D (Bayon-Auboyer et al., 1999, Arch. Virol. 144, 1091-1109; Eterradossi et al., 1995, Monbl. Veterinarmed. B. 42, 175- 186; Juhasz & Easton, 1994, J. Gen. Virol. 75, 2873-2880; Seal, 1998, Virus Res. 58, 45-52).
  • AMPV-C was first detected in the United States and is more closely related to hMPV than the other aMPV subgroups (Govindarajan & Samal, 2004, Virus Res.
  • metapneumoviruses lack the non- structural proteins NSl and NS2 and the order of the genes between the matrix (M) and large polymerase (L) genes is different; hMPV/aMPV, '3 le-N-P-M-F-M2-SH-G-L-tr 5', RSV-A2, '3 Ie-NS l-NS2-N-P-M-SH-G-F-M2-L-tr 5'.
  • the viral genome of all members of the Pneumovirinae subfamily is of antisense polarity and assembled in a ribonucleoprotein complex (RNP).
  • RNP ribonucleoprotein complex
  • This RNP contains the viral genomic RNA (vRNA) encapsidated by the nucleocapsid protein (N), the phosphoprotein (P) and the L protein.
  • vRNA viral genomic RNA
  • N nucleocapsid protein
  • P phosphoprotein
  • the L protein is responsible for the main catalytic activities of the viral polymerase complex (Grdzelishvili et al., 2005, J. Virol. 79, 7327-7337; Hercyk et al., 1988, Virology 163, 222-225; Ogino et al., 2005, J. Biol.
  • the assembly and polymerase cofactor P and the L protein form the minimal complex needed for viral polymerase activity (Mazumder & Barik, 1994, Virology 205, 104-1 11).
  • RSV RNA synthesis involves an additional viral protein, the M2.1 protein, a transcriptional elongation factor that enhances the synthesis of readthrough mRNAs (Collins et al., 1996, Proc. Natl. Acad. Sci. USA 93, 81-85; Fearns & Collins, 1999, J. Virol. 73, 5852-5864; Hardy & Wertz, 1998, J. Virol. 72, 520-526).
  • hMPV the function of M2.1 is not completely understood as recombinant hMPV can be recovered in the absence of M2.1 and viruses from which the M2.1 gene is deleted grow efficiently in vitro but not in vivo (Buchholz et al., 2005, J. Virol. 79, 6588-6597; Herfst et al., 2004, J. Virol. 78, 8264-8270).
  • the 3 '(leader) and 5 '(trailer) ends contain the viral promoters necessary for replication and transcription. Transcription of paramyxoviruses is further directed by gene start (GS) and gene end (GE) sequences flanking each of the open reading frames (ORFs) in the viral genome. Transcription of the viral genome results in a gradient of transcripts, steadily decreasing toward the 5 'end of the genome. Thus, the gene order roughly reflects the relative amount of gene products required for efficient virus replication.
  • LAVs may be useful to prime or boost hMPV-specif ⁇ c immune responses, since such viruses have the advantage of mimicking a natural infection, and thus could provide protection against subsequent infections without inducing enhanced disease.
  • Recently developed reverse genetics systems for hMPV (Biacchesi et al , 2004, Virology 321, 247-259; Herfst et al, 2004, J. Virol. 78, 8264-8270) facilitate the modification of viral genomes and thus provide a powerful tool to design LAVs.
  • hMPV deletion mutants chimeric viruses based on hMPV and avian metapneumovirus (aMPV), and a human/bovine parainfluenza virus type 3 (b/hPIV3) expressing the F protein of hMPV (Biacchesi et al, 2005, J. Virol. 79, 12608-12613; Pham et al, 2005, J. Virol. 79, 15114-15122; Tang et al, 2005, Vaccine 23, 1657-1667) have recently been described.
  • aMPV chimeric viruses based on hMPV and avian metapneumovirus
  • b/hPIV3 human/bovine parainfluenza virus type 3
  • the invention relates to mutants of mammalian metapneumovirus (mMPV).
  • the mammalian metapneumovirus is a human metapneumovirus (hMPV).
  • the mammalian MPV can be a variant Al, A2, Bl or B2 mammalian MPV.
  • the mutant mMPV or hMPV is attenuated and can be used as a vaccine.
  • the mutant mMPV or hMPV of the invention can be used in an immunogenic composition.
  • the mutant mMPV or hMPV is temperature-sensitive.
  • the invention also relates to an assay system to test the activity of a chimeric viral RNA polymerase complex that is composed of RNA polymerase subunits from different paramyxoviruses.
  • the invention provides for isolated mammalian MPV comprising genetic modifications.
  • an isolated mammalian MPV which comprises a genetic modification resulting in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: position 66 in the P protein; positions 9, 38, 52, and 132 in the M protein; positions 93, 101, 280, 471, 532, and 538 in the F protein; position 187 in the M2 protein; positions 139 and 164 in the G protein; and positions 235, 323, and 1453 in the L protein, with the proviso that the modification at position 101 in the F protein is not a substitution to Proline and that the modification at position 93 in the F protein is not a substitution to Lysine.
  • the genetic modification results in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: position 66 in the P protein; position 132 in the M protein; positions 101, 280, and 471 in the F protein; position 187 in the M2 protein; position 139 in the G protein; and positions 235, 323, and 1453 in the L protein, with the proviso that modification at position 101 in the F protein is not a substitution to Proline.
  • the genetic modification results in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: position 132 in the M protein; positions 101, 280, and 471 in the F protein; position 187 in the M2 protein; position 139 in the G protein; and position 1453 in the L protein, with the proviso that modification at position 101 in the F protein is not a substitution to Proline.
  • the genetic modification results in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: positions 235 and 323 in the L protein.
  • the isolated mammalian metapneumovirus comprises genetic modifications resulting in amino acid substitution, deletion, or insertion at amino acid positions 235 and 323 in the L protein.
  • the invention provides an isolated mammalian MPV comprising a genetic modification at one or more of the nucleotide positions selected from the group consisting of: position 197 in the P open reading frame; position 25, 1 13, 155, 336, 394, and 436 in the M open reading frame; positions 277, 301, 839, 1412, 1594, and 1612 in the F open reading frame; position 560 in the M2 open reading frame; position 415 and 491 in the G open reading frame; and positions 703, 967, and 4357 in the L open reading frame.
  • the isolated mammalian MPV of the invention further comprises a genetic modification that is a silent mutation, i.e., it does not result in an amino acid exchange.
  • the isolated mammalian MPV comprises a silent mutation at one or more of the nucleotide positions selected from the group consisting of: positions 336 and 436 in the M open reading frame.
  • the isolated mammalian MPV of the invention further comprises a genetic alteration that results in an amino acid exchange at amino acid 109 of the F protein.
  • the isolated mammalian MPV of the invention further comprises a mutation at nucleotide position 325 of the F protein that results in a serine at that position.
  • the isolated mammalian MPV of the invention further comprises a mutation at nucleotide position 325 of the F protein.
  • the invention provides an isolated mammalian MPV comprising a genetic modification at one or more of the nucleotide positions selected from the group consisting of: position 197 in the P open reading frame; position 25, 113, 155, 336, 394, and 436 in the M open reading frame; positions 277, 301, 839, 1412, 1594, and 1612 in the F open reading frame; position 560 in the M2 open reading frame; position 415 and 491 in the G open reading frame; and positions 703, 967, and 4357 in the L open reading frame, wherein the genetic modifications at positions 336 and 436 in the M open reading frame result in silent mutations.
  • the invention provides an isolated mammalian MPV comprising a genetic modification resulting in one or more amino acid changes selected from the group consisting of: (i) position 66 in the P gene is altered to VaI; (ii) position 9 in the M gene is altered to His; (iii) position 38 in the M gene is altered to Ser; (iv) position 52 in the M gene is altered to Pro; (v) position 132 in the M gene is altered to Pro; (vi) position 93 in the F gene is altered to Lys; (vii) position 280 in the F gene is altered to GIy; (viii) position 471 in the F gene is altered to Arg; (ix) position 532 in the F gene is altered to Tyr; (x) position 538 in the F gene is altered to Tyr; (xi) position 187 in the M2 gene is altered to He; (xii) position 139 in the G gene is altered to Pro; (xiii) position 164 in the G gene is altered to Pro; (xi) position 187 in the M2
  • the amino acid changes represented in (i) to (xvi) are combined with genetic modifications at one or more of the nucleotide positions selected from the group consisting of: positions 336 and 436 in the M open reading frame, wherein the genetic modifications at positions 336 and 436 in the M open reading frame result in silent mutations.
  • the isolated mammalian MPV may have at least two, at least three, at least four, at least five, at least six, at least seven or at least eight of the specified genetic modifications.
  • the invention provides for a recombinant mammalian
  • MPV comprising two or more genetic modifications, wherein the genetic modification is an amino acid substitution, deletion, or insertion amino acid position 456 of the L gene; position
  • the invention provides for a recombinant mammalian
  • MPV comprising an alteration in the gene start sequence of the M2 gene; an alteration in the
  • the mutant isolated mammalian MPV carries an amino acid exchange that is encoded by two or three nucleotide substitutions per codon, i.e., a stabilized codon.
  • the isolated mammalian MPV may be temperature-sensitive.
  • the isolated mammalian MPV may be a human MPV.
  • the isolated mammalian MPV may be hMPV variant Al, A2, Bl, or B2.
  • the isolated mammalian MPV may be hMPV strain NL/1/99, NL/17/00,
  • a method for stimulating the immune response against mammalian MPV in a mammal comprising administering to the mammal an isolated mammalian MPV of the invention including, but not limited to, the isolated mammalian MPV comprising the genetic modifications described above.
  • the mammal is a human.
  • the isolated mammalian MPV is a human MPV, wherein the the hMPV can in some aspects be hMPV variant Al, A2, Bl, or B2.
  • the hMPV can be hMPV strain NL/1/99, NL/1/00, NL/17/00, or NL/1/94.
  • the invention also provides for vaccine formulations comprising the isolated mammalian MPV of the invention including, but not limited to, the isolated mammalian
  • immunogenic compositions are provided, said immunogenic compositions comprising the isolated mammalian MPV of the invention including, but not limited to, the isolated mammalian MPV comprising the genetic modifications described above, along with a pharmaceutically acceptable excipient.
  • the isolated mammalian MPV of the invention including, but not limited to, the isolated mammalian MPV comprising the genetic modifications described above, can be used as a medicament.
  • the invention provides a recombinant nucleic acid comprising cDNA encoding the isolated mammalian MPV of the invention, including, but not limited to, the isolated mammalian MPV comprising the genetic modifications described above.
  • a vector is provided that comprises the recombinant nucleic acid comprising cDNA encoding the isolated mammalian MPV of the invention.
  • the invention provides a method of producing a mammalian MPV comprising: a) introducing recombinant nucleic acid comprising cDNA encoding an isolated mammalian MPV of the invention operatively linked to a promoter for DNA-directed RNA polymerase into a host cell, wherein the host cell expresses (i) the N, P, and L proteins of a mammalian MPV and (ii) the DNA-directed RNA polymerase; and b) isolating the virus produced by the cell.
  • the invention provides a method for producing a mammalian MPV comprising: a) introducing recombinant nucleic acid comprising cDNA encoding the isolated mammalian MPV of the invention operatively linked to a promoter for a DNA-directed RNA polymerase into a host cell, wherein the host cell expresses the DNA- directed RNA polymerase; b) introducing cDNA encoding the N, P, and L genes of a mammalian metapneumovirus into the host cell; and c) isolating the virus produced by the host cell.
  • the invention provides an infectious chimeric virus, wherein the chimeric virus comprises the genome of a mammalian MPV of a first variant, wherein one or more of the open reading frames in the genome of the mammalian MPV of the first variant have been replaced by the analogous open reading frame from a mammalian MPV of a second variant.
  • the invention provides an infectious chimeric virus, wherein the chimeric virus comprises the genome of a mammalian MPV of a first variant, wherein one or more of open reading frames of a mammalian MPV of a second variant are inserted into the genome of the mammalian MPV of the first variant.
  • the invention provides an infectious chimeric virus, wherein the chimeric virus comprises the genome of a mammalian MPV, wherein one or more of the open reading frames in the genome of the mammalian MPV have been replaced by an ORF which encodes one or more of (i) an avian MPV F protein; (ii) an avian MPV G protein (iii) an avian MPV SH protein; (iv) an avian MPV N protein (v) an avian MPV P protein; (vi) an avian MPV M2 protein;(vii) an avian MPV M2.1 protein; (viii) an avian MPV M2.2 protein; or (ix) an avian MPV L protein.
  • the invention provides an infectious chimeric virus, wherein the chimeric virus comprises the genome of an avian MPV, wherein one or more of the open reading frames in the genome of the avian MPV have been replaced by an ORF which encodes one or more of (i) a mammalian MPV F protein (ii) a mammalian MPV G protein; (iii) a mammalian MPV SH protein; (iv) a mammalian MPV N protein; (v) a mammalian MPV P protein; (vi) a mammalian MPV M2 protein; (vii) a mammalian MPV M2.1 protein; (viii) a mammalian MPV M2.2 protein; or (ix) a mammalian MPV L protein.
  • a mammalian MPV F protein ii) a mammalian MPV G protein
  • a mammalian MPV SH protein a mammalian MPV N protein
  • the invention provides a chimeric MPV wherein the N gene of an aMPV replaces the N gene of a hMPV.
  • the P gene of a hMPV is replaced by the P gene from an aMPV.
  • the L gene of a hMPV is replaced with the L gene of an aMPV.
  • the hMPV is serotype Bl.
  • the aMPV is from aMPV subgroup C.
  • the chimeric MPV is attenuated.
  • the invention provides an infectious chimeric or recombinant virus, wherein the chimeric or recombinant virus is rescued using an interspecies or intraspecies polymerase.
  • the invention provides a chimeric or recombinant virus, wherein the chimeric or recombinant virus is rescued using MPV polymerase.
  • the invention uses a polymerase from a virus different from the polymerase of the virus to be rescued, i.e., from a different clade, subtype, or other species.
  • the invention provides an infectious chimeric or recombinant virus, wherein the chimeric or recombinant virus is rescued using the polymerase from another virus, including, but not limited to the polymerase of PIV, AMPV or RSV.
  • RSV polymerase can be used to rescue MPV; MPV polymerase can be used to rescue RSV; or PIV polymerase can be used to rescue MPV.
  • the polymerase complex that is used to rescue the recombinant virus is encoded by polymerase proteins from different viruses.
  • the polymerase complex proteins are encoded by the N gene of MPV, the L gene of PIV, the P gene of RSV and the M2.1 gene of MPV.
  • the M2.1 gene is not a component of the polymerase complex.
  • the polymerase complex proteins are encoded by the N gene of RSV, the L gene of RSV, the P gene of AMPV, and the M2.1 gene of RSV.
  • the M2.1 gene is not required to rescue the recombinant virus of the invention.
  • the invention provides an infectious recombinant virus, wherein the recombinant virus is rescued using a chimeric polymerase complex.
  • the method comprises the steps of: a) introducing into a host cell cDNA encoding the MPV; b) introducing into the host cell cDNA encoding a chimeric polymerase complex comprising N, P, L, and M2.1 of a MPV, wherein N, P, L, and M2.1 are from at least two different MPV strains; and c) isolating the virus produced by the host cell.
  • the MPV is a human MPV.
  • the hMPV is variant Al, A2, Bl, or B2. In a specific aspect of this embodiment, the hMPV is variant Bl .
  • the chimeric polymerase complex comprises hMPV Bl and aMPV C, wherein at least one but not all of N, P, L, and M2.1 are from hMPV Bl and at least one but not all of N, P, L, and M2.1 are from aMPV C.
  • the invention provides an isolated chimeric viral RNA polymerase complex comprising RNA polymerase complex subunits from at least two different paramyxoviruses.
  • the RNA polymerase complex subunits are the N, P, L, and M2.1 proteins.
  • the two different paramyxoviruses are selected from the group consisting of RSV, PIV, aMPV, and mammalian MPV.
  • a method for determining the activity of a chimeric viral RNA polymerase complex comprising the steps: a) introducing into a host cell a cDNA encoding a reporter gene flanked by the genomic termini of a first paramyxoviridae; b) introducing into the host cell cDNAs encoding the RNA polymerase complex subunits from at least two different paramyxoviridae; and c) measuring the activity of the reporter gene.
  • the RNA polymerase complex subunits are heterologous to the first paramyxoviridae.
  • the invention provides an immunogenic composition, wherein the immunogenic composition comprises the infectious recombinant or chimeric virus of the invention.
  • the invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the infectious recombinant or chimeric virus of the invention.
  • the invention provides a method for detecting a mammalian MPV in a sample, wherein the method comprises amplifying or probing for MPV related nucleic acids, processed products, or derivatives thereof.
  • the invention provides polymerase chain reaction based methods for the detection of MPV in a sample.
  • the invention provides oligonucleotide probes that can be used to specifically detect the presence of MPV related nucleic acids, processed products, or derivatives thereof.
  • the invention provides diagnostic methods for the detection of MPV antibodies in a host that is infected with the virus.
  • the invention provides a method for treating or preventing a respiratory tract infection in a mammal, said method comprising administering a vaccine comprising a mammalian MPV.
  • the invention provides a method for treating or preventing a respiratory tract infection in a mammal, said method comprising administering a vaccine comprising the recombinant or chimeric mammalian MPV of the invention.
  • the invention provides a method for treating or preventing a respiratory tract infection in a mammal, said method comprising administering a vaccine comprising avian MPV.
  • the invention provides a method for treating or preventing a respiratory tract infection in a human, said method comprising administering a vaccine comprising avian MPV. In certain embodiments, the invention provides a method for treating or preventing a respiratory tract infection in a subject, said method comprising administering to the subject a composition of the invention.
  • HN hemagglutinin-neuraminidase glycoprotein
  • N, NP or NC nucleoprotein associated with RNA and required for polymerase activity
  • NA neuraminidase envelope glycoprotein
  • Position 1 Position of the first gene of the viral genome to be transcribed
  • Position 2 Position between the first and the second open reading frame of the native viral genome, or alternatively, the position of the second gene of the viral genome to be transcribed
  • Position 3 Position between the second and the third open reading frame of the native viral genome, or alternatively, the position of the third gene of the viral genome to be transcribed.
  • Position 4 Position between the third and the fourth open reading frame of the native viral genome, or alternatively, the position of the fourth gene of the viral genome to be transcribed.
  • Position 5 Position between the fourth and the fifth open reading frame of the native viral genome, or alternatively, the position of the fifth gene of the viral genome to be transcribed.
  • Position 6 Position between the fifth and the sixth open reading frame of the native viral genome, or alternatively, the position of the sixth gene of the viral genome to be transcribed. dpi days post-infection
  • FIG. 1 Replication kinetics in Vero cell cultures of wild-type hMPV and recombinant viruses in which cp-mutations were introduced.
  • Vero cells infected at a MOI of 0.1 with hMPV NL/1/99 (panel A), hMPV M8 (panel B), hMPV M ⁇ (panel C), hMPV M2 (panel D) or 1IMPV RSV3 (panel E) were washed and incubated for 6 to 8 days at 32°C (open diamond), 37°C (closed diamond), 38°C (open square), 39°C (closed square) or 4O 0 C (open triangle). Samples were collected every two days, and virus titers determined by plaque assay.
  • Figure 2 Infectious virus titers in (A) nasal turbinates and (B) lungs of Syrian golden hamsters inoculated with 10 6 TCID 50 of NL/1/99, hMPV M n or hMPV RSV 3. Nasal turbinates and lungs were collected at 4 dpi. Virus in tissues was quantified by serial dilution in Vero-1 18 monolayers. The lower limit of detection is indicated with the dotted line.
  • Figure 3 50 % Plaque reduction virus neutralization (PRVN) titers measured against NL/1/99, after immunization with NL/1/99, hMPV M n or hMPV RS v 3 - Blood samples were collected by orbital puncture at 21 dpi. Titers were calculated according to the method of Reed and Muench. The lower limit of detection is indicated with the dotted line.
  • Figure 4 Infectious virus titers in (A) nasal turbinates and (B) lungs of Syrian golden hamsters. Animals were immunized with PBS, NL/1/99, IIMPV MI i or 1IMPV RS V 3 .
  • mice were challenged with 10 7 TCID 5O of the heterologous virus hMPV NL/1/00. Animals were euthanized at 4 dpi. Virus present in tissues was quantified by serial dilution in Vero-1 18 monolayers. The lower limit of detection is indicated with the dotted line.
  • FIG. 5 Replication of vRNA-like molecules by polymerase complexes of homologous or heterologous viruses.
  • VRNA-like molecules were co-transfected into BSR- T7 cells with N, P, L and M2.1 expression plasmids and a plasmid expressing ⁇ - galactosidase. The means and standard deviations of three independent transfection experiments are given. CAT values are standardized to 10 ng ⁇ -galactosidase.
  • Figure 6 Replication of vRNA-like molecules by chimeric metapneumovirus polymerase complexes.
  • VRNA-like molecules were co-transfected into BSR-T7 cells with their own N, P, L and M2.1 expression plasmids (black bars) chimeric sets of expression plasmids (grey bars), or the heterologous set of expression plasmids (white bars) and a plasmid expressing ⁇ -galactosidase. Plasmids supplied from a heterologous virus species are indicated along the x-axis. The means and standard deviations of three independent transfection experiments are given. CAT values are standardized to 10 ng ⁇ -galactosidase. [0049] Figure 7: Replication kinetics of chimeric hMPV-Bl/hMPV-Al viruses. Vero- 118 cells, infected at a multiplicity of infection of 0.1 with hMPV-Bl( «), hMPV-Bl/N hM pv-Ai
  • hMPV-Bl /PhMPV-Ai *
  • hMPV-Bl/NP h MPv-Ai
  • hMPV-Bl/M2.1 hM pv-Ai ⁇
  • hMPV- B 1 /L hMPV - A i ( ⁇ ) and hMPV-Al(o) were washed and incubated. Supernatants were collected daily and virus titers were determined by plaque assay.
  • Figure 8 Replication kinetics of chimeric hMPV-Bl/aMPV-C viruses.
  • Vero-1 18 cells infected at a multiplicity of infection of 0.1 with hMPV-Bl (o), hMPV-Bl/N aM pv-c ( ⁇ ) , hMPV-Bl /PaMPV-c ("), hMPV-B 1/LaMPv-c ( ⁇ ), and aMPV-C (•) were washed and incubated. Supernatants were collected daily and virus titers were determined by plaque assay.
  • Figure 9 Evaluation of attenuation of hMPV-Bl /aMPV-C chimeric viruses in Syrian golden hamsters. Infectious virus titers were determined in (A) NT and (B) lungs of hamsters inoculated with hMPV-Bl, hMPV-Bl/N aM pv-c, hMPV-Bl/PaMPv-c, hMPV- Bl/LaMpv-c, and aMPV-C. NT and lungs were collected four days after inoculation. The lower limit of detection is indicated with the dotted line.
  • Figure 10 Alignment of hMPV-Al , hMPV-Bl, aMPV-C, aMPV-A, RSV, and PIV-3 leader and trailer sequences. Differences in sequence identity are underscored.
  • Figure 11 Titers of hMPV in the lungs (A) and nasal turbinates (B) of hamsters immunized with the F protein of hMPV NL/1/99 or NL/1/00. Animals in groups of 8 were immunized with 10 ⁇ g of the F protein of NL/1/99 (F 1/99) or NL/1/00 (F 1/00) along with Specol, with IM (iscom matrix), or without adjuvant.
  • Control groups consisting of 6 animals each were immunized with Specol alone, IM alone, or PBS. Immunizations were administered twice, with a 3 week interval between them. Three weeks after the second immunization, all animals were challenged with 10 6 TCID50 of hMPV strain NL/1/00. Four days following challenge, animals were sacrificed, and lungs and nasal turbinates were collected and subjected to virus titration on Vero cells.
  • Figure 12 Growth curve of hMPV isolate NL/1/00 (Al) in Vero cells. The Vero cells were infected at a MOI of 0.1.
  • Figure 13 Sequence of CAT-hMPV minireplicon construct. The function encoded by a segment of sequence is indicated underneath the sequence.
  • Figure 14 Leader and Trailer Sequence Comparison: Alignments of the leader and trailer sequences of different viruses as indicated are shown.
  • Figure 15 Expression of CAT from the CAT-hMPV minireplicon. The different constructs used for transfection are indicated on the x-axis; the amount of CAT expression is indicated on the y-axis. The Figure shows CAT expression 24 hours after transfection and CAT expression 48 hours after transfection. Standards were dilutions of CAT protein.
  • Figure 16 hMPV genome analysis: PCR fragments of hMPV genomic sequence relative to the hMPV genomic organization are shown. The position of mutations are shown underneath the vertical bars indicating the PCR fragments.
  • Figure 17 Restriction maps of hMPV isolate NL/1/00 (Al) and hMPV isolate
  • Figure 18 A and 18B hMPV cDNA assembly.
  • the diagram on top shows the genomic organization of hMPV, the bars underneath indicate the PCR fragments ⁇ see Figure
  • Figure 19 Results of RT-PCR assays on throat and nose swabs of 12 guinea pigs
  • Figure 20 Comparison of the use of the hMPV ELISA and the APV inhibition
  • Figure 21 Generation of M2 deletion mutants. To construct M2 deletions,
  • BspEl sites were constructed at nucleotides 4741 and 5444 and the intervening nucleotides were deleted.
  • M2-1 deletions Nhel sites were constructed at nucleotides 4744 and 5241 and the intervening nucleotides were deleted.
  • M2-2 deletions Swa ⁇ sites were constructed at nucleotides 531 1 and 5435 and the intervening nucleotides were deleted.
  • Figure 23 Replication of wild type and recombinant hMPV in the upper and lower respiratory tract of hamsters.
  • the invention relates to mutants of mammalian metapneumovirus (mMPV).
  • the mammalian metapneumovirus is a human metapneumovirus (hMPV).
  • the mammalian MPV can be a variant Al , A2, Bl or B2 mammalian MPV.
  • the mutant mMPV or hMPV is attenuated and can be used as a vaccine.
  • the mutant mMPV or hMPV of the invention can be used in an immunogenic composition.
  • the mutant mMPV or hMPV is temperature-sensitive.
  • the mutant viruses of the invention are generation using recombinant DNA technology.
  • a mutant mMPV of the invention alters the host specificity, replication efficiency, efficiency of infectivity, efficiency of viral mRNA transcription, efficiency of viral protein synthesis, efficiency of assembly and release of the mutant mMPV relative to wild type mMPV.
  • a recombinant virus is one derived from a mammalian MPV or an APV that is encoded by endogenous or native genomic sequences or non-native genomic sequences.
  • a non-native sequence is one that is different from the native or endogenous genomic sequence due to one or more mutations, including, but not limited to, point mutations, rearrangements, insertions, deletions etc., the genomic sequence that may or may not result in a phenotypic change.
  • a chimeric virus is a recombinant MPV or APV which further comprises a heterologous nucleotide sequence.
  • a chimeric virus may be encoded by a nucleotide sequence in which heterologous nucleotide sequences have been added to the genome or in which endogenous or native nucleotide sequences have been replaced with heterologous nucleotide sequences.
  • the replication rate of the recombinant virus of the invention is at most 5 %, at most 10 %, at most 20 %, at most 30 %, at most 40 %, at most 50 %, at most 75 %, at most 80 %, at most 90 % of the replication rate of the wild type virus from which the recombinant virus is derived under the same conditions.
  • the replication rate of the recombinant virus of the invention is at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 75 %, at least 80 %, at least 90 % of the replication rate of the wild type virus from which the recombinant virus is derived under the same conditions.
  • the replication rate of the recombinant virus of the invention is between 5 % and 20 %, between 10 % and 40 %, between 25 % and 50 %, between 40 % and 75 %, between 50 % and 80 %, or between 75 % and 90 % of the replication rate of the wild type virus from which the recombinant virus is derived under the same conditions.
  • the mutant viruses of the invention can be used in pharmaceutical compositions, in immunogenic compositions, and in vaccines.
  • the mutant viruses of the invention can be used as expression vectors of non-native nucleotide sequences (/. e. , non-native to the mMPV). See section 5.5.
  • Such expression vectors can be used to express protein in different expression systems or as immunogenic compositions to stimulate the immune system against the non-native protein.
  • an isolated mammalian MPV which comprises a genetic modification resulting in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: position 66 in the P protein; positions 9, 38, 52, and 132 in the M protein; positions 93, 101, 280, 471, 532, and 538 in the F protein; position 187 in the M2 protein; positions 139 and 164 in the G protein; and positions 235, 323, and 1453 in the L protein, with the proviso that the modification at position 101 in the F protein is not a substitution to Proline and that the modification at position 93 in the F protein is not a substitution to Lysine.
  • the genetic modification results in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: position 66 in the P protein; position 132 in the M protein; positions 101, 280, and 471 in the F protein; position 187 in the M2 protein; position 139 in the G protein; and positions 235, 323, and 1453 in the L protein, with the proviso that modification at position 101 in the F protein is not a substitution to Proline.
  • the genetic modification results in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: position 132 in the M protein; positions 101, 280, and 471 in the F protein; position 187 in the M2 protein; position 139 in the G protein; and position 1453 in the L protein, with the proviso that modification at position 101 in the F protein is not a substitution to Proline.
  • the genetic modification results in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: positions 235 and 323 in the L protein.
  • the isolated mammalian metapneumovirus comprises genetic modifications resulting in amino acid substitution, deletion, or insertion at amino acid positions 235 and 323 in the L protein.
  • the isolated mammalian MPV of the invention further comprises a genetic modification that is a silent mutation, i.e., it does not result in an amino acid exchange.
  • the isolated mammalian MPV comprises a silent mutation at one or more of the nucleotide positions selected from the group consisting of: positions 336 and 436 in the M open reading frame.
  • the isolated mammalian MPV of the invention further comprises a genetic alteration that results in an amino acid exchange at amino acid 109 of the F protein.
  • the isolated mammalian MPV of the invention further comprises a mutation at nucleotide position 325 of the F protein that results in a serine at that position.
  • the isolated mammalian MPV of the invention further comprises a mutation at nucleotide position 325 of the F protein.
  • the invention provides an isolated mammalian MPV comprising a genetic modification at one or more of the nucleotide positions selected from the group consisting of: position 197 in the P open reading frame; position 9, 1 13, 155, 336, 394, and 436 in the M open reading frame; positions 277, 301, 839, 1412, 1594, and 1612 in the F open reading frame; position 560 in the M2 open reading frame; position 415 and 491 in the G open reading frame; and positions 703, 967, and 4357 in the L open reading frame.
  • the invention provides an isolated mammalian MPV comprising a genetic modification resulting in one or more amino acid changes selected from the group consisting of: (i) position 66 in the P gene is altered to VaI; (ii) position 9 in the M gene is altered to His; (iii) position 38 in the M gene is altered to Ser; (iv) position 52 in the M gene is altered to Pro; (v) position 132 in the M gene is altered to Pro; (vi) position 93 in the F gene is altered to Lys; (vii) position 280 in the F gene is altered to GIy; (viii) position 471 in the F gene is altered to Arg; (ix) position 532 in the F gene is altered to Tyr; (x) position 538 in the F gene is altered to Tyr; (xi) position 187 in the M2 gene is altered to He; (xii) position 139 in the G gene is altered to Pro; (xiii) position 164 in the G gene is altered to Pro; (xi) position 187 in the M2
  • the invention provides an isolated mammalian MPV comprising a genetic modification resulting in one or more amino acid changes selected from the group consisting of: (i) position 66 in the P gene is altered to VaI; (ii) position 9 in the M gene is altered to His; (iii) position 38 in the M gene is altered to Ser; (iv) position 52 in the M gene is altered to Pro; (v) position 132 in the M gene is altered to Pro; (vi) position 93 in the F gene is altered to Lys; (vii) position 280 in the F gene is altered to GIy; (viii) position 471 in the F gene is altered to Arg; (ix) position 532 in the F gene is altered to Tyr; (x) position 538 in the F gene is altered to Tyr; (xi) position 187 in the M2 gene is altered to He; (xii) position 139 in the G gene is altered to Pro; (xiii) position 164 in the G gene is altered to Pro; (xiv
  • the isolated mammalian MPV may have at least two, at least three, at least four, at least five, at least six, at least seven or at least eight of the specified genetic modifications.
  • the invention provides for a recombinant mammalian MPV comprising two or more genetic modifications, wherein the genetic modification is an amino acid substitution, deletion, or insertion amino acid position 456 of the L gene; position 1094 of the L gene; or position 1246 of the L gene; or a nucleotide substitution, deletion, or insertion at the gene start sequence of the M2 gene.
  • the genetic modification is an amino acid substitution, deletion, or insertion amino acid position 456 of the L gene; position 1094 of the L gene; or position 1246 of the L gene; or a nucleotide substitution, deletion, or insertion at the gene start sequence of the M2 gene.
  • the invention provides for a recombinant mammalian MPV, comprising an alteration in the gene start sequence of the M2 gene; an alteration in the L gene such that Phe at amino acid position 456 is mutated to Leu; and an alteration of the L gene such that Met at amino acid position 1094 is mutated to VaI.
  • an isolated mammalian MPV which comprises a genetic modification resulting in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: amino acid position 129 of the M gene, amino acid positions 129, 231 , 294, 307, 475 and 488 of the F protein, amino acid position 35 of the SH protein, amino acid positions 1 13 and 133 of the G protein, and amino acid positions 403, 537, 1220, 1336, 1440 and 1997 of the L protein.
  • the isolated mammalian MPV comprises a genetic modification at one or more genomic nucleotide positions selected from the group consisting of: genomic nucleotide positions 6072 and 6076 in the gene end sequence of the SH protein.
  • the isolated mammalian MPV of the invention further comprises a genetic modification that is a silent mutation, i.e., it does not result in an amino acid exchange.
  • the isolated mammalian MPV comprises a silent mutation at one of the nucleotide positions in the codon of one or more of the amino acids positions selected from the group consisting of: amino acid position 177 in the G protein and amino acid positions 554, 568, 582, 819, and 1343 in the L protein.
  • the isolated mammalian MPV of the invention comprises all of these mutations.
  • the isolated mammalian MPV is strain NL/1/94.
  • an isolated mammalian MPV which comprises a genetic modification resulting in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: amino acid positions 341 and 465 of the F protein, amino acid position 119 of the M2.1 protein, and amino acid position 467 of the L protein.
  • the isolated mammalian MPV comprises a genetic modification at one or more genomic nucleotide positions selected from the group consisting of: genomic nucleotide position 27 in the leader sequence, genomic nucleotide position 4692 in the gene end sequence of the F protein, and genomic nucleotide position 6981 in the gene end sequence of the G protein.
  • the isolated mammalian MPV comprises a silent mutation at one of the nucleotide positions in the codon of one or more of the amino acids positions selected from the group consisting of: amino acid positions 1206, 1402, and 1407 in the L protein.
  • the isolated mammalian MPV of the invention comprises all of these mutations.
  • the isolated mammalian MPV is strain NL/17/00.
  • an isolated mammalian MPV which comprises a genetic modification resulting in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: amino acid position 130 of the M protein, amino acid positions 93, 100, and 101 of the F protein, amino acid position 10 of the G protein, and amino acid position 1138 of the L protein.
  • an isolated mammalian MPV which comprises a genetic modification resulting in an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: amino acid position 130 of the M protein, amino acid positions 100, 101 , 468, and 529 of the F protein, amino acid position 45 of the M2.2 protein, and amino acid position 10 of the G protein.
  • the isolated mammalian MPV comprises a genetic modification at one or more genomic nucleotide positions selected from the group consisting of: genomic nucleotide position 13306 in the trailer sequence.
  • the isolated mammalian MPV comprises a silent mutation at one of the nucleotide positions in the codon of one or more of the amino acids positions selected from the group consisting of: amino acid position 93 of the F protein, amino acid position 90 of the SH protein, and amino acid positions 270, 736, 689, and 1138 of the L protein..
  • the isolated mammalian MPV of the invention comprises all of these mutations.
  • the isolated mammalian MPV is strain NL/1/00.
  • the mutant isolated mammalian MPV carries an amino acid exchange that is encoded by two or three nucleotide substitutions per codon, i.e., a stabilized codon.
  • the isolated mammalian MPV may be temperature-sensitive.
  • the isolated mammalian MPV may be a human MPV.
  • the isolated mammalian MPV may be hMPV variant Al, A2, Bl, or B2.
  • the isolated mammalian MPV may be hMPV strain NL/1/99, NL/17/00, NL/1/00, or NL/1/94.
  • a method for stimulating the immune response against mammalian MPV in a mammal comprising administering to the mammal an isolated mammalian MPV of the invention including, but not limited to, the isolated mammalian MPV comprising the genetic modifications described above.
  • the mammal is a human.
  • the isolated mammalian MPV is a human MPV, wherein the the hMPV can in some aspects be hMPV variant Al, A2, Bl, or B2.
  • the hMPV can be hMPV strain NL/1/99, NL/1/00, NL/17/00, or NL/1/94.
  • the invention also provides an assay to determine the activity of an RNA-directed chimeric RNA polymerase complex.
  • This assay is also suited for determining the activity of an RNA polymerase complex that is from a virus other than the virus being replicated.
  • the RNA polymerase complex is from a virus different from the virus whose genomic termini are replicated. This assay can be used to determine the specificity of an RNA polymerase complex for a particular virus as substrate.
  • the invention provides an assay for determining the activity of an RNA-directed RNA polymerase complex wherein the substrate of the RNA polymerase complex is a minigenome (i.e., a reporter gene flanked by the genomic termini of a virus) with genomic termini of a virus different from the virus from which the RNA polymerase was obtained.
  • a minigenome i.e., a reporter gene flanked by the genomic termini of a virus
  • This assay can be used to determine which combinations of RNA polymerase subunits are suitable to replicate a virus at lower levels to result in a replication-competent, yet attenuated, virus.
  • the subunits of the RNA polymerase complex or the chimeric RNA polymerase complex can be mutated.
  • one or more subunits of the chimeric RNA polymerase complex are from mMPV.
  • one or more of the mMPV subunits carries one or more of the mutations of a virus of the invention (see Section 5.1).
  • the assay can be performed as discussed for the minireplicon constructs in section 5.8(a).
  • the subunits of the chimeric RNA polymerase complex are the N, P, L, and M2.1 proteins.
  • the individual components are from two, three, or four different viruses of the family of paramyxoviridae.
  • at least one subunit is from mMPV, RSV, PIV, measles virus, mumps virus, or avian metapneumo virus.
  • at least one RNA polymerase complex subunit is from a Mononegavirales other than a paramyxoviridae.
  • the different subunits are derived from different variants of mMPV, i.e., Al, A2, Bl, and/or B2.
  • the genomic termini of the substrate of the RNA polymerase complex are from a member of the paramyxoviridae, such as, but not limited to, mMPV, RSV, PIV, measles virus, mumps virus, or avian metapneumo virus.
  • a host cell is transfected with nucleic acids encoding the individual components of the viral RNA polymerase complex and with a nucleic acid encoding the minireplicon.
  • the subunits and the replicon can be transcribed by a DNA- directed RNA polymerase, such as, but not limited to T3, T7, or Sp6.
  • the host cell can be transiently transfected or stably transfected with DNA encoding the DNA-directed RNA polymerase, such as, but not limited to T3, T7, or Sp6.
  • Vero cells can be engineered to express T7 RNA polymerase under the control of a CMV or SV40 promoter.
  • the host cell is infected with Modified Vaccinia Virus Ankara (MVA) encoding T7 RNA polymerase.
  • VAA Modified Vaccinia Virus Ankara
  • the following cells can be used as hosts: Vero cells, LLC-MK-2 cells, HEp-2 cells, LF 1043 (HEL) cells, tMK cells, LLC-MK2, HUT 292, FRHL-2 (rhesus), FCL-I (green monkey), WI-38 (human), MRC-5 (human) cells, 293 T cells, QT 6 cells, QT 35 cells and CEF cells.
  • the reporter gene can be a viral gene, CAT (chloramphenicol acetyltransferase ⁇ transfers radioactive acetyl groups to chloramphenicol or detection by thin layer chromatography and autoradiography); GAL (b-galactosidase — hydrolyzes colorless galactosides to yield colored products); GUS (b-glucuronidase— hydrolyzes colorless glucuronides to yield colored products); LUC (luciferase— oxidizes luciferin, emitting photons); GFP (green fluorescent protein - fluorescent protein without substrate); SEAP (secreted alkaline phosphatase-luminescence reaction with suitable substrates or with substrates that generate chromophores); HRP (horseradish peroxidase— in the presence of hydrogen oxide, oxidation of 3,3',5,5'-tetramethylbenzidine to form a colored complex); and AP (alkaline phosphatase— luminescence reaction with suitable
  • the amount of reporter gene expressed or the activity of the expressed reporter gene can be determined by any method known to the skilled artisan.
  • transcribed RNA can be detected and quantified by Northern blotting, PCR analysis, real time PCR analysis, molecular beacons etc.
  • Expressed protein can te detected and quantified by, e.g. , Western blotting and immunoprecipitation.
  • Peptide tags can also be used to quantify the expressed reporter gene.
  • the activity of the expressed reporter gene can be detected and quantified based on the enzymatic properties of the reporter gene. See section 5.8(b).
  • the amount/activity of the expressed reporter gene is a measure for the activity of the RNA-directed RNA polymerase complex or the chimeric RNA-directed RNA polymerase complex.
  • the lower the amount/activity of the expressed reporter gene the lower the activity of the RNA-directed RNA polymerase complex or the chimeric RNA-directed RNA polymerase complex.
  • the specificity (attributes to heterologous viruses) and the effect of the terminal residues of the leader (attributes to homologous virus) of the minireplicon system can also be tested by superinfecting the minireplicon-transfected cells with hMPV polymerase components (NL/1/00 and NL/1/99) or polymerase components from APV-A, APV-C, RSV or PIV.
  • hMPV polymerase components NL/1/00 and NL/1/99
  • polymerase components from APV-A, APV-C, RSV or PIV.
  • the different amount of each of the six plasmids can also be tested in order to determine the optimal conditions.
  • Any mammalian metapneumovirus can be used for the generation of the mutant viruses of the invention, for the chimeric viruses and RNA polymerase complexes of the invention, and for the methods of the invention.
  • human metapneumovirus hMPV
  • hMPV human metapneumovirus
  • mMPV is an isolated negative-sense single stranded RNA virus MPV belonging to the sub-family Pneumovirinae of the family Paramyxoviridae and identifiable as phylogenetically corresponding to the genus Metapneumovirus, wherein the virus is phylogenetically more closely related to a virus isolate deposited as 1-2614 with CNCM, Paris than to turkey rhinotracheitis virus, the etiological agent of avian rhinotracheitis.
  • mMPV can be devided into two subgroups: subgroup A and subgroup B.
  • the mammalian MPVs can be a variant Al , A2, Bl or B2 mammalian MPV.
  • a mammalian MPV can be identified as a member of subgroup A if it is phylogenetically closer related to the isolate NL/1/00 (SEQ ID NO:2) than to the isolate NL/1/99 (SEQ ID NO:1).
  • a mammalian MPV can be identified as a member of subgroup B if it is phylogenetically closer related to the isolate NL/1/99 (SEQ ID NO: 1) than to the isolate NL/1/00 (SEQ ID NO:2).
  • the isolate NL/1/00 (SEQ ID NO:2) is an example of the variant Al of mammalian MPV.
  • the isolate NL/1/99 (SEQ ID NO:1) is an example of the variant Bl of mammalian MPV.
  • An isolate of mammalian MPV is classified as a variant Bl if it is phylogenetically closer related to the viral isolate NL/1/99 (SEQ ID NO:1) than it is related to any of the following other viral isolates: NL/1/00 (SEQ ID NO:2), NL/17/00 (SEQ ID NO:3) and NL/1/94 (SEQ ID NO:4).
  • An isolate of mammalian MPV is classified as a variant Al if it is phylogenetically closer related to the viral isolate NL/1/00 (SEQ ID NO:2) than it is related to any of the following other viral isolates: NL/1/99 (SEQ ID NO:1), NL/17/00 (SEQ ID NO:3) and NL/1/94 (SEQ ID NO:4).
  • An isolate of mammalian MPV is classified as a variant A2 if it is phylogenetically closer related to the viral isolate NL/17/00 (SEQ ID NO:3) than it is related to any of the following other viral isolates: NL/1/99 (SEQ ID NO:1), NL/1/00 (SEQ ID NO:2) and NL/1/94 (SEQ ID NO:4).
  • An isolate of mammalian MPV is classified as a variant B2 if it is phylogenetically closer related to the viral isolate NL/1/94 (SEQ ID NO:4) than it is related to any of the following other viral isolates: NL/1/99 (SEQ ID NO:1), NL/1/00 (SEQ ID NO:2) and NL/17/00 (SEQ ID NO:3).
  • the classification of an mMPV into one of the variants, Al, A2, Bl, and B2, can be based on nucleotide sequence of amino acid sequence identity of one or more genes, non- coding regions, and/or proteins.
  • the N, P, M, F, M2, SH, G, or L protein of an Al mMPV is at least 90%, 95%, 98%, 99% or 99.5% identical to the corresponding protein of NL/1/00 (SEQ ID NO:2).
  • the N, P, M, F, M2, SH, G, or L protein of an A2 mMPV is at least 90%, 95%, 98%, 99% or 99.5% identical to the corresponding protein of NL/17/00 (SEQ ID NO:3).
  • the N, P, M, F, M2, SH, G, or L protein of a Bl mMPV is at least 90%, 95%, 98%, 99% or 99.5% identical to the corresponding protein of NL/1/94 (SEQ ID NO:4).
  • the N, P, M, F, M2, SH, G, or L protein of a B2 mMPV is at least 90%, 95%, 98%, 99% or 99.5% identical to the corresponding protein of NL/1/99 (SEQ ID NO: 1).
  • mMPV such as hMPV
  • methods for identifying mMPV such as hMPV, are disclosed in International Patent Application PCT/NL02/00040, published as WO 02/057302, which is incorporated by reference in its entirety herein.
  • PCT/NL02/00040 discloses nucleic acid sequences that are suitable for phylogenetic analysis at page 12, line 27 to page 19, line 29, which are incorporated by reference herein.
  • a mutant MPV of the invention further comprises a non- native nucleotide sequence.
  • a chimeric virus may be encoded by a nucleotide sequence in which the non-native nucleotide sequence has been added to the genome or in which an endogenous or native nucleotide sequence has been replaced with heterologous nucleotide sequence.
  • the non-native nucleotide sequence can be from a different strains of mMPV.
  • the non-native nucleotide sequence can encode a polypeptide, or it may be a non-coding sequence.
  • Non-native nucleotide sequences to be incorporated into the viral genome include sequences obtained or derived from different strains of metapneumovirus, a different variant of MPV, i.e., variant Al, A2, Bl, or B2, strains of avian pneumovirus, and other negative strand RNA viruses, including, but not limited to, RSV, PIV and influenza virus, HIV (e.g., the gpl60 protein), and other viruses, including morbillivirus.
  • a non-native sequence may encode a tag or marker or a biological response modifier, examples of which include, lymphokines, interleukines, granulocyte macrophage colony stimulating factor and granulocyte colony stimulating factor, cytokines, interferon type 1 , gamma interferon, colony stimulating factors, and interleukin -1, -2, -4, -5, -6, -12, or a chimeric F or G protein of RSV, PIV, APV or hMPV.
  • a tag or marker or a biological response modifier examples of which include, lymphokines, interleukines, granulocyte macrophage colony stimulating factor and granulocyte colony stimulating factor, cytokines, interferon type 1 , gamma interferon, colony stimulating factors, and interleukin -1, -2, -4, -5, -6, -12, or a chimeric F or G protein of RSV, PIV, APV or hMPV.
  • the mutant virus of the invention that carries a non-native sequence may express a protein from a different virus or organism.
  • Such chimeric mutant mMPV can be used as immunogenic compositions or as vaccines to stimulate an immune response against the mMPV and against the the other virus or organism.
  • the expression products and/or recombinant or chimeric virions obtained in accordance with the invention may advantageously be utilized in vaccine formulations.
  • the expression products and chimeric virions of the present invention may be engineered to create vaccines against a broad range of pathogens, including viral and bacterial antigens, tumor antigens, allergen antigens, and auto antigens involved in autoimmune disorders.
  • the chimeric virions of the present invention may be engineered to create vaccines for the protection of a subject from infections with PIV, RSV, and/or metapneumo virus.
  • Non-native gene sequences that can be expressed into the recombinant viruses of the invention include but are not limited to antigenic epitopes and glycoproteins of viruses which result in respiratory disease, such as influenza glycoproteins, in particular hemagglutinin H5, H7, respiratory syncytial virus epitopes, New Castle Disease virus epitopes, Sendai virus and infectious Laryngotracheitis virus (ILV).
  • Non-native nucleotide sequences can be from a RSV or PIV.
  • Non-native gene sequences that can be engineered into the chimeric viruses of the invention include, but are not limited to, viral epitopes and glycoproteins of viruses, such as hepatitis B virus surface antigen, hepatitis A or C virus surface glycoproteins of Epstein Barr virus, glycoproteins of human papilloma virus, simian virus 5 or mumps virus, West Nile virus, Dengue virus, glycoproteins of herpes viruses, VPI of poliovirus, and sequences derived from a lentivirus, preferably, but not limited to human immunodeficiency virus (HIV) type 1 or type 2.
  • viruses such as hepatitis B virus surface antigen, hepatitis A or C virus surface glycoproteins of Epstein Barr virus, glycoproteins of human papilloma virus, simian virus 5 or mumps virus, West Nile virus, Dengue virus, glycoproteins of herpes viruses, VPI of poliovirus, and sequences derived from a
  • heterologous gene sequences that can be engineered into chimeric viruses of the invention include, but are not limited to, Marek's Disease virus (MDV) epitopes, epitopes of infectious Bursal Disease virus (IBDV), epitopes of Chicken Anemia virus, infectious laryngotracheitis virus (ILV), Avian Influenza virus (AIV), rabies, feline leukemia virus, canine distemper virus, vesicular stomatitis virus, and swinepox virus ⁇ see Fields et al., (ed.), 1991, Fundamental Virology, Second Edition. Raven Press, New York, incorporated by reference herein in its entirety).
  • MDV Marek's Disease virus
  • IBDV infectious Bursal Disease virus
  • IVBDV epitopes of Chicken Anemia virus
  • ILV infectious laryngotracheitis virus
  • AIV Avian Influenza virus
  • rabies feline leukemia virus
  • canine distemper virus canine distemper
  • the non-native nucleotide sequence encodes an F protein or a G protein or a fragment of an F protein or a G protein.
  • the F-gene and/or the G-gene of human metapneumovirus have been replaced with the F-gene and/or the G-gene of avian pneumovirus to construct chimeric hMPV/APV virus.
  • viral vectors contain sequences derived from APV and mammalian MPV, such that a chimeric APV/hMPV virus is encoded by the viral vector.
  • the F-gene and/or the G-gene of avian pneumovirus have been replaced with the F-gene and/or the G-gene of human metapneumovirus to construct the chimeric APV/hMPV virus.
  • the chimeric virions of the present invention may be engineered to create anti-HIV vaccines, wherein an immunogenic polypeptide from gpl60, and/or from internal proteins of HIV is engineered into the glycoprotein HN protein to construct a vaccine that is able to elicit both vertebrate humoral and cell-mediated immune responses.
  • the invention relates to recombinant metapneumoviral vectors and viruses which are engineered to encode mutant antigens.
  • a mutant antigen has at least one amino acid substitution, deletion or addition relative to the wild-type viral protein from which it is derived.
  • such sequences may include, but are not limited to sequences derived from the env gene (/ ' . e, , sequences encoding all or part of gpl60 5 gpl20, and/or gp41), the pol gene (i.e., sequences encoding all or part of reverse transcriptase, endonuclease, protease, and/or integrase), the gag gene (i.e., sequences encoding all or part of p7, p6, p55, pl7/18, p24/25) tat, rev, nef, vif, vpu, vpr, and/or vpx.
  • the env gene i.e., sequences encoding all or part of gpl60 5 gpl20, and/or gp41
  • the pol gene i.e., sequences encoding all or part of reverse transcriptase, endonuclease,
  • Mutant mMPV of the invention may be engineered to express tumor-associated antigens (TAAs), including but not limited to, human tumor antigens recognized by T cells (Robbins and Kawakami, 1996, Curr. Opin. Immunol.
  • TAAs tumor-associated antigens
  • melanocyte lineage proteins including gplOO, MART-1/MelanA, TRP-I (gp75), tyrosinase; Tumor-specific widely shared antigens, MAGE-I , MAGE-3, BAGE, GAGE-I, GAGE-I , N-acetylglucosaminyltransferase-V, pi 5; Tumor-specific mutated antigens, ⁇ -catenin, MUM-I, CDK4; Nonmelanoma antigens for breast, ovarian, cervical and pancreatic carcinoma, HER-2/neu, human papillomavirus -E6, -E7, MUC-I .
  • melanocyte lineage proteins including gplOO, MART-1/MelanA, TRP-I (gp75), tyrosinase; Tumor-specific widely shared antigens, MAGE-I , MAGE-3, BAGE, GAGE-I, GAGE-I , N-
  • the non-native sequence can be from Salmonella, Shigella, Chlamydia, Helicobacter, Yersinia, Bordatella, Pseudomonas, Neisseria, Vibrio, Haemophilus, Mycoplasma, Streptomyces, Treponema, Coxiella, Ehrlichia, Brucella, Streptobacillus, Fusospiro réelle, Spirillum, Ureaplasma, Spirochaeta, Mycoplasma, Actinomycetes, Borrelia, Bacteroides, Trichomoras, Branhamella, Pasteurella, Clostridium, Corynebacterium, Listeria, Bacillus, Erysipelothrix, Rhodococcus, Escherichia, Klebsiella, Pseudomanas, Enterobacter, Serratia, Staphylococcus, Streptococcus, Legionella, Mycobacterium, Proteus,
  • E. coli P. cepacia
  • S. epidermis E. faecalis
  • S. pneumonias S. aureus
  • N. meningitidis S. pyogenes
  • Pasteurella multocida Treponema pallidum, and P. mirabilis.
  • non-native gene sequences derived from pathogenic fungi include, but are not limited to, antigens derived from fungi such as Cryptococcus neoformans; Blastomyces dermatitidis; Aiellomyces dermatitidis; Histoplasma capsulatum; Coccidioides immitis; Candida species, including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii and C. krusei, Aspergillus species, including A. fumigatus, A. flavus and A. niger, Rhizopus species; Rhizomucor species; Cunninghammella species; Apophysomyces species, including A.
  • antigens derived from fungi such as Cryptococcus neoformans; Blastomyces dermatitidis; Aiellomyces dermatitidis; Histoplasma capsulatum; Coccidioides immitis; Candida species, including C. alb
  • non-native gene sequences derived from parasites include, but are not limited to, antigens derived from members of the Apicomplexa phylum such as, for example, Babesia, Toxoplasma, Plasmodium, Eimeria, Isospora, Atoxoplasma, Cystoisospora, Hammondia, Besniotia, Sarcocystis, Frenkelia, Haemoproteus, Leucocytozoon, Theileria, Perkinsus and Gregarina spp.; Pneumocystis carinii; members of the Microspora phylum such as, for example, Nosema, Enterocytozoon, Encephalitozoon, Septata, Mrazekia, Amblyospora, Ameson, Glugea, Pleistophora and Microsporidium spp.; and members of the Ascetospora phylum such as
  • a chimeric virus may be of particular use for the generation of recombinant vaccines protecting against two or more viruses (Tao et al., J. Virol. 72, 2955-2961 ; Durbin et al., 2000, J.Virol. 74, 6821-6831 ; Skiadopoulos et al., 1998, J. Virol. 72, 1762-1768; Teng et al., 2000, J.Virol. 74, 9317-9321).
  • a MPV or APV virus vector expressing one or more proteins of another negative strand RNA virus e.g., RSV or a RSV vector expressing one or more proteins of MPV will protect individuals vaccinated with such vector against both virus infections.
  • RSV negative strand RNA virus
  • a similar approach can be envisaged for PIV or other paramyxoviruses.
  • Attenuated and replication-defective viruses may be of use for vaccination purposes with live vaccines as has been suggested for other viruses. (See, PCT WO 02/057302, at pp. 6 and 23, incorporated by reference herein).
  • one or more sequences, intergenic regions, termini sequences, or portions or entire ORF have been substituted with a non-native sequence.
  • the non-native nucleotide sequence is inserted or added at Position 1 of the viral genome. In another preferred embodiment, the non-native nucleotide sequence is inserted or added at Position 2 of the viral genome. In even another preferred embodiment, the non-native nucleotide sequence is inserted or added at Position 3 of the viral genome. Insertion or addition of nucleic acid sequences at the lower-numbered positions of the viral genome results in stronger or higher levels of expression of the non-native nucleotide sequence compared to insertion at higher-numbered positions due to a transcriptional gradient across the genome of the virus.
  • non-native nucleotide sequences at lower-numbered positions is the preferred embodiment of the invention if high levels of expression of the heterologous nucleotide sequence is desired.
  • the non-native sequence can be inserted at Postion 1 , 2, 3, 4, 5, or 6.
  • the position of insertion or addition of the non-native sequence affects the replication rate of the virus. The higher rates of replication can be achieved if the non- native sequence is inserted or added at Position 2 or Position 1 of the viral genome. The rate of replication is reduced if the non-native sequence is inserted or added at Position 3, Position 4, Position 5, or Position 6.
  • the position of the insertion and the length of the intergenic region of the inserted heterologous nucleotide sequence can be determined by various indexes including, but not limited to, replication kinetics and protein or mRNA expression levels, measured by following non-limiting examples of assays: plaque assay, fluorescent- focus assay, infectious center assay, transformation assay, endpoint dilution assay, efficiency of plating, electron microscopy, hemagglutination, measurement of viral enzyme activity, viral neutralization, hemagglutination inhibition, complement fixation, immunostaining, immunoprecipitation and immunoblotting, enzyme-linked immunosorbent assay, nucleic acid detection (e.g., Southern blot analysis, Northern blot analysis, Western blot analysis), growth curve, employment of a reporter gene (e.g., using a reporter gene, such as Green Fluorescence
  • the non-native sequence is inserted into the region of the G-ORF that encodes for the ectodomain, such that it is expressed on the surface of the viral envelope.
  • the non-native sequence may be inserted within the antigenic site without deleting any viral sequences.
  • non-native sequences replaces sequences of the G-ORF.
  • Expression products of such constructs may be useful in vaccines against the foreign antigen, and may indeed circumvent problems associated with propagation of the recombinant virus in the vaccinated host.
  • An intact G molecule with a substitution only in antigenic sites may allow for G function and thus allow for the construction of a viable virus. Therefore, this virus can be grown without the need for additional helper functions.
  • the size of the intergenic region between the viral gene and the non-native sequence further determines rate of replication of the virus and expression levels of the heterologous sequence.
  • the viral vector of the invention contains two or more different non-native nucleotide sequences.
  • Standard recombinant DNA technology can be used to generate a cDNA encoding a mutant virus of the invention.
  • the cDNA can optionally contain one or more non-native nucleotide sequences. See Section 5.4.
  • the starting material is a cDNA of the sequence of SEQ ID NO: 1 , 2, 3, or 4. Mutations can be introduced into the cDNA by any method known to the skilled artisan. Such methods include PCR amplification using primers encoding the mutation. Exemplary mutagenic primers are provided as SEQ ID NOs: 123-140.
  • Non-native gene coding sequences flanked by the complement of the viral polymerase binding site/promoter e.g., the complement of 3'-hMPV virus terminus, or the complements of both the 3'- and 5'-hMPV virus termini may be constructed using techniques known in the art.
  • a recombinant virus of the invention contains the leader and trailer sequence of hMPV or APV.
  • the intergenic regions are obtained from hMPV or APV.
  • the resulting RNA templates may be of the negative-polarity and contain appropriate terminal sequences which enable the viral RNA-synthesizing apparatus to recognize the template.
  • RNA templates which contain appropriate terminal sequences which enable the viral RNA- synthesizing apparatus to recognize the template, may also be used.
  • Recombinant DNA molecules containing these hybrid sequences can be cloned and transcribed by a DNA- directed RNA polymerase, such as bacteriophage T7, T3, the SP6 polymerase or eukaryotic polymerase such as polymerase I and the like, to produce in vitro or in vivo the recombinant RNA templates which possess the appropriate viral sequences that allow for viral polymerase recognition and activity.
  • the RNA polymerase is fowlpox virus T7 RNA polymerase or a MVA T7 RNA polymerase.
  • An illustrative approach for constructing these hybrid molecules is to insert the non-native nucleotide sequence into a DNA complement of an hMPV, APV, APV/hMPV or hMPV/ APV genome, so that the heterologous sequence is flanked by the viral sequences required for viral polymerase activity; i.e., the viral polymerase binding site/promoter, hereinafter referred to as the viral polymerase binding site, and a polyadenylation site.
  • the heterologous coding sequence is flanked by the viral sequences that comprise the replication promoters of the 5' and 3' termini, the gene start and gene end sequences, and the packaging signals that are found in the 5' and/or the 3' termini.
  • oligonucleotides encoding the viral polymerase binding site e.g. , the complement of the 3 '-terminus or both termini of the virus genomic segment can be ligated to the heterologous coding sequence to construct the hybrid molecule.
  • Suitable restriction enzyme sites can readily be placed anywhere within a viral cDNA through the use of site-directed mutagenesis (e.g., see, for example, the techniques described by Kunkel, 1985, Proc. Natl. Acad. Sci. U.S.A. 82;488). Variations in polymerase chain reaction (PCR) technology also allow for the specific insertion of sequences (i.e., restriction enzyme sites) and allow for the facile construction of hybrid molecules. Alternatively, PCR reactions could be used to prepare recombinant templates without the need of cloning.
  • PCR polymerase chain reaction
  • PCR reactions could be used to prepare double-stranded DNA molecules containing a DNA-directed RNA polymerase promoter (e.g., bacteriophage T3, T7 or SP6) and the hybrid sequence containing the heterologous gene and the hMPV polymerase binding site.
  • RNA templates could then be transcribed directly from this recombinant DNA.
  • the recombinant RNA templates may be prepared by ligating RNAs specifying the negative polarity of the heterologous gene and the viral polymerase binding site using an RNA ligase.
  • nucleotides can be added in the untranslated region to adhere to the "Rule of Six" which may be important in obtaining virus rescue.
  • the "Rule of Six” applies to many paramyxoviruses and states that the RNA nucleotide genome must be divisible by six to be functional.
  • the addition of nucleotides can be accomplished by techniques known in the art such as using a commercial mutagenesis kits such as the QuikChange mutagenesis kit (Stratagene). After addition of the appropriate number of nucleotides, the correct DNA fragment can then be isolated by digestion with appropriate restriction enzyme and gel purification. Sequence requirements for viral polymerase activity and constructs which may be used in accordance with the invention are described in the subsections below.
  • the leader and or trailer sequence of the virus are modified relative to the wild type virus.
  • the lengths of the leader and/or trailer are altered.
  • the sequence(s) of the leader and/or trailer are mutated relative to the wild type virus.
  • vRNA negative sense viral RNA
  • cRNA complementary copy thereof
  • vRNA or cRNA can be isolated from infectious virus, produced upon in- vitro transcription, or produced in cells upon transfection of nucleic acids.
  • the production of recombinant negative strand virus relies on a functional polymerase complex.
  • the polymerase complex of pneumoviruses consists of N, P, L and possibly M2 proteins, but is not necessarily limited thereto.
  • Polymerase complexes or components thereof can be isolated from virus particles, isolated from cells expressing one or more of the components, or produced upon transfection of specific expression vectors.
  • Infectious copies of MPV can be obtained when the above mentioned vRNA, cRNA, or vectors expressing these RNAs are replicated by the above mentioned polymerase complex 16 (Schnell et al, 1994, EMBO J 13: 4195-4203; Collins, et al., 1995, PNAS 92: 11563-11567; Hoffmann, et al., 2000, PNAS 97: 6108-6113; Bridgen, et al., 1996, PNAS 93: 15400-15404; Palese, et al., 1996, PNAS 93: 11354-11358; Peeters, et al., 1999, J.Virol. 73: 5001-5009; Durbin, et al., 1997, Virology 235: 323-332).
  • the invention also provides a host cell comprising a nucleic acid or a vector according to the invention.
  • Plasmid or viral vectors containing the polymerase components of MPV are generated in prokaryotic cells for the expression of the components in relevant cell types. Plasmid or viral vectors containing full-length or partial copies of the MPV genome will be generated in prokaryotic cells for the expression of viral nucleic acids in-vitro or in-vivo.
  • eukaryotic cells transiently or stably expressing one or more full-length or partial MPV proteins can be used. Such cells can be made by transfection (proteins or nucleic acid vectors), infection (viral vectors) or transduction (viral vectors) and may be useful for complementation of mentioned wild type, attenuated, replication-defective or chimeric viruses.
  • Bicistronic mRNA could be constructed to permit internal initiation of translation of viral sequences and allow for the expression of foreign protein coding sequences from the regular terminal initiation site.
  • a bicistronic mRNA sequence may be constructed wherein the viral sequence is translated from the regular terminal open reading frame, while the foreign sequence is initiated from an internal site.
  • Certain internal ribosome entry site (IRES) sequences may be utilized.
  • the IRES sequences which are chosen should be short enough to not interfere with MPV packaging limitations. Thus, it is preferable that the IRES chosen for such a bicistronic approach be no more than 500 nucleotides in length.
  • the IRES is derived from a picornavirus and does not include any additional picornaviral sequences.
  • Specific IRES elements include, but are not limited to the mammalian BiP IRES and the hepatitis C virus IRES.
  • a foreign protein may be expressed from a new internal transcriptional unit in which the transcriptional unit has an initiation site and polyadenylation site.
  • the foreign gene is inserted into a MPV gene such that the resulting expressed protein is a fusion protein.
  • the cDNA encoding the mMPV encodes the wild-type leader sequence of the virus. In certain embodiments, the cDNA encoding the mMPV encodes the C4A mutation in the leader sequence of the virus, i.e., a nucleotide substitution at position 4 of the leader sequence that results in an A in place of the C.
  • a cDNA encoding the genome of a recombinant or chimeric virus of the invention in the plus or minus sense may be used to transfect a host cell which provide viral proteins and functions required for replication and rescue.
  • cells may be transfected with helper virus before, during, or after transfection by the DNA or RNA molecule coding for the recombinant virus of the invention.
  • the synthetic recombinant plasmid DNAs and RNAs of the invention can be replicated and rescued into infectious virus particles by any number of techniques known in the art, as described, e.g., in U.S.
  • synthetic recombinant viral RNAs may be prepared that contain the non-coding regions (leader and trailer) of the negative strand virus RNA which are essential for the recognition by viral polymerases and for packaging signals necessary to generate a mature virion.
  • the recombinant RNAs are synthesized from a recombinant DNA template and reconstituted in vitro with purified viral polymerase complex to form recombinant ribonucleoproteins (RNPs) which can be used to transfect cells.
  • the viral polymerase proteins are present during transcription of the synthetic RNAs either in vitro or in vivo.
  • the synthetic RNAs may be transcribed from cDNA plasmids which are either co-transcribed in vitro with cDNA plasmids encoding the polymerase proteins, or transcribed in vivo in the presence of polymerase proteins, i.e., in cells which transiently or constitutively express the polymerase proteins.
  • infectious chimeric or recombinant virus may be replicated in host cell systems that express a metapneumoviral polymerase protein (e.g., in virus/host cell expression systems; transformed cell lines engineered to express a polymerase protein, etc.), so that infectious chimeric or recombinant virus are replicated and rescued.
  • helper virus need not be utilized since this function is provided by the viral polymerase proteins expressed.
  • viral proteins may be supplied in the form of wildtype virus, helper virus, purified viral proteins or recombinantly expressed viral proteins.
  • the viral proteins may be supplied prior to, during or post transcription of the synthetic cDNAs or RNAs encoding the chimeric virus. The entire mixture may be used to transfect host cells.
  • viral proteins and functions required for replication may be supplied prior to or during transcription of the synthetic cDNAs or RNAs encoding the chimeric virus.
  • viral proteins and functions required for replication are supplied in the form of wildtype virus, helper virus, viral extracts, synthetic cDNAs or RNAs which express the viral proteins are introduced into the host cell via infection or transfection. This infection/transfection takes place prior to or simultaneous to the introduction of the synthetic cDNAs or RNAs encoding the chimeric virus genome.
  • Helper viruses that may be used in accordance with the invention, include those that express the polymerase viral proteins natively, such as MPV or APV. Alternatively, helper viruses may be used that have been recombinantly engineered to provide the polymerase viral proteins
  • the host cell expresses components of the viral polymerase constitutively. In other aspects, the expression of the viral polymerase components is induced. In certain aspects, the host cell is transiently transfected with the plasmids encoding the viral polymerase components. In other aspects, the host cell is a stable cell line with the nucleotide sequences encoding the viral polymerase components. In other embodiments, the host cell is infected with a helper virus that provides the RNA polymerase.
  • viral proteins and functions required for replication may be supplied as genetic material in the form of synthetic cDNAs or RNAs so that they are co-transcribed with the synthetic cDNAs or RNAs encoding the chimeric virus. Plasmids that encode express the virus and the viral polymerase and/or other viral functions are co- transfected into host cells. Alternatively, rescue of the recombinant viruses of the invention may be accomplished by the use of Modified Vaccinia Virus Ankara (MVA) encoding T7 RNA polymerase, or a combination of MVA and plasmids encoding the polymerase proteins (N, P, and L).
  • MVA Modified Vaccinia Virus Ankara
  • MVA-T7 or Fowl Pox-T7 can be infected into Vero cells, LLC- MK-2 cells, HEp-2 cells, LF 1043 (HEL) cells, tMK cells, LLC-MK2, HUT 292, FRHL-2 (rhesus), FCL-I (green monkey), WI-38 (human), MRC-5 (human) cells, 293 T cells, QT 6 cells, QT 35 cells and CEF cells.
  • a full length antigenomic or genomic cDNA encoding the recombinant virus of the invention may be transfected into the cells together with the N, P, L, and M2.1 encoding expression plasmids.
  • the polymerase may be provided by plasmid transfection.
  • the cells and cell supernatant can subsequently be harvested and subjected to a single freeze-thaw cycle.
  • the resulting cell lysate may then be used to infect a fresh Vero cell monolayer in the presence of 1-beta-D-arabinofuranosylcytosine (ara C), a replication inhibitor of vaccinia virus, to generate a virus stock.
  • ara C 1-beta-D-arabinofuranosylcytosine
  • the supernatant and cells from these plates can then be harvested, freeze-thawed once and the presence of recombinant virus particles of the invention can be assayed by immunostaining of virus plaques using antiserum specific to the particular virus.
  • Another approach to propagating the chimeric or recombinant virus may involve co-cultivation with wild-type virus. This could be done by simply taking recombinant virus and co-infecting cells with this and another wild-type virus.
  • the wild-type virus should complement for the defective virus gene product and allow growth of both the wild-type and recombinant virus.
  • leader and trailer sequences In order to achieve replication and packaging of the viral genome, it is important that the leader and trailer sequences retain the signals necessary for viral polymerase recognition.
  • the leader and trailer sequences for the viral RNA genome can be optimized or varied to improve and enhance viral replication and rescue. Alternatively, the leader and trailer sequences can be modified to decrease the efficiency of viral replication and packaging, resulting in a rescued virus with an attenuated phenotype. Examples of different leader and trailer sequences, include, but are not limited to, leader and trailer sequences of a paramyxovirus. In a specific embodiment of the invention, the leader and trailer sequence is that of a wild type or mutated hMPV.
  • the leader and trailer sequence is that of a PIV, APV, or an RSV.
  • the leader and trailer sequence is that of a combination of different virus origins.
  • the leader and trailer sequence can be a combination of any of the leader and trailer sequences of hMPV, PIV, APV, RSV, or any other paramyxovirus.
  • leader and trailer sequences examples include varying the spacing relative to the viral promoter, varying the sequence, e.g., varying the number of G residues (typically 0 to 3), and defining the 5' or 3' end using ribozyme sequences, including, Hepatitis Delta Virus (HDV) ribozyme sequence, Hammerhead ribozyme sequences, or fragments thereof, which retain the ribozyme catalytic activity, and using restriction enzymes for run-off RNA produced in vitro.
  • HDV Hepatitis Delta Virus
  • the efficiency of viral replication and rescue may be enhanced if the viral genome is of hexamer length.
  • the 5' or 3' end may be defined using ribozyme sequences, including, Hepatitis Delta Virus (HDV) ribozyme sequence, Hammerhead ribozyme sequences, or fragments thereof, which retain the ribozyme catalytic activity, and using restriction enzymes for run-off RNA produced in vitro.
  • HDV Hepatitis Delta Virus
  • the genetic material encoding the viral genome and for the genetic material encoding the RNA polymerase components to be transcribed the genetic material is engineered to be placed under the control of appropriate transcriptional regulatory sequences, e.g., promoter sequences recognized by a polymerase.
  • the promoter sequences are recognized by a T7, Sp6 or T3 polymerase.
  • the promoter sequences are recognized by cellular DNA dependent RNA polymerases, such as RNA polymerase I (Pol I) or RNA polymerase II (Pol II).
  • the genetic material encoding the viral genome may be placed under the control of the transcriptional regulatory sequences, so that either a positive or negative strand copy of the viral genome is transcribed.
  • the genetic material encoding the viral genome is recombinantly engineered to be operatively linked to the transcriptional regulatory sequences in the context of an expression vector, such as a plasmid based vector, e.g.
  • a plasmid with a pol II promoter such as the immediate early promoter of CMV, a plasmid with a T7 promoter, or a viral based vector, e.g., pox viral vectors, including vaccinia vectors, MVA-T7, and Fowl pox vectors.
  • the genetic material encoding the viral genome may be modified to enhance expression by the polymerase of choice, e.g., varying the number of G residues (typically 0 to 3) upstream of the leader or trailer sequences to optimize expression from a T7 promoter.
  • Replication and packaging of the viral genome occurs intracellular ⁇ in a host cell permissive for viral replication and packaging.
  • the host cell can be engineered to provide sufficient levels of the viral polymerase and structural proteins necessary for replication and packaging, including, host cells infected with an appropriate helper virus, host cells engineered to stably or constitutively express the viral polymerase and structural proteins, or host cells engineered to transiently or inducibly express the viral polymerase and structural proteins.
  • Protein function required for MPV viral replication includes, but not limited to, the polymerase proteins P, N, L, and M2.1.
  • polymerase proteins In order to achieve efficient viral replication and packaging, high levels of expression of the polymerase proteins is preferred. Such levels are obtained using 100-200 ng L/pCITE, 200-400 ng N/pCITE, 200-400 ng P/pCITE, and 100-200 ng M2.1/pCITE plasmids encoding paramyxovirus proteins together with 2 - 4 ug of plasmid encoding the full-length viral cDNA transfected into cells infected with MVA-T7.
  • 0.1 - 2.0 ⁇ g of pSH25 (CAT expressing), 0.1 - 3.0 ⁇ g of pRF542 (expressing T7 polymerase), 0.1 - 0.8 ⁇ g pCITE vector with N cDNA insert, and 0.1 - 1.0 ⁇ g of each of three pCITE vectors containing P, L and M2.1 cDNA insert are used.
  • one or more polymerase and structural proteins can be introduced into the cells in conjunction with the genetic material by transfecting cells with purified ribonucleoproteins. Host cells that are permissive for MPV viral replication and packaging are preferred. Examples of preferred host cells include, but are not limited to, 293T, Vero, tMK, and BHK.
  • host cells include, but are not limited to, LLC-MK-2 cells, Hep-2 cells, LF 1043 (HEL) cells, LLC-MK2, HUT 292, FRHL-2 (rhesus), FCL-I (green monkey), WI-38 (human), MRC-5 (human) cells, QT 6 cells, QT 35 cells and CEF cells.
  • the host cells can be treated using a number of methods in order to enhance the level of transfection and /or infection efficiencies, protein expression, in order to optimize viral replication and packaging.
  • treatment methods include, but are not limited to, sonication, freeze/thaw, and heat shock.
  • standard techniques known to the skilled artisan can be used to optimize the transfection and/ or infection protocol, including, but are not limited to, DEAE-dextran- mediated transfection, calcium phosphate precipitation, lipofectin treatment, liposome- mediated transfection and electroporation. The skilled artisan would also be familiar with standard techniques available for the optimization of transfection/infection protocols.
  • viruses of the invention can be propagated using any technique known to the skilled artisan.
  • the viruses are propagated in serum-free medium as described in International Patent Application No. PCT/US04/12724 (published as WO 04/096993; Section 5.6).
  • additional mutations can be introduced into the mutant mMPV, such as hMPV.
  • the additional mutations contribute to an attenuated phenotype of the viruses of the invention.
  • the additional mutation is a deletion of an entire open reading frame, or a deletion that reduces the function of the affected gene.
  • the additional mutation can be a deletion of (or in) the M2.2 gene or the SH gene.
  • the recombinant viruses of the invention exhibit an attenuated phenotype in a subject to which the virus is administered as a vaccine. Attenuation can be achieved by any method known to a skilled artisan. Without being bound by theory, the attenuated phenotype of the recombinant virus can be caused, e.g., by using a virus that naturally does not replicate well in an intended host (e.g., using an APV in human), by reduced replication of the viral genome, by reduced ability of the virus to infect a host cell, or by reduced ability of the viral proteins to assemble to an infectious viral particle relative to the wild type strain of the virus.
  • the viability of certain sequences of the virus, such as the leader and the trailer sequence can be tested using a minigenome assay (see section 5.8).
  • the attenuated phenotypes of a recombinant virus of the invention can be tested by any method known to the artisan (see, e.g., section 5.8).
  • a candidate virus can, for example, be tested for its ability to infect a host or for the rate of replication in a cell culture system.
  • a mimi-genome system is used to test the attenuated virus when the gene that is altered is N, P, L, M2, F, G, M2.1 , M2.2 or a combination thereof.
  • growth curves at different temperatures are used to test the attenuated phenotype of the virus.
  • an attenuated virus is able to grow at 35 0 C, but not at 39 0 C or 4O 0 C.
  • different cell lines can be used to evaluate the attenuated phenotype of the virus.
  • an attenuated virus may only be able to grow in monkey cell lines but not the human cell lines, or the achievable virus titers in different cell lines are different for the attenuated virus.
  • viral replication in the respiratory tract of a small animal model is used to evaluate the attenuated phenotypes of the virus.
  • the immune response induced by the virus including but not limited to, the antibody titers (e.g., assayed by plaque reduction neutralization assay or ELISA) is used to evaluate the attenuated phenotypes of the virus.
  • the plaque reduction neutralization assay or ELISA is carried out at a low dose.
  • the ability of the recombinant virus to elicit pathological symptoms in an animal model can be tested.
  • a reduced ability of the virus to elicit pathological symptoms in an animal model system is indicative of its attenuated phenotype.
  • the candidate viruses are tested in a monkey model for nasal infection, indicated by mucous production.
  • the viruses of the invention can be attenuated such that one or more of the functional characteristics of the virus are impaired.
  • attenuation is measured in comparison to the wild type strain of the virus from which the attenuated virus is derived.
  • attenuation is determined by comparing the growth of an attenuated virus in different host systems.
  • an APV is said to be attenuated when grown in a human host if the growth of the APV in the human host is reduced compared to the growth of the APV in an avian host.
  • the attenuated virus of the invention is capable of infecting a host, is capable of replicating in a host such that infectious viral particles are produced.
  • the attenuated strain grows to lower titers or grows more slowly. Any technique known to the skilled artisan can be used to determine the growth curve of the attenuated virus and compare it to the growth curve of the wild type virus. For exemplary methods see Example section, infra.
  • the attenuated virus grows to a titer of less than 10 5 pfu/ml, of less than 10 4 pfu/ml, of less than 10 3 pfu/ml, or of less than 10 2 pfu/ml in Vero cells.
  • the attenuated hMPV of the invention cannot replicate in human cells as well as the wild type virus ⁇ e.g., wild type mammalian MPV) does.
  • the attenuated virus can replicate well in a cell line that lack interferon functions, such as Vero cells.
  • the attenuated virus of the invention is capable of infecting a host, of replicating in the host, and of causing proteins of the virus of the invention to be inserted into the cytoplasmic membrane, but the attenuated virus does not cause the host to produce new infectious viral particles.
  • the attenuated virus infects the host, replicates in the host, and causes viral proteins to be inserted in the cytoplasmic membrane of the host with the same efficiency as the wild type mammalian virus.
  • the ability of the attenuated virus to cause viral proteins to be inserted into the cytoplasmic membrane into the host cell is reduced compared to the wild type virus.
  • the ability of the attenuated mammalian virus to replicate in the host is reduced compared to the wild type virus. Any technique known to the skilled artisan can be used to determine whether a virus is capable of infecting a mammalian cell, of replicating within the host, and of causing viral proteins to be inserted into the cytoplasmic membrane of the host. For illustrative methods see section 5.8. [00162] In certain embodiments, the attenuated virus of the invention is capable of infecting a host. In contrast to the wild type mammalian MPV, however, the attenuated mammalian MPV cannot be replicated in the host.
  • the attenuated mammalian virus can infect a host and can cause the host to insert viral proteins in its cytoplasmic membranes, but the attenuated virus is incapable of being replicated in the host. Any method known to the skilled artisan can be used to test whether the attenuated mammalian MPV has infected the host and has caused the host to insert viral proteins in its cytoplasmic membranes.
  • the ability of the attenuated mammalian virus to infect a host is reduced compared to the ability of the wild type virus to infect the same host. Any technique known to the skilled artisan can be used to determine whether a virus is capable of infecting a host. For illustrative methods see section 5.8.
  • mutations are introduced into the genome of the virus to generated a virus with an attenuated phenotype.
  • Mutations e.g., missense mutations
  • a single amino acid deletion mutation for the N, P, L, F, G, M2.1, M2.2 or M2 proteins is introduced, which can be screened for functionality in the mini-genome assay system and be evaluated for predicted functionality in the virus.
  • the missense mutation is a cold-sensitive mutation.
  • the missense mutation is a heat-sensitive mutation.
  • major phosphorylation sites of P protein of the virus is removed.
  • a mutation or mutations are introduced into the L gene of the virus to generate a temperature sensitive strain.
  • the cleavage site of the F gene is mutated in such a way that cleavage does not occur or occurs at very low efficiency.
  • the motif with the amino acid sequence RQSR at amino acid postions 99 to 102 of the F protein of hMPV is mutated.
  • a mutation can be, but is not limited to, a deletion of one or more amino acids, an addition of one or more amino acids, a substitution (conserved or non-conserved) of one or more amino acids or a combination thereof.
  • the cleavage site is RQPR (see Example "PlOlS").
  • the cleavage site with the amino acid sequence is RQPR is mutated.
  • the cleavage site of the F protein of hMPV is mutated such that the infectivity of hMPV is reduced.
  • the infectivity of hMPV is reduced by a factor of at least 5, 10, 50, 100, 500, 10 3 , 5x10 3 , 10 4 , 5xlO 4 , 10 5 , 5xlO 5 , or at least 10 6 .
  • the infectivity of hMPV is reduced by a factor of at most 5, 10, 50, 100, 500, 10 3 , 5x10 3 , 10 4 , 5x10 4 , 10 5 , 5x10 5 , or at most 10 6 .
  • deletions are introduced into the genome of the recombinant virus.
  • a deletion can be introduced into the N- gene, the P-gene, the M-gene, the F-gene, the M2-gene, the SH-gene, the G-gene or the L- gene of the recombinant virus.
  • the deletion is in the M2-gene of the recombinant virus of the present invention.
  • the deletion is in the SH-gene of the recombinant virus of the present invention.
  • both the M2-gene and the SH-gene are deleted.
  • the intergenic region of the recombinant virus is altered. In one embodiment, the length of the intergenic region is altered. In another embodiment, the intergenic regions are shuffled from 5' to 3' end of the viral genome.
  • the genome position of a gene or genes of the recombinant virus is changed.
  • the F or G gene is moved to the 3' end of the genome.
  • the N gene is moved to the 5' end of the genome.
  • attenuation of the virus is achieved by replacing a gene of the wild type virus with the analogous gene of a virus of a different species (e.g., of RSV, APV, PIV3 or mouse pneumovirus), of a different subgroup, or of a different variant.
  • the N-gene, the P-gene, the M-gene, the F-gene, the M2-gene, the SH-gene, the G-gene or the L-gene of a mammalian MPV is replaced with the N-gene, the P- gene, the M-gene, the F-gene, the M2-gene, the SH-gene, the G-gene or the L-gene, respectively, of an APV.
  • the N-gene, the P-gene, the M- gene, the F-gene, the M2-gene, the SH-gene, the G-gene or the L-gene of APV is replaced with the N-gene, the P-gene, the M-gene, the F-gene, the M2-gene, the SH-gene, the G-gene or the L-gene, respectively, of a mammalian MPV.
  • attenuation of the virus is achieved by replacing one or more polymerase associated genes (e.g., N, P, L or M2) with genes of a virus of a different species.
  • Attenuation of the virus is achieved by replacing one or more specific domains of a protein of the wild type virus with domains derived from the corresponding protein of a virus of a different species.
  • the ectodomain of a F protein of APV is replaced with an ectodomain of a F protein of a mammalian MPV.
  • one or more specific domains of L, N, or P protein are replaced with domains derived from corresponding proteins of a virus of a different species.
  • attenuation of the virus is achieved by deleting one or more specific domains of a protein of the wild type virus.
  • the transmembrane domain of the F-protein is deleted.
  • the leader and/or trailer sequence of the recombinant virus of the invention can be modified to achieve an attenuated phenotype.
  • the leader and/or trailer sequence is reduced in length relative to the wild type virus by at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides or at least 6 nucleotides.
  • the sequence of the leader and/or trailer of the recombinant virus is mutated.
  • the leader and the trailer sequence are 100% complementary to each other.
  • 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides are not complementary to each other where the remaining nucleotides of the leader and the trailer sequences are complementary to each other.
  • the non-complementary nucleotides are identical to each other. In certain other embodiments, the non-complementary nucleotides are different from each other. In other embodiments, if the non-complementary nucleotide in the trailer is purine, the corresponding nucleotide in the leader sequence is also a purine.
  • the leader and/or trailer sequence of the recombinant virus of the invention can be replaced with the leader and/or trailer sequence of a another virus, e.g., with the leader and/or trailer sequence of RSV, APV, PIV3, mouse pneumovirus, or with the leader and/or trailer sequence of a human metapneumovirus of a subgroup or variant different from the hMPV metapneumovirus from which the protein-encoding parts of the recombinant virus are derived.
  • a live attenuated vaccine When a live attenuated vaccine is used, its safety must also be considered. Preferably the vaccine does not cause disease. Any techniques known in the art that can make a vaccine safe may be used in the present invention. In addition to attenuation techniques, other techniques may be used. One non-limiting example is to use a soluble heterologous gene that cannot be incorporated into the virion membrane. For example, a single copy of the soluble RSV F gene, a version of the RSV gene lacking the transmembrane and cytosolic domains, can be used. Since it cannot be incorporated into the virion membrane, the virus tropism is not expected to change.
  • sucrose gradients and neutralization assays can be used to test the safety.
  • a sucrose gradient assay can be used to determine whether a heterologous protein is inserted in a virion. If the heterologous protein is inserted in the virion, the virion should be tested for its ability to cause symptoms even if the parental strain does not cause symptoms. Without being bound by theory, if the heterologous protein is incorporated in the virion, the virus may have acquired new, possibly pathological, properties.
  • one or more genes are deleted from the hMPV genome to generate an attenuated virus.
  • the M2.2 ORF, the M2.1 ORF, the M2 gene, the SH gene and/or the G2 gene is deleted.
  • small single amino acid deletions are introduced in genes involved in virus replication to generate an attenuated virus.
  • a small single amino acid deletion is introduced in the N, L, or the P gene.
  • one or more of the following amino acids are mutated in the L gene of a recombinant hMPV: Phe at amino acid position 456, GIu at amino acid position 749, Tyr at amino acid position 1246, Met at amino acid position 1094 and Lys at amino acid position 746 to generate an attenuated virus.
  • a mutation can be, e.g., a deletion or a substitution of an amino acid.
  • An amino acid substitution can be a conserved amino acid substitution or a non- conserved amino acid substitution.
  • amino acid substitutions that maintain structural and/or functional properties of the amino acids' side-chains, e.g., an aromatic amino acid is substituted for another aromatic amino acid, an acidic amino acid is substituted for another acidic amino acid, a basic amino acid is substituted for another basic amino acid, and an aliphatic amino acid is substituted for another aliphatic amino acid.
  • non-conserved amino acid exchanges are amino acid substitutions that do not maintain structural and/or functional properties of the amino acids' side-chains, e.g., an aromatic amino acid is substituted for a basic, acidic, or aliphatic amino acid, an acidic amino acid is substituted for an aromatic, basic, or aliphatic amino acid, a basic amino acid is substituted for an acidic, aromatic or aliphatic amino acid, and an aliphatic amino acid is substituted for an aromatic, acidic or basic amino acid.
  • Phe at amino acid position 456 is replaced by a Leu.
  • one nucleic acid is substituted to encode one amino acid exchange.
  • two or three nucleic acids are substituted to encode one amino acid exchange. It is preferred that two or three nucleic acids are substituted to reduce the risk of reversion to the wild type protein sequence.
  • small single amino acid deletions are introduced in genes involved in virus assembly to generate an attenuated virus.
  • a small single amino acid deletion is introduced in the M gene or the M2 gene.
  • the M gene is mutated.
  • the gene order in the genome of the virus is changed from the gene order of the wild type virus to generate an attenuated virus.
  • the F, SH, and/or the G gene is moved to the 3' end of the viral genome.
  • the N gene is moved to the 5' end of the viral genome.
  • one or more gene start sites are mutated or substituted with the analogous gene start sites of another virus (e.g., RSV, PIV3, APV or mouse pneumovirus) or of a human metapneumovirus of a subgroup or a variant different from the human metapneumovirus from which the protein-encoding parts of the recombinant virus are derived.
  • the gene start site of the N-gene, the P-gene, the M- gene, the F-gene, the M2-gene, the SH-gene, the G-gene and/or the L-gene is mutated or replaced with the start site of the N-gene, the P-gene, the M-gene, the F-gene, the M2-gene, the SH-gene, the G-gene and/or the L-gene, respectively, of another virus (e.g., RSV, PIV3, APV or mouse pneumovirus) or of a human metapneumovirus of a subgroup or a variant different from the human metapneumovirus from which the protein-encoding parts of the recombinant virus are derived.
  • another virus e.g., RSV, PIV3, APV or mouse pneumovirus
  • Attenuation is achieved by replacing one or more of the genes of a virus with the analogous gene of a different virus, different strain, or different viral isolate.
  • one or more of the genes of a metapneumovirus such as a mammalian metapneumovirus, e.g., hMPV, or APV, is replaced with the analogous gene(s) of another paramyxovirus.
  • the N- gene, the P-gene, the M-gene, the F-gene, the M2-gene, the M2.1 ORF, the M2.2 ORF, the SH-gene, the G-gene or the L-gene or any combination of two or more of these genes of a mammalian metapneumovirus, e.g., hMPV, is replaced with the analogous gene of another viral species, strain or isolate, wherein the other viral species can be, but is not limited to, another mammalian metapneumovirus, APV, or RSV.
  • one or more of the genes of human metapneumovirus are replaced with the analogous gene(s) of another isolate of human metapneumovirus.
  • the analogous gene(s) of another isolate of human metapneumovirus E.g., the N-gene, the P-gene, the M-gene, the F-gene, the M2-gene, the M2.1 ORF, the M2.2 ORF, the SH-gene, the G-gene or the L-gene or any combination of two or more of these genes of isolate NL/1/99, NL/1/00, NL/17/00, or NL/1/94 is replaced with the analogous gene or combination of genes, i.e., the N-gene, the P-gene, the M-gene, the F- gene, the M2-gene, the M2.1 ORF, the M2.2 ORF, the SH-gene, the G-gene or the L-gene, of a different isolate, e.g., NL/1/99, NL
  • one or more regions of the genome of a virus is/are replaced with the analogous region(s) from the genome of a different viral species, strain or isolate.
  • the region is a region in a coding region of the viral genome.
  • the region is a region in a non-coding region of the viral genome.
  • two regions of two viruses are analogous to each other if the two regions support the same or a similar function in the two viruses.
  • two regions of two viruses are analogous if the two regions provide the same of a similar structural element in the two viruses.
  • two regions are analogous if they encode analogous protein domains in the two viruses, wherein analogous protein domains are domains that have the same or a similar function and/or structure.
  • one or more of regions of a genome of a metapneumovirus such as a mammalian metapneumovirus, e.g., hMPV, or APV, is/are replaced with the analogous region(s) of the genome of another paramyxovirus.
  • one or more of regions of the genome of a paramyxovirus is/are replaced with the analogous region(s) of the genome of a mammalian metapneumovirus, e.g., hMPV, or APV.
  • a region of the N-gene, the P-gene, the M-gene, the F- gene, the M2-gene, the M2.1 ORF, the M2.2 ORF, the SH-gene, the G-gene or the L-gene or any combination of two or more regions of these genes of a mammalian metapneumovirus, e.g., hMPV, is replaced with the analogous region of another viral species, strain or isolate.
  • Another viral species can be, but is not limited to, another mammalian metapneumovirus, APV, or RSV.
  • one or more regions of human metapneumovirus are replaced with the analogous region(s) of another isolate of human metapneumovirus.
  • one or more region(s) of the N-gene, the P-gene, the M-gene, the F-gene, the M2-gene, the M2.1 ORF, the M2.2 ORF, the SH-gene, the G-gene or the L-gene or any combination of two or more regions of isolate NL/ 1/99, NL/ 1/00, NL/ 17/00, or NL/ 1/94 is replaced with the analogous region(s) of a different isolate of hMPV, e.g., NL/1/99, NL/1/00, NL/17/00, or NL/1/94.
  • the region is at least 5 nucleotides (nt) in length, at least 10 nt, at least 25 nt, at least 50 nt, at least 75 nt, at least 100 nt, at least 250 nt, at least 500 nt, at least 750 nt, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 4 kb, or at least 5 kb in length.
  • nt nucleotides
  • the region is at most 5 nucleotides (nt) in length, at most 10 nt, at most 25 nt, at most 50 nt, at most 75 nt, at most 100 nt, at most 250 nt, at most 500 nt, at most 750 nt, at most 1 kb, at most 1.5 kb, at most 2 kb, at most 2.5 kb, at most 3 kb, at most 4 kb, or at most 5 kb in length.
  • nt nucleotides
  • a number of assays may be employed in accordance with the present invention in order to determine the rate of growth of a chimeric or recombinant virus in a cell culture system, an animal model system or in a subject.
  • a number of assays may also be employed in accordance with the present invention in order to determine the requirements of the chimeric and recombinant viruses to achieve infection, replication and packaging of virions.
  • Expression levels of non-native sequence in a chimeric virus of the invention can be determined by infecting cells in culture with a virus of the invention and subsequently measuring the level of protein expression by, e.g., Western blot analysis or ELISA using antibodies specific to the gene product of the heterologous sequence, or measuring the level of RNA expression by, e.g., Northern blot analysis using probes specific to the heterologous sequence.
  • expression levels of the heterologous sequence can be determined by infecting an animal model and measuring the level of protein expressed from the heterologous sequence of the recombinant virus of the invention in the animal model.
  • the protein level can be measured by obtaining a tissue sample from the infected animal and then subjecting the tissue sample to Western blot analysis or ELISA, using antibodies specific to the gene product of the heterologous sequence. Further, if an animal model is used, the titer of antibodies produced by the animal against the gene product of the heterologous sequence can be determined by any technique known to the skilled artisan, including but not limited to, ELISA.
  • the heterologous sequence encodes a reporter gene. Once the optimal parameters are determined, the reporter gene is replaced by a heterologous nucleotide sequence encoding an antigen of choice. Any reporter gene known to the skilled artisan can be used with the methods of the invention.
  • the rate of replication of the recombinant virus can be determined by any standard technique known to the skilled artisan.
  • the rate of replication is represented by the growth rate of the virus and can be determined by plotting the viral titer over the time post infection.
  • the viral titer can be measured by any technique known to the skilled artisan.
  • a suspension containing the virus is incubated with cells that are susceptible to infection by the virus.
  • Cell types that can be used with the methods of the invention include, but are not limited to, Vero cells, LLC-MK-2 cells, Hep-2 cells, LF 1043 (HEL) cells, MRC-5 cells, WI-38 cells, tMK cells, 293 T cells, QT 6 cells, QT 35 cells, or chicken embryo fibroblasts (CEF).
  • HEL LF 1043
  • WI-38 WI-38 cells
  • tMK cells 293 T cells
  • QT 6 cells QT 35 cells
  • CEF chicken embryo fibroblasts
  • the virus comprises a heterologous nucleotide sequence encoding for eGFP, and the number of cells expressing eGFP, i.e., the number of cells infected with the virus, is determined using FACS.
  • the assays described herein may be used to assay viral titre over time to determine the growth characteristics of the virus.
  • the viral titre is determined by obtaining a sample from the infected cells or the infected subject, preparing a serial dilution of the sample and infecting a monolayer of cells that are susceptible to infection with the virus at a dilution of the virus that allows for the emergence of single plaques.
  • the plaques can then be counted and the viral titre express as plaque forming units per milliliter of sample.
  • the growth rate of a virus of the invention in a subject is estimated by the titer of antibodies against the virus in the subject.
  • the antibody titer in the subject reflects not only the viral titer in the subject but also the antigenicity. If the antigenicity of the virus is constant, the increase of the antibody titer in the subject can be used to determine the growth curve of the virus in the subject.
  • the growth rate of the virus in animals or humans is best tested by sampling biological fluids of a host at multiple time points postinfection and measuring viral titer.
  • the expression of heterologous gene sequence in a cell culture system or in a subject can be determined by any technique known to the skilled artisan.
  • the expression of the heterologous gene is measured by quantifying the level of the transcript.
  • the level of the transcript can be measured by Northern blot analysis or by RT-PCR using probes or primers, respectively, that are specific for the transcript.
  • the transcript can be distinguished from the genome of the virus because the virus is in the antisense orientation whereas the transcript is in the sense orientation.
  • the expression of the heterologous gene is measured by quantifying the level of the protein product of the heterologous gene.
  • the level of the protein can be measured by Western blot analysis using antibodies that are specific to the protein.
  • the heterologous gene is tagged with a peptide tag.
  • the peptide tag can be detected using antibodies against the peptide tag.
  • the level of peptide tag detected is representative for the level of protein expressed from the heterologous gene.
  • the protein expressed from the heterologous gene can be isolated by virtue of the peptide tag.
  • the amount of the purified protein correlates with the expression level of the heterologous gene.
  • Such peptide tags and methods for the isolation of proteins fused to such a peptide tag are well known in the art.
  • peptide tags known in the art may be used in the modification of the heterologous gene, such as, but not limited to, the immunoglobulin constant regions, polyhistidine sequence (Petty, 1996, Metal-chelate affinity chromatography, in Current Protocols in Molecular Biology, volume 1-3 (1994-1998). Ed. by Ausubel, F. M., Brent, R., Kunststoffon, R.E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. Published by John Wiley and sons, Inc., USA, Greene Publish. Assoc. & Wiley Interscience), glutathione S-transferase (GST; Smith, 1993, Methods MoI. Cell Bio.
  • peptide tags As will be appreciated by those skilled in the art, many methods can be used to obtain the coding region of the above-mentioned peptide tags, including but not limited to, DNA cloning, DNA amplification, and synthetic methods. Some of the peptide tags and reagents for their detection and isolation are available commercially.
  • Samples from a subject can be obtained by any method known to the skilled artisan.
  • the sample consists of nasal aspirate, throat swab, sputum or broncho-alveolar lavage.
  • reporter genes that can be used include, but are not limited to, genes that encode GFP, HRP, LUC, and AP.
  • reporter gene that is used encodes CAT.
  • the reporter gene is flanked by leader and trailer sequences.
  • the leader and trailer sequences that can be used to flank the reporter genes are those of any negative-sense virus, including, but not limited to, MPV, RSV, and APV.
  • the reporter gene can be flanked by the negative-sense hMPV or APV leader linked to the hepatitis delta ribozyme (Hep-d Ribo) and T7 polymerase termination (T-T7) signals, and the hMPV or APV trailer sequence preceded by the T7 RNA polymerase promoter.
  • the plasmid encoding the minireplicon is transfected into a host cell.
  • hMPV is rescued in a host cell expressing T7 RNA polymerase, the N gene, the P gene, the L gene, and the M2.1 gene.
  • the host cell is transfected with plasmids encoding T7 RNA polymerase, the N gene, the P gene, the L gene, and the M2.1 gene.
  • the plasmid encoding the minireplicon is transfected into a host cell and the host cell is infected with a helper virus.
  • the hMPV minireplicon can be rescued using a number of polymerases, including, but not limited to, interspecies and intraspecies polymerases.
  • the hMPV minireplicon is rescued in a host cell expressing the minimal replication unit necessary for hMPV replication.
  • hMPV can be rescued from a cDNA using a number of polymerases, including, but not limited to, the polymerase of RSV, APV, MPV, or PIV.
  • hMPV is rescued using the polymerase of an RNA virus.
  • hMPV is rescued using the polymerase of a negative stranded RNA virus.
  • hMPV is rescued using RSV polymerase.
  • hMPV is rescued using APV polymerase.
  • hMPV is rescued using an MPV polymerase.
  • hMPV is rescued using PIV polymerase.
  • hMPV is rescued from a cDNA using a complex of hMPV polymerase proteins.
  • the hMPV minireplicon can be rescued using a polymerase complex consisting of the L, P, N, and M2.1 proteins.
  • the polymerase complex consists of the L, P, and N proteins.
  • the hMPV minireplicon can be rescued using a polymerase complex consisting of polymerase proteins from other viruses, such as, but not limited to, RSV, PIV, and APV.
  • the hMPV minireplicon can be rescued using a polymerase complex consisting of the L, P, N, and M2.1 proteins of RSV, PIV, or APV.
  • the polymerase complex used to rescue the hMPV minireplicon consists of the L, P, and N proteins of RSV, PIV, or APV.
  • different polymerase proteins from various viruses can be used to form the polymerase complex.
  • the polymerase used to rescue the hMPV minireplicon can be formed by different components of the RSV, PIV, or APV polymerases.
  • the N protein can be encoded by the N gene of RSV, APV, or PIV
  • the L protein is encoded by the L gene of RSV, APV, or PIV
  • P protein can be encoded by the P gene of RSV, APV, or PIV.
  • One skilled in the art would be able to determine the possible combinations that may be used to form the polymerase complex necessary to rescue the hMPV minireplicon.
  • the expression of a reporter gene is measured in order to confirm the successful rescue of the virus and also to characterize the virus.
  • the expression level of the reporter gene and/or its activity can be assayed by any method known to the skilled artisan, such as, but not limited to, the methods described in section 5.8.2.
  • the minireplicon comprises the following elements, in the order listed: T7 RNA Polymerase or RNA polymerase I, leader sequence, gene start, GFP, trailer sequence, Hepatitis delta ribozyme sequence or RNA polymerase I termination sequence. If T7 is used as RNA polymerase, Hepatitis delta ribozyme sequence should be used as termination sequence. If RNA polymerase I is used, RNA polymerase I termination sequence may be used as a termination signal. Dependent on the rescue system, the sequence of the minireplicon can be in the sense or antisense orientation.
  • the leader sequence can be modified relative to the wild type leader sequence of hMPV.
  • the leader sequence can optionally be preceded by an AC.
  • the T7 promoter sequence can be with or without a G-doublet or triplet, where the G-doublet or triplet provides for increased transcription.
  • assays for measurement of reporter gene expression in tissue culture or in animal models can be used with the methods of the invention.
  • the nucleotide sequence of the reporter gene is cloned into the virus, such as APV, hMPV, hMPV/APV or APV/hMPV, wherein (i) the position of the reporter gene is changed and (ii) the length of the intergenic regions flanking the reporter gene are varied. Different combinations are tested to determine the optimal rate of expression of the reporter gene and the optimal replication rate of the virus comprising the reporter gene.
  • the reporter gene can also be inserted into a minireplicon, see above.
  • the amount of reporter gene expression is representative of the activity of the minireplicon system or the virulence of the virus.
  • the biochemical activity of the reporter gene product represents the expression level of the reporter gene.
  • the total level of reporter gene activity depends also on the replication rate of the recombinant virus of the invention. Thus, to determine the true expression level of the reporter gene from the recombinant virus, the total expression level should be divided by the titer of the recombinant virus in the cell culture or the animal model.
  • Reporter genes that can be used with the methods of invention include, but are not limited to: CAT (chloramphenicol acetyltransferase ⁇ transfers radioactive acetyl groups to chloramphenicol or detection by thin layer chromatography and autoradiography); GAL (b- galactosidase — hydrolyzes colorless galactosides to yield colored products); GUS (b- glucuronidase— hydrolyzes colorless glucuronides to yield colored products); LUC (luciferase— oxidizes luciferin, emitting photons); GFP (green fluorescent protein ⁇ fluorescent protein without substrate); SEAP (secreted alkaline phosphatase— luminescence reaction with suitable substrates or with substrates that generate chromophores); HRP (horseradish peroxidase— in the presence of hydrogen oxide, oxidation of 3, 3', 5,5'- tetramethylbenzidine to form a colored complex); and AP (
  • the abundance of the reporter gene can be measured by, inter alia, Western blot analysis or Northern blot analysis or any other technique used for the quantification of transcription of a nucleotide sequence, the abundance of its mRNA its protein (seeSho ⁇ t Protocols in Molecular Biology, Ausubel et al., (editors), John Wiley & Sons, Inc., 4 th edition, 1999).
  • the activity of the reporter gene product is measured as a readout of reporter gene expression from the recombinant virus.
  • biochemical characteristics of the reporter gene product can be employed. The methods for measuring the biochemical activity of the reporter gene products are well-known to the skilled artisan. A more detailed description of illustrative reporter genes is set forth below.
  • the incidence of infection can be determined by any method well-known in the art, for example, but not limited to, clinical samples (e.g., nasal swabs) can be tested for the presence of a virus of the invention by immunofluorescence assay (IFA) using an anti-APV- antigen antibody, an anti-hMPV-antigen antibody, an anti-APV-antigen antibody, and/or an antibody that is specific to the gene product of the heterologous nucleotide sequence, respectively.
  • IFA immunofluorescence assay
  • samples containing intact cells can be directly processed, whereas isolates without intact cells should first be cultured on a permissive cell line (e.g. HEp-2 cells).
  • a permissive cell line e.g. HEp-2 cells.
  • cultured cell suspensions should be cleared by centrifugation at, e.g., 300xg for 5 minutes at room temperature, followed by a PBS, pH 7.4 (Ca++ and Mg++ free) wash under the same conditions. Cell pellets are resuspended in a small volume of PBS for analysis. Primary clinical isolates containing intact cells are mixed with PBS and centrifuged at 300xg for 5 minutes at room temperature.
  • Mucus is removed from the interface with a sterile pipette tip and cell pellets are washed once more with PBS under the same conditions. Pellets are then resuspended in a small volume of PBS for analysis. Five to ten microliters of each cell suspension are spotted per 5 mm well on acetone washed 12-well HTC supercured glass slides and allowed to air dry. Slides are fixed in cold (-2O 0 C) acetone for 10 minutes. Reactions are blocked by adding PBS - 1% BSA to each well followed by a 10 minute incubation at room temperature. Slides are washed three times in PBS - 0.1% Tween-20 and air dried.
  • Antibody serum titer can be determined by any method well-known in the art, for example, but not limited to, the amount of antibody or antibody fragment in serum samples can be quantitated by a sandwich ELISA. Briefly, the ELISA consists of coating microtiter plates overnight at 4 0 C with an antibody that recognizes the antibody or antibody fragment in the serum. The plates are then blocked for approximately 30 minutes at room temperature with PBS-Tween-0.5% BSA. Standard curves are constructed using purified antibody or antibody fragment diluted in PBS-TWEEN-BSA, and samples are diluted in PBS-BSA. The samples and standards are added to duplicate wells of the assay plate and are incubated for approximately 1 hour at room temperature.
  • the non-bound antibody is washed away with PBS-TWEEN and the bound antibody is treated with a labeled secondary antibody (e.g., horseradish peroxidase conjugated goat-anti-human IgG) for approximately 1 hour at room temperature.
  • a labeled secondary antibody e.g., horseradish peroxidase conjugated goat-anti-human IgG
  • Binding of the labeled antibody is detected by adding a chromogenic substrate specific for the label and measuring the rate of substrate turnover, e.g., by a spectrophotometer.
  • the concentration of antibody or antibody fragment levels in the serum is determined by comparison of the rate of substrate turnover for the samples to the rate of substrate turnover for the standard curve at a certain dilution, (e) SEROLOGICAL TESTS
  • the presence of antibodies that bind to a component of a mammalian MPV is detected.
  • the presence of antibodies directed to a protein of a mammalian MPV can be detected in a subject to diagnose the presence of a mammalian MPV in the subject. Any method known to the skilled artisan can be used to detect the presence of antibodies directed to a component of a mammalian MPV.
  • serological tests can be conducted by contacting a sample, from a host suspected of being infected with MPV, with an antibody to an MPV or a component thereof, and detecting the formation of a complex.
  • the serological test can detect the presence of a host antibody response to MPV exposure.
  • the antibody that can be used in the assay of the invention to detect host antibodies or MPV components can be produced using any method known in the art. Such antibodies can be engineered to detect a variety of epitopes, including, but not limited to, nucleic acids, amino acids, sugars, polynucleotides, proteins, carbohydrates, or combinations thereof.
  • serological tests can be conducted by contacting a sample from a host suspected of being infected with MPV, with an a component of MPV, and detecting the formation of a complex. Examples of such methods are well known in the art, including but are not limited to, direct immunofluoresence, ELISA, western blot, immunochromatography.
  • components of mammalian MPV are linked to a solid support.
  • the component of the mammalian MPV can be, but is not limited to, the F protein or the G protein.
  • the material that is to be tested for the presence of antibodies directed to mammalian MPV is incubated with the solid support under conditions conducive to the binding of the antibodies to the mammalian MPV components.
  • the solid support is washed under conditions that remove any unspecifically bound antibodies. Following the washing step, the presence of bound antibodies can be detected using any technique known to the skilled artisan.
  • the mammalian MPV protein-antibody complex is incubated with detectably labeled antibody that recognizes antibodies that were generated by the species of the subject, e.g., if the subject is a cotton rat, the detectably labeled antibody is directed to rat antibodies, under conditions conducive to the binding of the detectably labeled antibody to the antibody that is bound to the component of mammalian MPV.
  • the detectably labeled antibody is conjugated to an enzymatic activity.
  • the detectably labeled antibody is radioactively labeled.
  • the complex of mammalian MPV protein-antibody-detectably labeled antibody is then washed, and subsequently the presence of the detectably labeled antibody is quantified by any technique known to the skilled artisan, wherein the technique used is dependent on the type of label of the detectably labeled antibody.
  • microneutralization assay The ability of antibodies or antigen-binding fragments thereof to neutralize virus infectivity is determined by a microneutralization assay.
  • This microneutralization assay is a modification of the procedures described by Anderson et al., (1985, J. Clin. Microbiol. 22:1050-1052, the disclosure of which is hereby incorporated by reference in its entirety). The procedure is also described in Johnson et al., 1999, J. Infectious Diseases 180:35-40, the disclosure of which is hereby incorporated by reference in its entirety.
  • Antibody dilutions are made in triplicate using a 96-well plate.
  • TCID 50 of a mammalian MPV are incubated with serial dilutions of the antibody or antigen-binding fragments thereof to be tested for 2 hours at 37°C in the wells of a 96-well plate.
  • Cells susceptible to infection with a mammalian MPV such as, but not limited to Vero cells (2.5 x 10 4 ) are then added to each well and cultured for 5 days at 37 0 C in 5% CO 2 . After 5 days, the medium is aspirated and cells are washed and fixed to the plates with 80% methanol and 20% PBS. Virus replication is then determined by viral antigen, such as F protein expression.
  • Biotin-conjugated anti-viral antigen such as anti-F protein monoclonal antibody (e.g., pan F protein, C-site-specific MAb 133- IH) washed and horseradish peroxidase conjugated avidin is added to the wells. The wells are washed again and turnover of substrate TMB (thionitrobenzoic acid) is measured at 450 nm.
  • the neutralizing titer is expressed as the antibody concentration that causes at least 50% reduction in absorbency at 450 nm (the OD 450 ) from virus-only control cells.
  • the cells can be infected with the respective virus for four hours prior to addition of antibody and the read-out is in terms of presence of absence of fusion of cells (Taylor et al., 1992, J. Gen. Virol. 73:2217-2223).
  • Any method or approach using nucleotide or peptide sequence information to compare mammalian MPV isolates can be used to establish phylogenetic relationships, including, but not limited to, distance, maximum likelihood, and maximum parsimony methods or approaches. Any method known in the art can be used to analyze the quality of phylogenetic data, including but not limited to bootstrapping. Alignment of nucleotide or peptide sequence data for use in phylogenetic approaches, include but are not limited to, manual alignment, computer pairwise alignment, and computer multiple alignment. One skilled in the art would be familiar with the preferable alignment method or phylogenetic approach to be used based upon the information required and the time allowed.
  • a DNA maximum likehood method is used to infer relationships between hMPV isolates.
  • bootstrapping techniques are used to determine the certainty of phylogenetic data created using one of said phylogenetic approaches.
  • jumbling techniques are applied to the phylogenetic approach before the input of data in order to minimize the effect of sequence order entry on the phylogenetic analyses.
  • a DNA maximum likelihood method is used with bootstrapping.
  • a DNA maximum likelihood method is used with bootstrapping and jumbling.
  • a DNA maximum likelihood method is used with 50 bootstraps.
  • a DNA maximum likelihood method is used with 50 bootstraps and 3 jumbles.
  • nucleic acid or peptide sequence information from an isolate of hMPV is compared or aligned with sequences of other hMPV isolates.
  • the amino acid sequence can be the amino acid sequence of the L protein, the M protein, the N protein, the P protein, or the F protein.
  • nucleic acid or peptide sequence information from an hMPV isolate or a number of hMPV isolates is compared or aligned with sequences of other viruses.
  • phylogenetic approaches are applied to sequence alignment data so that phylogenetic relationships can be inferred and/or phylogenetic trees constructed. Any method or approach that uses nucleotide or peptide sequence information to compare hMPV isolates can be used to infer said phylogenetic relationships, including, but not limited to, distance, maximum likelihood, and maximum parsimony methods or approaches.
  • PCT/NL02/00040 discloses nucleic acid sequences that are suitable for phylogenetic analysis at page 12, line 27 to page 19, Iine29, which is incorporated herein by reference.
  • a very useful outgroup isolate can be obtained from avian pneumovirus serotype C (APV-C).
  • ClustalW is used in conjunction with DNA maximum likelihood methods with 100 bootstraps and 3 jumbles in order to generate phylogenetic relationships.
  • Nasopharyngeal aspirateples from patients suffering from RTI can be analyzed by DIF as described (Rothbarth et. al, 1999, J. of Virol. Methods 78:163-169). Samples are stored at -70°C. In short, nasopharyngeal aspirates are diluted with 5 ml Dulbecco MEM (BioWhittaker, Walkersville, MD) and thoroughly mixed on a vortex mixer for one minute. The suspension is centrifuged for ten minutes at 840 x g.
  • DIF DIRECT IMMUNOFLUORESENCE ASSAY
  • the sediment is spread on a multispot slide (Nutacon, Leimuiden, The Netherlands) and the supernatant is used for virus isolation. After drying, the cells are fixed in acetone for one minute at room temperature. After the slides are washed, they are incubated for 15 minutes at 37°C with commercially available FITC-labeled anti-sera specific for viruses such as influenza A and B, hRSV and hPIV 1 to 3 (Dako, Glostrup, Denmark). After three washings in PBS and one in tap water, the slides are submerged in a glycerol/PBS solution (Citifluor, UKO, Canterbury, UK) and covered. The slides are then analyzed using a Axioscop fluorescence microscope, (i) VIRUS CULTURE OF MPV
  • the detection of the virus in a cultivated sample from a host is a direct indication of the host's current and/or past exposure or infection with the virus.
  • Samples that display CPE after the first passage are used to inoculate sub-confluent mono-layers of tMK cells in media in 24 well plates. Cultures are checked for CPE daily and the media is changed once a week. Since CPE can differ for each isolate, all cultures are tested at day 12 to 14 with indirect IFA using ferret antibodies against the new virus isolate. Positive cultures are freeze- thawed three times, after which the supernatants are clarified by low-speed centrifugation, aliquoted and stored frozen at -70°C.
  • tissue culture infectious doses (TCID 5 o) of virus in the culture supernatants are determined as described (Lennette, D. A. et al. In: DIAGNOSTIC PROCEDURES FOR VIRAL, RICKETTSIAL, AND CHLAMYDIAL INFECTIONS, 7th ed. (eds. Lennette, E.H., Lennette, D.A. & Lennette, E.T.) 3-25; 37-138; 431-463; 481-494; 539- 563 (American Public Health Association, Washington, 1995)).
  • Antibodies can be used to visualize viral proteins in infected cells or tissues.
  • Indirect immunofluorescence assay (IFA) is a sensitive approach in which a second antibody coupled to a fluorescence indicator recognizes a general epitope on the virus-specific antibody. IFA is more advantageous than DIF because of its higher level of sensitivity.
  • IFA Indirect immunofluorescence assay
  • collected specimens are diluted with 5 ml Dulbecco MEM medium (BioWhittaker, Walkersville, MD) and thoroughly mixed on a vortex mixer for one minute. The suspension is then centrifuged for ten minutes at 840 x g. The sediment is spread on a multispot slide. After drying, the cells are fixed in acetone for 1 minute at room temperature.
  • virus is cultured on tMK cells in 24 well slides containing glass slides. These glass slides are washed with PBS and fixed in acetone for 1 minute at room temperature.
  • Two indirect IFAs can be performed.
  • slides containing infected tMK cells are washed with PBS, and then incubated for 30 minutes at 37 0 C with virus specific antisera.
  • Monoclonal antibodies against influenza A, B and C, hPIV type 1 to 3, and hRSV can be used.
  • hPIV type 4 mumps virus, measles virus, sendai virus, simian virus type 5, and New-Castle Disease virus, polyclonal antibodies (RIVM) and ferret and guinea pig reference sera are used. After three washings with PBS and one wash with tap water, the slides are stained with secondary antibodies directed against the sera used in the first incubation.
  • Secondary antibodies for the polyclonal antisera are goat-anti-ferret (KPL, Guilford, UK, 40 fold diluted), mouse-anti -rabbit (Dako, Glostrup, Denmark, 20 fold diluted), rabbit-anti-chicken (KPL, 20 fold dilution) and mouse-anti-guinea pig (Dako, 20 fold diluted).
  • the slides are incubated for 30 minutes at 37 0 C with 20 polyclonal antibodies at a dilution of 1 :50 to 1 :100 in PBS. Immunized ferrets and guinea pigs are used to obtain polyclonal antibodies, but these antibodies can be raised in various animals, and the working dilution of the polyclonal antibody can vary for each immunization. After three washes with PBS and one wash with tap water, the slides are incubated at 37 0 C for 30 minutes with FITC labeled goat-anti-ferret antibodies (KPL, Guilford, UK, 40 fold diluted).
  • the slides After three washes in PBS and one in tap water, the slides are included in a glycerol/PBS solution (Citifluor, UKO, Canterbury, UK) and covered. The slides are analyzed using an Axioscop fluorescence microscope (Carl Zeiss B.V., Weesp, the Netherlands).
  • virus Different characteristics of a virus can be utilized for the detection of the virus. For example, many virus contain proteins that can bind to erythrocytes resulting in a lattice. This property is called hemagglutination and can be used in hemagglutination assays for detection of the virus. Virus may also be visualized under an electron microscope (EM) or detected by PCR techniques.
  • EM electron microscope
  • virus can be concentrated from infected cell culture supernatants in a micro-centrifuge at 4°C at 17000 x g, after which the pellet is resuspended in PBS and inspected by negative contrast EM.
  • Specific antibodies to viruses are formed during the course of infection/illness. Thus, detection of virus-specific antibodies in a host is an indicator of current and/or past infections of the host with that virus.
  • the indirect enzyme immunoassay can be used to detect the IgG class of hMPV antibodies.
  • This assay is performed in microtitre plates essentially as described previously (Rothbarth et al, 1999, J. of Vir. Methods 78:163-169). Briefly, concentrated hMPV is solubilized by treatment with 1% Triton X-100. After determination of the optimal working dilution by checkerboard titration, it is coated for 16 hr at room temperature into microtitre plates in PBS. Subsequently, 100 ul volumes of 1:100 diluted human serum samples in EIA buffer are added to the wells and incubated for 1 hour at 37 0 C.
  • Binding of human IgG is detected by adding a goat anti-human IgG peroxidase conjugate (Biosource, USA), adding TMB as substrate developed plates and Optical Density (OD) is measured at 450 nm. The results are expressed as the S(ignal)/N(egative) ratio of the OD. A serum is considered positive for IgG if the S/N ratio was beyond the negative control plus three times the standard.
  • the hMPV antibodies of the IgM and IgA classes can be detected in sera by capture EIA essentially as described previously (Rothbarth et al. , 1999, J Vir Methods 78:163-169).
  • capture EIA essentially as described previously (Rothbarth et al. , 1999, J Vir Methods 78:163-169).
  • Sera can be diluted 1 :100. After incubation of 1 hour at 37 0 C, an optimal working dilution of hMPV is added to each well (100 ⁇ l) before incubation for 1 hour at 37 0 C.
  • AVP antibodies are detected in an AVP inhibition assay.
  • the protocol for the APV inhibition test is included in the APV-Ab SVANOVIR® enzyme immunoassay that is manufactured by SVANOVA Biotech AB, Uppsala Science Park Glunten SE-751 83 Uppsala Sweden.
  • the results are expressed as the S(ignal)/N(egative ratio of the OD.
  • a serum is considered positive for IgG, if the S/N ratio was beyond the negative control plus three times the standard.
  • infected tMK cells with MPV can be fixed with acetone on coverslips (as described above), washed with PBS and incubated 30 minutes at 37°C with serum samples at a 1 to 16 dilution. After two washes with PBS and one with tap water, the slides are incubated for 30 minutes at 37 0 C with FITC-labeled secondary antibodies to the species used (Dako). Slides are processed as described above. Antibodies can be labeled directly with a fluorescent dye, which will result in a direct immunofluorescence assay. FITC can be replaced with any fluorescent dye. (n) VIRUS NEUTRALIZATION ASSAY
  • VN Virus neutralization assays
  • Neutralizing antibodies can be used to define type-specific antigens on the virus particle, e.g., neutralizing antibodies could be used to define serotypes of a virus. Additionally, broadly neutralizing antibodies may also exist.
  • VN assays can be performed with serial two-fold dilutions of human and animal sera starting at an eight- fold dilution. Diluted sera are incubated for one hour with 100 TCID 50 of virus before inoculation of tMK cells grown in 96 well plates, after which the plates can be centrifuged at 840 x g. The media is changed after three and six days and IFA was conducted with FTIC-labeled ferret antibodies against MPV 8 days after inoculation. The VN titre can be defined as the lowest dilution of the serum sample resulting in negative IFA and inhibition of CPE in cell cultures, (o) RNA ISOLATION
  • RNA can be isolated from the supernatants of infected cell cultures or sucrose gradient fractions using a High Pure RNA Isolation kit, according to instructions from the manufacturer (Roche Diagnostics, Ahnere, The Netherlands). RNA can also be isolated following other procedures known in the art ⁇ see, e.g., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, volume 1-3 (1994-1998). Ed. by Ausubel, F. M. et ah, Published by John Wiley and sons, Inc., USA), (p) RT-PCR TO DETECT/DIAGNOSE MPV
  • Detection of the virus in a biological sample can be done using methods that copy or amplify the genomic material of the virus.
  • a one-step RT-PCR can be performed in 50 ⁇ l reactions containing 50 mM Tris.HCl pH 8.5, 50 mM NaCl, 4 mM MgCl 2 , 2 mM dithiotreitol, 200 ⁇ M each dNTP, 10 units recombinant RNAsin (Promega, Leiden, the Netherlands), 10 units AMV RT (Promega, Leiden, The Netherlands), 5 units Amplitaq Gold DNA polymerase (PE Biosystems, Nieuwerkerk aan de Ijssel, The Netherlands) and 5 ⁇ l RNA.
  • Cycling conditions can be 45 min. at 42 0 C and 7 min. at 95 0 C once, 1 min at 95 0 C, 2 min. at 42°C and 3 min. at 72 0 C repeated 40 times and 10 min. at 72°C once, (q) RAP PCR
  • RAP-PCR can be performed essentially as described (Welsh et al. , 1992, NAR 20:4965-4970). Essentially, the RAP PCR can be performed as follows: For the RT reaction, 2 ⁇ l of RNA are used in a 10 ⁇ l reaction containing 10 ng/ ⁇ l oligonucleotide, 10 mM dithiotreitol, 500 ⁇ m each dNTP, 25 mM Tris-HCl pH 8.3, 75 mM KCl and 3 mM MgCl 2 . The reaction mixture is incubated for 5 minutes at 70 0 C and 5 minutes at 37°C, after which 200 units Superscript RT enzyme (LifeTechnologies) are added.
  • the incubation at 37 0 C is continued for 55 minutes and the reaction is terminated by a 5 minute incubation at 72 0 C.
  • the RT mixture is diluted to give a 50 ⁇ l PCR reaction containing 8 ng/ ⁇ l oligonucleotide, 300 ⁇ l each dNTP, 15 mM Tris-HCl pH 8.3,65 mM KCl, 3.0 mM MgCL 2 and 5 units Taq DNA polymerase (FE Biosystems). Cycling conditions are 5 minutes at 94 0 C, 5 minutes at 40 0 C, and 1 minute at 72 0 C once, followed by 1 minute at 94 0 C, 2 minutes at 56 0 C and 1 minute at 72 0 C repeated 40 times, and 5 minutes at 72°C once. (r) CAPTURE ANTI-MPV IgM EIA USING A RECOMBINANT NUCLEOPROTEIN.
  • an immunological assay that detects the presence of the antibodies in a variety of hosts.
  • antibodies to the N protein are used because it is the most abundant protein that is produced. This feature is due the transciptional gradient that occurs across the genome of the virus.
  • a capture IgM EIA using the recombinant nucleoprotein or any other recombinant protein as antigen can be performed by modification of assays as previously described by
  • Affinity purified anti-human IgM capture antibody (or against other species), such as that obtained from Dako, is added to wells of a microtiter plate in a concentration of 250 ng per well in 0.1 M carbonate buffer pH 9.6. After overnight incubation at room temperature, the plates are washed two times with PBS/0.05% Tween. 100 ⁇ l of test serum diluted 1 :200 to.1 :1000 in ELISA buffer is added to triplicate wells and incubated for 1 hour at 37 0 C. The plates are then washed two times with in PBS/0.05%T ween.
  • Uninfected cell lysate serves as a negative control and is run in duplicate wells.
  • the plates are then washed three times in PBS/0.05% Tween and incubated for 1 hour at 37°C with 100 ⁇ l of a polyclonal antibody against MPV in a optimal dilution in ELISA buffer. After 2 washes with PBS/0.05% Tween , the plates are incubated with horseradish peroxide labeled secondary antibody (such as rabbit anti ferret), and the plates are incubated 20 minutes at
  • glycoproteins G and F are the two transmembraneous envelope glycoproteins of the MPV virion and represent the major neutralisation and protective antigens.
  • the expression of these glycoproteuns in a vector virus system sych as a baculovinus system provides a source of recombinant antigens for use in assays for detection of MPV specific antibodies.
  • their use in combination with the nucleoprotein, for instance, further enhances the sensitivity of enzyme immunoassays in the detection of antibodies against MPV.
  • broncheo alveolar lavages and throat swabs preferable from but not limited to humans, carnivores (dogs, cats, seals etc.), horses, ruminants (cattle, sheep, goats etc.), pigs, rabbits, birds (poultry, ostridges, etc) can be examined. From birds, cloaca and intestinal swabs and droppings can be examined as well. For all samples, serology (antibody and antigen detection etc.), virus isolation and nucleic acid detection techniques can be performed for the detection of virus.
  • Monoclonal antibodies can be generated by immunizing mice (or other animals) with purified MPV or parts thereof (proteins, peptides) and subsequently using established hybridoma technology (Current Protocols in Immunology, Published by John Wiley and sons, Inc., USA). Alternatively, phage display technology can be used for this purpose (Current Protocols in Immunology, Published by John Wiley and sons, Inc., USA). Similarly, polyclonal antibodies can be obtained from infected humans or animals, or from immunised humans or animals (Current Protocols in Immunology, Published by John Wiley and sons, Inc., USA).
  • the detection of the presence or absence of NSl and NS2 proteins can be performed using western-blotting, IFA, immuno precipitation techniques using a variety of antibody preparations.
  • the detection of the presence or absence of NSl and NS2 genes or homologues thereof in virus isolates can be performed using PCR with primer sets designed on the basis of known NSl and/or NS2 genes as well as with a variety of nucleic acid hybridisation techniques.
  • a pharmaceutical composition comprising a virus, a nucleic acid, a proteinaceous molecule or fragment thereof, an antigen and/or an antibody according to the invention can for example be used in a method for the treatment or prevention of a MPV infection and/or a respiratory illness comprising providing an individual with a pharmaceutical composition according to the invention.
  • This is most useful when said individual comprises a human, specifically when said human is below 5 years of age, since such infants and young children are most likely to be infected by a human MPV as provided herein.
  • the compositions of the invention can be used for the treatment of immuno-compromised individuals including cancer patients, transplant recipients and the elderly.
  • the vaccine of the invention comprises mutant mMPV, or, more specifically, a mutant hMPV.
  • the mammalian metapneumovirus to be used in a vaccine formulation has an attenuated phenotype.
  • the invention provides vaccine formulations for the prevention and treatment of infections with PIV, RSV, APV, and/or hMPV.
  • the vaccine of the invention comprises recombinant and chimeric viruses of the invention.
  • the vaccine comprises APV and the vaccine is used for the prevention and treatment for hMPV infections in humans.
  • infection with APV will result in the production of antibodies in the host that will cross-react with hMPV and protect the host from infection with hMPV and related diseases.
  • the vaccine comprises hMPV and the vaccine is used for the prevention and treatment for APV infection in birds, such as, but not limited to, in turkeys.
  • APV infection in birds, such as, but not limited to, in turkeys.
  • the invention encompasses the use of recombinant and chimeric APV/hMPV viruses which have been modified in vaccine formulations to confer protection against APV and/or hMPV.
  • APV/hMPV is used in a vaccine to be administered to birds, to protect the birds from infection with APV.
  • the replacement of the APV gene or nucleotide sequence with a hMPV gene or nucleotide sequence results in an attenuated phenotype that allows the use of the chimeric virus as a vaccine.
  • the APV/hMPV chimeric virus is administered to humans.
  • the APV viral vector provides the attenuated phenotype in humans and the expression of the hMPV sequence elicits a hMPV specific immune response.
  • the invention encompasses the use of recombinant and chimeric hMPV/ APV viruses which have been modified in vaccine formulations to confer protection against APV and/or hMPV.
  • hMPV/APV is used in a vaccine to be administered to humans, to protect the human from infection with hMPV.
  • the replacement of the hMPV gene or nucleotide sequence with a APV gene or nucleotide sequence results in an attenuated phenotype that allows the use of the chimeric virus as a vaccine.
  • the hMPV/APV chimeric virus is administered to birds.
  • the hMPV backbone provides the attenuated phenotype in birds and the expression of the APV sequence elicits an APV specific immune response.
  • a vaccine formulation contains a virus comprising a heterologous nucleotide sequence derived from an avian pneumovirus type A, and the vaccine formulation is used to protect from infection by avian pneumovirus type A and avian pneumovirus type B.
  • the invention encompasses vaccine formulations to be administered to humans and animals which are useful to protect against APV, including APV-C and APV-D, hMPV, PIV, influenza, RSV, Sendai virus, mumps, laryngotracheitis virus, simianvirus 5, human papillomavirus, measles, mumps, as well as other viruses and pathogens and related diseases.
  • the invention further encompasses vaccine formulations to be administered to humans and animals which are useful to protect against human metapneumovirus infections and avian pneumovirus infections and related diseases.
  • the invention encompasses vaccine formulations which are useful against domestic animal disease causing agents including rabies virus, feline leukemia virus (FLV) and canine distemper virus.
  • the invention encompasses vaccine formulations which are useful to protect livestock against vesicular stomatitis virus, rabies virus, rinderpest virus, swinepox virus, and further, to protect wild animals against rabies virus.
  • the recombinant virus is non-pathogenic to the subject to which it is administered.
  • the use of genetically engineered viruses for vaccine purposes may desire the presence of attenuation characteristics in these strains.
  • the introduction of appropriate mutations (e.g., deletions) into the templates used for transfection may provide the novel viruses with attenuation characteristics.
  • specific missense mutations which are associated with temperature sensitivity or cold adaption can be made into deletion mutations. These mutations should be more stable than the point mutations associated with cold or temperature sensitive mutants and reversion frequencies should be extremely low.
  • the mutant mMPV of the invention may further have "suicide" characteristics. Such viruses would go through only one or a few rounds of replication within the host. When used as a vaccine, the recombinant virus would go through limited replication cycle(s) and induce a sufficient level of immune response but it would not go further in the human host and cause disease. Recombinant viruses lacking one or more of the genes of wild type APV and hMPV, respectively, or possessing mutated genes as compared to the wild type strains would not be able to undergo successive rounds of replication. Defective viruses can be produced in cell lines which permanently express such a gene(s).
  • Viruses lacking an essential gene(s) will be replicated in these cell lines but when administered to the human host will not be able to complete a round of replication.
  • Such preparations may transcribe and translate —in this abortive cycle — a sufficient number of genes to induce an immune response.
  • larger quantities of the strains could be administered, so that these preparations serve as inactivated (killed) virus vaccines.
  • the heterologous gene product be expressed as a viral component, so that the gene product is associated with the virion.
  • the advantage of such preparations is that they contain native proteins and do not undergo inactivation by treatment with formalin or other agents used in the manufacturing of killed virus vaccines.
  • recombinant virus of the invention made from cDNA may be highly attenuated so that it replicates for only a few rounds.
  • a mutant mMPV of the invention can be effective as a vaccine even if the attenuated virus is incapable of causing a cell to generate new infectious viral particles because the viral proteins are inserted in the cytoplasmic membrane of the host thus stimulating an immune response.
  • inactivated vaccine formulations may be prepared using conventional techniques to "kill" the chimeric viruses.
  • Inactivated vaccines are "dead” in the sense that their infectivity has been destroyed. Ideally, the infectivity of the virus is destroyed without affecting its immunogenicity.
  • the chimeric virus may be grown in cell culture or in the allantois of the chick embryo, purified by zonal ultracentrifugation, inactivated by formaldehyde or ⁇ -propiolactone, and pooled. The resulting vaccine is usually inoculated intramuscularly.
  • Inactivated viruses may be formulated with a suitable adjuvant in order to enhance the immunological response.
  • suitable adjuvants may include but are not limited to mineral gels, e.g., aluminum hydroxide; surface active substances such as lysolecithin, pluronic polyols, polyanions; peptides; oil emulsions; and potentially useful human adjuvants such as BCG,
  • Many methods may be used to introduce the vaccine formulations described above, these include but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, percutaneous, and intranasal and inhalation routes. It may be preferable to introduce the chimeric virus vaccine formulation via the natural route of infection of the pathogen for which the vaccine is designed.
  • the invention relates to immunogenic compositions.
  • the immunogenic compositions comprise a mammalian MPV.
  • the immunogenic composition comprises a human MPV.
  • the immunogenic composition comprises an attenuated mammalian MPV or an attenuated human MPV.
  • the immunogenic composition further comprises a pharmaceutically acceptable carrier.
  • administering is combined with heptad repeats of the F protein of the mMPV.
  • heptad repeats for the inhibition of virus-cell fusion, see section 5.16 of International Patent Application No. PCT/US04/12724 (published as WO 04/096993).
  • the present invention provides vaccines and immunogenic preparations comprising the mutant mMPV of the invention.
  • the vaccines or immunogenic formulations of the invention provide protection against or reduce the symptoms of a respiratory tract infections in a host.
  • a recombinant virus and/or a vaccine or immunogenic formulation of the invention can be administered alone or in combination with other vaccines.
  • a vaccine or immunogenic formulation of the invention is administered in combination with other vaccines or immunogenic formulations that provide protection against respiratory tract diseases, such as but not limited to, respiratory syncytial virus vaccines, influenza vaccines, measles vaccines, mumps vaccines, rubella vaccines, pneumococcal vaccines, rickettsia vaccines, staphylococcus vaccines, whooping cough vaccines or vaccines against respiratory tract cancers.
  • the virus and/or vaccine of the invention is administered concurrently with pediatric vaccines recommended at the corresponding ages.
  • the virus and/or vaccine of the invention can be administered concurrently with DtaP (IM), Hib (IM), Polio (IPV or OPV) and Hepatitis B (IM).
  • IM DtaP
  • Hib IM
  • Polio IPV or OPV
  • IM Hepatitis B
  • the virus and/or vaccine of the invention can be administered concurrently with Hib (IM), Polio (IPV or OPV), MMRII® (SubQ); Varivax® (SubQ), and hepatitis B (IM).
  • the vaccines that can be used with the methods of invention are reviewed in various publications, e.g., The Jordan Report 2000, Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States, the content of which is incorporated herein by reference in its entirety.
  • a vaccine or immunogenic formulation of the invention may be administered to a subject per se or in the form of a pharmaceutical or therapeutic composition.
  • Pharmaceutical compositions comprising an adjuvant and an immunogenic antigen of the invention may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the immunogenic antigen of the invention into preparations which can be used pharmaceutically.
  • a vaccine or immunogenic composition of the invention comprises adjuvants or is administered together with one or more adjuvants
  • the adjuvants that can be used include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particulate adjuvants, microparticulate adjuvants, mucosal adjuvants, and immunostimulatory adjuvants.
  • adjuvants include, but are not limited to, aluminum hydroxide, aluminum phosphate gel, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, squalene or squalane oil-in- water adjuvant formulations, biodegradable and biocompatible polyesters, polymerized liposomes, triterpenoid glycosides or saponins (e.g., QuilA and QS- 21, also sold under the trademark STIMULON, ISCOPREP), N-acetyl-muramyl-L-threonyl- D-isoglutamine (Threonyl-MDP, sold under the trademark TERMURTIDE), LPS, monophosphoryl Lipid A (3D-MLAsold under the trademark MPL).
  • aluminum hydroxide aluminum phosphate gel
  • Freund's Complete Adjuvant Freund's Incomplete Adjuvant
  • squalene or squalane oil-in- water adjuvant formulations examples include, but are not limited to, aluminum hydroxide,
  • the subject to which the vaccine or an immunogenic composition of the invention is administered is preferably a mammal, most preferably a human, but can also be a non- human animal, including but not limited to, primates, cows, horses, sheep, pigs, fowl (e.g., chickens, turkeys), goats, cats, dogs, hamsters, mice and rodents.
  • a mammal most preferably a human, but can also be a non- human animal, including but not limited to, primates, cows, horses, sheep, pigs, fowl (e.g., chickens, turkeys), goats, cats, dogs, hamsters, mice and rodents.
  • fowl e.g., chickens, turkeys
  • Many methods may be used to introduce the vaccine or the immunogenic composition of the invention, including but not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, percutaneous, intranasal and inhalation routes, and via scarification (scratching through the top layers of skin, e.g., using a bifurcated needle).
  • the vaccine or immunogenic preparations of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.
  • the preparation for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the vaccine or immunogenic preparations may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' s solution, Ringer's solution, or physiological saline buffer.
  • the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the proteins may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • An effective dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models to achieve an induction of an immunity response using techniques that are well known in the art.
  • Dosage amount and interval may be adjusted individually.
  • a suitable dose is an amount of the composition that when administered as described above, is capable of eliciting an antibody response.
  • the vaccine or immunogenic formulations of the invention may be administered in about 1 to 3 doses for a 1-36 week period.
  • a suitable dose is an amount of the vaccine formulation that, when administered as described above, is capable of raising an immunity response in an immunized animal sufficient to protect the animal from an infection for at least 4 to 12 months.
  • the amount of the antigen present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 ⁇ g. Suitable dose range will vary with the route of injection and the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
  • the viruses and/or vaccines of the invention are administered at a starting single dose of at least 10 3 TCIDs 0 , at least 10 4 TCID 5 O, at least 10 5 TCID 50 , at least 10 6 TCID 50 .
  • the virus and/or vaccines of the invention are administered at multiple doses.
  • a primary dosing regimen at 2, 4, and 6 months of age and a booster dose at the beginning of the second year of life are used. More preferably, each dose of at least 10 5 TCID 50 , or at least 10 6 TCID 50 is given in a multiple dosing regimen.
  • This assay is used to determine the ability of the recombinant viruses of the invention and of the vaccines of the invention to prevent lower respiratory tract viral infection in an animal model system, such as, but not limited to, cotton rats or hamsters.
  • the recombinant virus and/or the vaccine can be administered by intravenous (IV) route, by intramuscular (IM) route or by intranasal route (IN).
  • IV intravenous
  • IM intramuscular
  • IN intranasal route
  • the recombinant virus and/or the vaccine can be administered by any technique well-known to the skilled artisan.
  • This assay is also used to correlate the serum concentration of antibodies with a reduction in lung titer of the virus to which the antibodies bind.
  • mice On day 0, groups of animals, such as, but not limited to, cotton rats (Sigmodon hispidis, average weight 100 g) cynomolgous macacques (average weight 2.0 kg) are administered the recombinant or chimeric virus or the vaccine of interest or BSA by intramuscular injection, by intravenous injection, or by intranasal route. Prior to, concurrently with, or subsequent to administration of the recombinant virus or the vaccine of the invention, the animals are infected with wild type virus wherein the wild type virus is the virus against which the vaccine was generated.
  • cotton rats Sigmodon hispidis, average weight 100 g
  • cynomolgous macacques average weight 2.0 kg
  • the animals Prior to, concurrently with, or subsequent to administration of the recombinant virus or the vaccine of the invention, the animals are infected with wild type virus wherein the wild type virus is the virus against which the vaccine was generated.
  • the animals are infected with the wild type virus at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, 1 week or 1 or more months subsequent to the administration of the recombinant virus and/or the vaccine of the invention.
  • the target population for the therapeutic and diagnostic methods of the invention is defined by age.
  • the target population for the therapeutic and/or diagnostic methods of the invention is characterized by a disease or disorder in addition to a respiratory tract infection.
  • the target population encompasses young children, below 2 years of age.
  • the children below the age of 2 years do not suffer from illnesses other than respiratory tract infection.
  • the target population encompasses patients above 5 years of age.
  • the patients above the age of 5 years suffer from an additional disease or disorder including cystic fibrosis, leukaemia, and non- Hodgkin lymphoma, or recently received bone marrow or kidney transplantation.
  • the target population encompasses subjects in which the hMPV infection is associated with immunosuppression of the hosts.
  • the subject is an immunocompromised individual.
  • the target population for the methods of the invention encompasses the elderly.
  • the subject to be treated or diagnosed with the methods of the invention was infected with hMPV in the winter months, (c) CLINICAL TRIALS
  • Vaccines of the invention or fragments thereof tested in in vitro assays and animal models may be further evaluated for safety, tolerance and pharmacokinetics in groups of normal healthy adult volunteers.
  • the volunteers are administered intramuscularly, intravenously or by a pulmonary delivery system a single dose of a recombinant virus of the invention and/or a vaccine of the invention.
  • Each volunteer is monitored at least 24 hours prior to receiving the single dose of the recombinant virus of the invention and/or a vaccine of the invention and each volunteer will be monitored for at least 48 hours after receiving the dose at a clinical site.
  • volunteers are monitored as outpatients on days 3, 7, 14, 21, 28, 35, 42, 49, and 56 postdose.
  • Blood samples are collected via an indwelling catheter or direct venipuncture using 10 ml red-top Vacutainer tubes at the following intervals: (1) prior to administering the dose of the recombinant virus of the invention and/or a vaccine of the invention; (2) during the administration of the dose of the recombinant virus of the invention and/or a vaccine of the invention; (3) 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, and 48 hours after administering the dose of the recombinant virus of the invention and/or a vaccine of the invention; and (4) 3 days, 7 days 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administering the dose of the recombinant virus of the invention and/or a vaccine of the invention.
  • the concentration of antibody levels in the serum of volunteers are corrected by subtracting the predose serum level (background level) from the serum levels at each collection interval after administration of the dose of recombinant virus of the invention and/or a vaccine of the invention.
  • the pharmacokinetic parameters are computed according to the model-independent approach (Gibaldi et al., eds., 1982, Pharmacokinetics, 2nd edition, Marcel Dekker, New York) from the corrected serum antibody or antibody fragment concentrations.
  • Diagnosis of an mMPV infection can be performed using any method known to the skilled artisan.
  • Descriptions of detection and diagnosis methods for mMPV, such as hMPV can be found in International Patent Application No PCT/US03/05271 (published as WO 03/072719) and International Patent Application No. PCT/US04/12724 (published as WO 04/096993), both of which are incorporated herein by reference in their entireties.
  • these international patent application publications describe the detection and diagnosis of mMPV variants Al, A2, Bl, and B2.
  • the virus can detected or diagnosed by virtue of the presence of its components, such as viral protein or viral nucleic acids in a sample (e.g., in the sample from a patient).
  • a sample e.g., in the sample from a patient.
  • detection is performed using antibodies or nucleic acids that react specifically with these components of mMPV.
  • antibodies that are formed in a mammal against an mMPV can be detected in a sample from the mammal.
  • the invention relates to nucleic acid sequences encoding the mammalian MPV of the invention, proteins of the mutant mMPV, and antibodies against proteins of the mutant mMPV.
  • the invention provides an mMPV protein carrying one or more of the genetic modifications set forth in section 5.1.
  • the invention also provides nucleic acids encoding these mutated proteins.
  • the invention provides an isolated mammalian MPV protein with an amino acid substitution, deletion, or insertion at one or more amino acid positions selected from the group consisting of: position 66 in the P protein; positions 9, 38, 52, and 132 in the M protein; positions 93, 101, 280, 471, 532, and 538 in the F protein; position 187 in the M2 protein; positions 139 and 164 in the G protein; and positions 235, 323, and 1453 in the L protein, with the proviso that the modification at position 101 in the F protein is not a substitution to Proline and that the modification at position 93 in the F protein is not a substitution to Lysine.
  • the invention provides mMPV L protein with an amino acid substitution, deletion, or insertion at amino acid positions 235 and 323.
  • the isolated mammalian MPV of the invention further comprises a genetic modification that is a silent mutation, i.e., it does not result in an amino acid exchange.
  • the isolated mammalian MPV comprises a silent mutation at one or more of the nucleotide positions selected from the group consisting of: positions 336 and 436 in the M open reading frame.
  • the invention provides nucleic acids encoding a protein of mMPV with the following genetic modifications: position 197 in the P open reading frame; position 9, 113, 155, 336, 394, and 436 in the M open reading frame; positions 277, 301, 839, 1412, 1594, and 1612 in the F open reading frame; position 560 in the M2 open reading frame; position 415 and 491 in the G open reading frame; and positions 703, 967, and 4357 in the L open reading frame.
  • a nucleotide that is next to one of the recited positions is also mutated resulting in a stabilized codon.
  • the invention provides mMPV proteins with the following genetic modifications: (i) position 66 in the P gene is altered to VaI; (ii) position 9 in the M gene is altered to His; (iii) position 38 in the M gene is altered to Ser; (iv) position 52 in the M gene is altered to Pro; (v) position 132 in the M gene is altered to Pro; (vi) position 93 in the F gene is altered to Lys; (vii) position 280 in the F gene is altered to GIy; (viii) position 471 in the F gene is altered to Arg; (ix) position 532 in the F gene is altered to Tyr; (x) position 538 in the F gene is altered to Tyr; (xi) position 187 in the M2 gene is altered to He; (xii) position 139 in the G gene is altered to Pro; (xiii) position 164 in the G gene is altered to Pro; (xiv) position 235 in the L gene is altered to Arg; (xv) position 235 in the L gene is altered
  • the invention also provides for vaccines, immunogenic compositions, and pharmaceutical compositions.
  • the vaccines, immunogenic compositions, and pharmaceutical compositions comprise the isolated mutated mammalian MPV of the invention and pharmaceutically acceptable excipient.
  • the vaccines, immunogenic compositions, and pharmaceutical compositions comprise a mutated mMPV protein of the invention and pharmaceutically acceptable excipient.
  • the invention provides an isolated chimeric viral RNA polymerase complex comprising RNA polymerase complex subunits from at least two different paramyxoviruses.
  • the RNA polymerase complex subunits are the N, P, L, and M2.1 proteins.
  • the two different paramyxoviruses are selected from the group consisting of RSV, PIV, aMPV, and mammalian MPV.
  • a virus of the invention is inactivated and used for vaccination. In other embodiments, a fragments of a virus of the invention is used for vaccination.
  • Virus adaptation to replication at low temperatures was used to attenuate hMPV, and the associated sequence-changes in the viral genome were identified.
  • Recombinant viruses containing hMPV or RSV cp-mutations were generated by reverse genetics. These recombinant viruses were found to be temperature-sensitive (ts) in vitro, attenuated for replication in hamsters, yet highly immunogenic in this animal model.
  • Hamsters vaccinated with cp/ts-hMPV strains were protected against heterologous virus infection in the lower respiratory tract (LRT), and had significantly reduced virus titers in the URT.
  • LRT lower respiratory tract
  • IIMPVM H A virus with only 1 1 of the 19 mutations, IIMPVM H turned out to have a ts- phenotype in-vitro (Fig. Ic). Ten of these 11 mutations were non-silent, and were located in the P, M, F, M2, G, and L genes.
  • Vero cells were grown in Iscove's Modified Dulbecco's Medium (IMDM, BioWhittaker, Verviers, Belgium) supplemented with 10% fetal calf serum (FCS, Greiner Bio-One, Alphen aan den Rijn, The Netherlands), 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin and 2mM glutamine.
  • FCS fetal calf serum
  • Subclone 83 of WHO Vero cells was selected for virus passaging at low temperatures, and subclone 1 18 (Kuiken et al., 2004, Am J Pathol 164:1893-900) for all other experiments.
  • virus strains were grown in infection medium consisting of IMDM supplemented with 4% bovine serum albumin fraction V (Invitrogen, Breda, The Netherlands), 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin, 2mM glutamine and 3.75 ⁇ g/ml trypsin until 70 - 90 % of the cells displayed cytopathic effects. After one freeze-thaw cycle, cell-free supernatants were purified and concentrated using a 30 - 60 % (w/w) sucrose gradient.
  • bovine serum albumin fraction V Invitrogen, Breda, The Netherlands
  • hMPV isolate NL/1/99 (passage 3 at 37°C) was serially passaged in Vero-83 cells at decreasing temperatures. Virus was cultured at 34°C, 31 0 C, 28 0 C and 25°C for 3, 3, 2 and 2 passages respectively. When the temperature was decreased further to 22 0 C or 20°C, virus replication was seriously impaired, and passaging was thus continued at 25°C until passage 35 was reached. Cultures were harvested from every passage approximately 7 days after inoculation and stored in 25 % sucrose at -70 0 C.
  • Genome sequences of RSV strains containing mutations responsible for temperature sensitivity in vitro and attenuation in vivo were aligned with the full-length sequence of hMPV NL/1/99 using BioEdit software (Hall, 1999, Nucleic Acids Symposium Series 41 :95-98). Regions containing known ts-mutations in the RSV genome were compared with their counterparts of hMPV, to determine whether RSV ts-mutations could be introduced in the hMPV genome.
  • mice were immunized by virus inoculation as described above, with 10 6 TCID 50 of LAV or NL/1/99, or PBS as challenge control.
  • animals were challenged intranasally with 10 7 TCIDs 0 of NL/ 1/00 virus.
  • lungs, nasal turbinates and blood samples were collected for further processing.
  • VN antibody titers were determined in serum samples by a plaque reduction virus neutralization (PRVN) assay as described previously (de Graaf et al., 2007, J Virol Methods 143: 169-74). Briefly, serum samples were diluted and incubated for 30 minutes at 37°C with approximately 50 plaque forming units (pfu) of NL/ 1/00 or NL/1/99, expressing the enhanced green fluorescent protein (eGFP). Subsequently, the virus-serum mixtures were added to Vero-118 cells in 24 well plates, and incubated at 37°C. After two hours, the supernatants were replaced by a mixture of equal amounts of infection medium and 2% methyl cellulose.
  • PRVN plaque reduction virus neutralization
  • VN antibody titers are expressed as the dilution resulting in 50% reduction of the number of plaques, calculated according to the method of Reed and Muench (Reed and Muench, 1938, J Hyg 27:493-497). Per assay, each serum was tested in duplicate against hMPV NL/1/00 and NL/1/99.
  • Tissues from the inoculated hamsters were homogenized using a Polytron homogenizer (Kinematica AG, Littau-Lucerne, Switzerland) in infection media. After removal of tissue debris by centrifugation, supernatants were used for virus titration in Vero- 118 cells. Titrations were performed with 10-fold serial dilutions in 96-well plates (Greiner Bio-One). Confluent monolayers of Vero-118 cells were spin-inoculated (15 min., 2000 X g) with 100 ⁇ l often fold serial dilutions of each sample and incubated at 37°C. Two hours after the spin-inoculation, the inoculum was replaced with fresh infection media.
  • hMPV isolate NL/1/99 was serially passaged in Vero-83 cells at slowly decreasing temperatures until a temperature of 25 0 C was reached. When the temperature was further decreased to 22°C or 20 0 C, virus replication was severely impaired and virus yield was very poor. Therefore, passaging was continued at 25°C until passage 35 was reached.
  • Viral RNA of cp-NL/1/99 obtained after 35 passages was subjected to RT-PCR, followed by direct sequencing. Analysis of the full viral genome sequence and comparison with the original NL/1/99 genome revealed the presence of 19 nucleotide changes, resulting in 17 amino acid substitutions (Table 2). Analysis of virus genome sequences after fewer passages (passage 14, 23, and 29) indicated the gradual accumulation of these mutations.
  • AATA AACA -- -- 4 aPosition is specified as the amino acid number of the L gene of RSV.
  • nt nucleotide
  • wt wild-type
  • cp cold-passaged
  • aa amino acid
  • L large polymerase protein
  • GS-M2 gene-start sequence of the M2 gene. Nucleotides changes in each codon or nucleotide sequence are underlined.
  • Wild-type recombinant hMPV NL/1/99 was used as a backbone for the introduction of mutations as listed in Tables 2 and 3.
  • Three different viruses containing all mutations or subsets of cp-hMPV mutations were constructed. These viruses containing 19, 8 or 11 nucleotide substitutions were named 1IMPVMI9, hMPV M 8 and IIMPV M H respectively, based on the number of mutations that were introduced (Table 2).
  • Mutant virus 1IMPV M I 9 could not be rescued by reverse genetics after three attempts.
  • the parental virus obtained after 35 passages at 25 0 C also replicated very poorly, to low virus titers. Therefore, we next attempted to rescue recombinant viruses that contained only a selection of the cp-mutations, 8 and 11 respectively, and that were generated as cloning intermediates during the cloning of hMPV M i9.
  • virus growth curves were generated at different temperatures. Vero cells in 25 cm 2 flasks were inoculated at an MOI of 0.1, after which the cultures were incubated at 32°C, 37°C, 38 0 C, 39°C or 40 0 C. Plaque assays were performed to determine the viral titers in the supernatants of samples that were collected daily. Wild-type hMPV was able to replicate at all temperatures, with the highest virus titer obtained at 37°C. At 40 0 C, the virus titer was more than 100-fold reduced compared to the optimal temperature of 37°C (Fig. Ia).
  • HMPVM 8 which was an intermediate virus in the cloning procedure of 1IMPV M I9 , also replicated at all temperatures, however with higher titers as compared to wild-type hMPV, and an optimal replication temperature of 32°C (Fig. Ib). Although this virus was not temperature sensitive, it displayed faster replication kinetics in Vero cells and reached high maximum virus titers. Mutant IIMPV MH also displayed optimal virus growth at a temperature of 32°C, and virus titers at 6 dpi were even higher as compared to IIMPV MS (Fig. Ic). This virus did not replicate at 39°C and 40 0 C, demonstrating that this virus was temperature-sensitive.
  • IIMPVM I i and IIMPV MS were two mutations in the L gene and one mutation in the P gene (Table 2). Since for RSV most mutations causing temperature-sensitivity were located in the L gene, ITMPV M2 was constructed which only had two L mutations (nt 7826 and 8090, Table 2) as compared to wild-type NL/1/99. The replication kinetics of hMPV M 2 was most similar to that of the wild- type NL/1/99 virus (compare Fig. Ia and Id), suggesting that the mutation in the P gene (nt 1458) contributed to the temperature-sensitive phenotype of IIMPV M I I .
  • hMPV RS v 3 The only viable NL/1/99 with cp-RSV mutations, hMPV RS v 3 , replicated slowly and to low virus titers at 32 0 C and 37°C. At 38°C, no virus was detected until 4 dpi, and at 39°C and 4O 0 C the virus did not replicate at all. Thus, hMPV R sv 3 appeared to be temperature- sensitive in vitro (Fig. Ie).
  • mean virus titers ranged from 10 2 and 10 4 TCID 50 / gram NT, indicating that virus replication was -10.000 fold reduced in the URT.
  • the mean virus titer was 10 2 2 TCID 50 / gram lung, while in the animals inoculated with IIMPV MH or IIMPV RSV3 virus titers were below the detection limit of 10 1 2 TCID 50 , with the exception of a single animal in the WVIPV MH inoculate group (10 1 3 TCID 50 ).
  • both viruses appeared to be highly attenuated in hamsters; virus replication was restricted to the URT, where virus titers were ⁇ 10.000 fold reduced compared to wild-type hMPV.
  • serum samples were collected and subjected to a PRVN assay to determine virus neutralizing antibody titers against hMPV NL/1/99, induced by the candidate LAVs (Fig. 3).
  • the PRVN titers in the wild-type hMPV inoculated animals were slightly higher than those observed in the IIMPV M I I or 1IMPV RS V 3 inoculated animals (mean VN antibody titers of 90, 25, and 28 respectively, not statistically significant, Mann- Whitney test).
  • virus titers upon challenge reached >10 TCID 5 0 / gram tissue in the NT samples. These virus titers were more than 1.000-fold reduced in animals immunized with hMPVRsv3, and >10.000-fold reduced in the animals immunized with IIMPV MI 1 or wild-type hMPV.
  • the mean virus titers after challenge infection was 10 4 3 TCID 50 / gram lung tissue. Virus was undetectable in all animals immunized with IIMPV M ⁇ , IIMPV RSV3 , and wild-type hMPV NL/1/99 (Mann- Whitney test, P ⁇ 0,05).
  • hMPV M ⁇ and hMPV RSV3 are attenuated in hamsters, yet induce an hMPV-specific immune response which is sufficient to provide protective immunity to prevent hMPV lower respiratory tract infection.
  • Vaccinated animals were completely protected from hMPV LRT infection, and virus titers in the URT were reduced to the same extend as seen in hamsters exposed to wild- type hMPV.
  • a tool frequently used for the analysis of cis- and trans- acting elements influencing viral RNA synthesis are minireplicon systems. In such systems all components of the viral polymerase complex are transfected and the replication and transcription of a synthetic vRNA-like molecule is measured using reporter genes.
  • VRNA-like molecules of morbilliviruses are efficiently replicated by polymerase complex proteins of other morbilliviruses but not or less efficiently by polymerase complexes consisting of proteins of two different morbilliviruses (Bailey et al., 2007, Virus Res. 126:250-5; Brown et al., 2005, J Gen Virol 86, 1077-81).
  • vRNA-like molecules based on aMPV-A were replicated by the polymerase complex proteins of hRSV (Marriott et al., 2001, J Virol 75, 6265-72).
  • An aMPV-C minireplicon system was generated and used in combination with minireplicon systems for hMPV-Al and hMPV-Bl .
  • Each of these sets of metapneumovirus polymerase complex proteins was able to replicate synthetic vRNA-like molecules of hMPV- Al and Bl, aMPV-A and C and hRSV but not human parainfluenza virus type 3 (bPIV-3).
  • vRNA-like molecules were co-transfected with different combinations of N, P, L and M2.1 expression plasmids revealing that chimeric polymerase complexes were functional but with different efficiencies.
  • several chimeric viruses were created which contained polymerase complex genes of hMPV-Al and Bl or hMPV-Bl and aMPV-C. Most of these chimeric viruses replicated with similar efficiency as the wild type viruses in vitro. A subset of these was tested for attenuation in hamsters and replicated to lower titers than the wild type viruses.
  • Chimeric polymerase complexes of members of the Paramyxoviridae family vary in their ability to replicate vRNA-like molecules or rescue recombinant virus (Bailey et al., 2007, Virus Res. 126:250-5; Brown et al., 2005, J Gen Virol 86, 1077-81 ; Govindarajan et al., 2006, J Virol 80, 5790-7).
  • Exchanging polymerase genes between two related viruses with different host range is a method frequently used for the design of live attenuated vaccine strains (Bailly et al., 2000, J Virol 74, 3188-95; Govindarajan et al., 2006, J Virol 80, 5790-7; Pham et al., 2005, J Virol 79, 151 14-22; Skiadopoulos et al., 2003, J Virol 77, 1 141-8).
  • Vero-118 (Kuiken et al., 2004, Am J Pathol 164, 1893-900) cells were cultured in Iscove's Modified Dulbecco's medium (BioWhittaker, Verviers, Belgium) supplemented with 10% FCS, 100 IU of penicillin/ml, 100 ⁇ g of streptomycin/ml, and 2mM glutamine as described previously.
  • Vero-118 cells and BSR-T7 cells were co-cultured in Dulbecco's Modified Eagle medium supplemented with 3% Fetal Calf Serum (FCS), 100 IU of penicillin/ml, 100 ⁇ g of streptomycin/ml, 2mM glutamine, and 0,25mg of trypsin/ml.
  • FCS Fetal Calf Serum
  • Vero-118 cells were grown in Iscove's Modified Dulbecco's medium supplemented with 4% bovine serum albumin fraction V (Invitrogen, Breda, the Netherlands), 100 IU of penicillin, 2mM glutamine, and 3.75 ⁇ g of trypsin.
  • Baby hamster kidney cells stably expressing T7 RNA polymerase (Buchholz et al., 1999, J Virol 73, 251-9) were grown in Dulbecco's Modified Eagle medium (BioWhittaker, Verviers, Belgium) supplemented with 10% FCS, nonessential amino acids, 100 IU of Penicillin/ml, 100 ⁇ g of streptomycin/ml, 2 mM glutamine and supplemented with 0,5 mg of G418 (Life Technologies, Breda, The Netherlands).
  • minireplicon systems of hMPV-Al and Bl have been described previously (Herfst et al., 2004, J Virol 78, 8264-70).
  • the minireplicon system for aMPV-C was constructed using the same vectors, with primers designed on the basis of the published sequence of aMPV-C (Gene bank accession no. AY57978).
  • the leader and the GS of N and the trailer and GE of L were amplified by PCR and ligated, separated by two BsmBI sites.
  • This fragment was ligated in a plasmid containing T7 RNA polymerase promoter (P ⁇ 7) and terminator (Tj 7 ) sequences and a hepatitis delta ribozyme (pSP72-P T 7-£-T T7 , (Herfst et al., 2004, J Virol 78, 8264-70) to yield pSP72-P T7 -Tr-Le- ⁇ 5-T T7 .
  • the ORF of CAT was amplified by PCR and cloned in the BsmBI sites between the GS of N and GE of L to yield pSP72-P T7 -Tr-CAT-Le- ⁇ -T T7
  • the N, P, and M2.1 ORFs of aMPV-C were amplified by PCR using primers spanning the start and stop codons and flanked by Ncol and Xhol sites, respectively, and were cloned in the multiple cloning site of pCITE (Novagen) to yield plasmids pCITE-N, pCITE-P, and pCITE-M2.1.
  • Constructs encoding the L gene of aMPV-C were assembled from overlapping PCR fragments using restriction sites in the L gene and were cloned in pCITE.
  • the restriction sites used were Ncol (introduced at nt 6935 before the start codon of L), Seal (nt 8557), Ndel (nt 9770), and BcII (nt 11535) and Xhol (introduced at nt 13135 after the trailer).
  • the minireplicon system of aMPV-A was a kind gift of Dr A. Easton.
  • the minireplicon systems of bPIV-3 and hRSV are published in Jin et al., 1998, Virology 251, 206-14.
  • the N and P ORFs of aMPV-C and the N, N and P, P and M2.1 ORFs of hMPV- Al were amplified by PCR using primers spanning GS and GE flanked by type II restriction sites.
  • the L ORF and GS and GE of hMPV-Al was assembled from overlapping PCR fragments using unique restriction sites in the L ORF and type II sites flanking GS and GE.
  • fragments of hMPV-Bl were amplified by PCR and cloned in pBluescript SK + (Stratagene).
  • the L ORF of aMPV-C was assembled from overlapping PCR fragments using unique restriction sites in the L ORF and type II sites flanking GS and GE were introduced. Using unique restriction sites the fragments containing the desired ORF were swapped back into the full-length hMPV-Bl cDNA plasmids. All plasmid inserts were sequenced to ensure the absence of mutations.
  • BSR-T7 cells grown to 80-95% confluence in six-well plates were transfected with l ⁇ g of the vector expressing the vRNA-like molecule, l ⁇ g pCITE-N, 0.5 ⁇ g pCITE-P, 0.5 ⁇ g pCITE-L, 0.5 ⁇ g pCITE-M2.1 and 0.4 ⁇ g of pTS27, a vector expressing ⁇ - galactosidase under the control of a cytomegalovirus immediate-early (CMV IE) promoter (a kind gift of Dr M. Malim).
  • CMV IE cytomegalovirus immediate-early
  • the hMPV-B 1 polymerase expression plasmid set was used for the recovery of all chimeric hMPV-Bl/hMPV-Al and hMPV-B 1/aMPV-C viruses. After transfection, the media was replaced with fresh media supplemented with trypsin. Three days after transfection, the BSR-T7 cells were scraped and cocultured with Vero-118 cells for 8 days.
  • Viruses were propagated in Vero-118 cells and virus titers were determined as described previously (Herfst et al., 2004, J Virol 78, 8264-70). Confluent monolayers of Vero-1 18 cells in 96-well plates (Greiner Bio-One) were spin-inoculated (15 min., 2000 X g) with 100 ⁇ l often fold serial dilutions of each sample and incubated at 37 0 C. After 2 hours and again after 3-4 days, the inoculum was replaced with fresh infection media.
  • infected wells were identified by immunofluorescence assays (IFA) with hMPV-specific polyclonal antiserum raised in guinea pigs, as described previously (van den Hoogen et al., 2001 , Nat Med 7, 719-24). Titers expressed as 50% tissue culture infectious dose (TCID 50 ) were calculated as described by Reed and Muench (Reed & Muench, 1938, J Hyg 27, 493-497).
  • Plaque assays were performed as described previously (Herfst et al., 2004, J Virol 78, 8264-70), with minor adjustments. Twenty-four-well plates containing 95% confluent monolayers of Vero-1 18 cells were inoculated with 10-fold serial virus dilutions for 1 h at 37°C, after which the media was replaced by 0.5 ml of fresh media and 0.5 ml of 2% methyl cellulose (MSD, Haarlem, the Netherlands) and cells were incubated at 37°C for 4 days. Methyl cellulose overlays were removed and cells were fixed with 80% acetone.
  • Tissues from the inoculated hamsters were homogenized using a Polytron homogenizer (Kinematica AG, Littau-Lucerne, Switzerland) in infection media. After removal of tissue debris by centrifugation, supernatants were used for virus titration in Vero-118 cells. Titers were calculated per gram tissue, with a detection limit of 10 1 6 and 10 1 2 TCID 50 per gram of tissue for NT and lung samples respectively.
  • vRNA-like molecules that contained a CAT ORF in antisense orientation flanked by the genomic termini of hMPV-Al and Bl, aMPV-A and C, hRSV and bPIV-3 were used.
  • Each of these plasmids was co- transfected in BSR-T7 cells with four plasmids expressing the N, P, L, and M2.1 proteins of hMPV-Al, Bl, or aMPV-C.
  • the M2.1 expression plasmid was omitted as the virus does not need M2.1 for efficient replication and transcription (Durbin et al., 1997, Virology 234, 74-83).
  • the reporter gene CAT was expressed efficiently (Fig. 5).
  • Polymerase complex proteins of hMPV-Al and Bl and aMPV-C could replicate the vRNA-like molecules of hMPV-Al and Bl, aMPV-A and C, and hRSV but not bPIV-3.
  • the bPIV-3 polymerase complex only replicated the homologous vRNA-like molecule.
  • the metapneumovirus polymerase complexes revealed little substrate specificity as they replicated heterologous metapneumovirus vRNA-like molecules with similar efficiency as homologous molecules.
  • VRNA-like molecules based on the hRSV genome were replicated less efficiently than the metapneumovirus vRNA-like molecules by the human metapneumoviruses polymerase complexes.
  • hMPV-Al /Bl polymerase complexes were functional and replicated vRNA-like molecules equally efficient as the homologous complex protein sets (Fig. 6A and C).
  • Chimeric polymerase complexes consisting of hMPV-Al and aMPV-C or hMPV-Bl and aMPV-C components were functional but differed in their replication efficiency (6B, D - F).
  • hMPV-Al and hMPV-Bl polymerase complex proteins appeared to be highly conserved as they caused similar increases and decreases in replication efficiency when exchanged with those of aMPV-C (compare Fig. 6B and 6D or 6E and 6F).
  • Chimeric hMPV- Al Fig.
  • M2.1 expression plasmid of pneumo virus and metapneumo virus minireplicon systems can be omitted, without significant effects on the levels of CAT (Collins et al., 1995, Proc Natl Acad Sci USA 92, 11563-7; Collins et al., 1996, Proc Natl AcadSci USA 93, 81-5; Herfst et al., 2004, J Virol 78, 8264-70; Naylor et al., 2004, J Gen Virol 85, 3219-27).
  • hMPV-Bl cDNA plasmid was co-transfected into BSR-T7 cells with the N, P, L and M2.1 expression plasmids of hMPV-Bl or aMPV-C or sets in which the hMPV-Bl N, P, L and M2.1 expression plasmids were individually exchanged with those of aMPV-C. It was possible to rescue hMPV-Bl by the hMPV-Bl, aMPV-C and all chimeric hMPV-Bl /aMPV-C polymerase complexes without significant differences in efficiency.
  • N, P, N and P, M2.1 and L genes of hMPV-Bl were replaced with those of hMPV-Al resulting in hMPV-Bl /N hM pv-Ai, hMPV-Bl/P h MPv-Ai, hMPV-Bl/NP h MPv-Ai, hMPV-Bl/M2.1 hM pv-Ai and hMPV-Bl /L hMPV- Ai respectively.
  • Standard multi-step growth curves were generated to compare the growth of the chimeric viruses with those of the parental viruses hMPV-Al and Bl .
  • Vero-118 cells were infected at a multiplicity of infection (MOI) of 0.1 with the parental and chimeric viruses after which supernatant samples were collected daily and virus titers were determined by plaque assay (Fig. 7). No apparent differences in replication kinetics could be observed, indicating that the viruses containing chimeric polymerase complexes are fully functional in vitro, in agreement with the minireplicon assays.
  • MOI multiplicity of infection
  • Vero-118 cells were infected at a MOI of 0.1 with parental and chimeric viruses after which supernatants were collected daily and virus titers were determined by plaque assay (Fig. 8). This revealed that aMPV-C replicated faster than its human counterpart hMPV-Bl. Furthermore, hMPV-B 1/LaMPv-c and hMPV-B 1/N 3 MPV-C grew to similar titers as the backbone virus hMPV-Bl . In contrast the hMPV-B 1/PaMPV-c grew to higher titers than hMPV-Bl .
  • AMPV-C replicated to 100-fold higher titers in the lungs, but 10-fold lower titers in the NT compared to hMPV-Bl .
  • the hMP VB 1 -NaMPV-c and hMPV-Bl /L 3 MPv-C chimeric viruses did not replicate in the lungs and slightly less efficiently in the NT compared to hMPV-Bl .
  • HMPV-B 1/P aMP v-c does not replicate in the lungs and resulted in 10.000-fold lower titers in the NT compared to hMPV- Bl .
  • the F proteins from hMPV strains NL/1/99 and NL/1/00 were used to vaccinate Syrian Golden Hamsters and to determine whether the proteins themselves might work as subunit vaccines, inducing protective immunity to prevent subsequent hMPV challenge infection.
  • Animals were inoculated with 10 ⁇ g of the F protein from NL/1/99 or NL/1/00 in the presence or absence of adjuvant.
  • the animals were immunized twice, with a 3 week interval between immunizations. Three weeks after immunization, the animals were challenged with 10 6 TCID 50 of hMPV strain NL/1/00.
  • Plaque reduction virus neutralization assays were performed generally as described in section 6.1(a)(viii). Virus neutralizing antibody titers were high in sera obtained from animals that were vaccinated with the F protein from hMPV strain NL/1/99 or NL/1/00 in the presence of adjuvant, as evidenced by the high dilutions possible to cause a 50% reduction in the number of plaques formed in the assay (Table 4). Antibodies generated following vaccination with the F protein from hMPV NL/1/99 were effective in reducing the number of plaques from the strain NL/ 1/00, although were much more effective at neutralizing the parent strain. Similarly, antibodies generated following vaccination with the F protein from hMPV NL/1/00 were capable of reducing plaque formation by the strain NL/1/99, but much more capable of neutralizing NL/1/00 (Table 4).
  • hMPV strains NL/1/94 (passage 3 at 37 0 C), NL/17/00 (passage 3 at 37 0 C), and NL/1/00 (passage 10 at 37 0 C) were cold-passaged and analyzed as described in sections 6.1(a)(ii) and 6.1(a)(iii). Briefly, virus was serially passaged in Vero-83 cells at 34°C, 31 0 C, 28°C and 25°C for 3, 3, 2 and 2 passages respectively. When the temperature was decreased further to 22 0 C or 2O 0 C, virus replication was seriously impaired, and passaging was thus continued at 25°C until passage 35 was reached.
  • hMPV strain NL/17/00 (A2) accumulated 9 nucleotide changes by passage 35 as well as a deletion of the nucleotide at position 4692 of the original NL/17/00 genome. These nucleotide changes corresponded to 4 amino acid substitutions (Table 6). Analysis of virus genome sequences after fewer passages (passage 29 and 35) indicated the gradual accumulation of these mutations.
  • Table 7 Overview of mutations in NL/1/00 after cold-passaging
  • mMPVs can be cultured in different cell lines in order to examine the characteristics of each virus. For example, the infectivity of different viruses can be characterized and distinguished on the basis of titer levels measured in culture. Alternatively, cells can be used to propagate or amplify strains of the virus in culture for further analysis. [00344] In one example, tertiary monkey kidney cells were used to amplify hMPV. However, tertiary monkey kidney cells are derived from primary cells which may only be passaged a limited number of times and have been passaged three times in vivo.
  • Vero cells and LLC-MK2 cells were the cell substrates most suitable for hMPV virus replication, resulting in virus stock titers of 10 6 - 10 7 pfu/ml. These titers were similar to those obtained from tMK cells. The addition of trypsin at a concentration of 0.01 mg/ml did not increase virus titers appreciably (Table 8). TABLE 8: HMPV VIRUS GROWTH IN DIFFERENT CELL LINES
  • Gelatinized plasticware was used to prevent cells from detaching throughout the transfection procedure. Plates or flasks were covered with 0.1 % gelatinin (1 :20 dilution of 2 % stock) for 10 minuted and rinsed one time with PBS once. To achieve the correct cell density; cells were used at a concentration of 1 xlO 7 cells per T75 flask or 100 mm plate (in 10 ml) or 1x10 6 cells per well of a 6-well plate (in 2 ml). [00348] Transfection lasted for a minimum of 7 hours, however, it was preferable to allow the transfection to occur overnight.
  • the cells appeared to be covered with little specks (the precipitate).
  • the transfection media was removed from the cells, and the cells were rinsed carefully with PBS, and then replaced with fresh media.
  • the cells were incubated in a 37 0 C CO 2 atmosphere until needed, usually between 8 - 24 hours.
  • a 10x stock of HBS was prepared with with 8.18 % NaCl, 5.94 % Hepes and 0.2 % Na 2 HPO 4 (all w/v). The solution was filter sterilized and stored at 4 0 C.
  • a 2x solution was prepared by diluting the 1Ox stock with H 2 O and adjusting the pH to 7.12 with 1 M NaOH. The solution was stored in aliquots at -2O 0 C. Care was taken to exactly titrate the pH of the solution. The pH was adjusted immediately before the solution was used for the transfection procedure.
  • Minireplicon constructs can be generated to contain an antisense reporter gene.
  • An example of a minireplicon, CAT-hMPV is shown in Figure 13.
  • the leader and trailer sequences that were used for the generation of the minireplicon construct are shown in Figure 14.
  • an alignment of APV, RSV and PIV3 leader and trailer sequences are also shown in Figure 14.
  • This experiment was entirely plasmid driven: the minireplicon was cotransfected with a T7 polymerase plasmid, and the N, P, L, M2.1 genes were expressed from pCITE-2a/3a (the pCite plasmids have a T7 promoter followed by the IRES element derived from the encephalomyocarditis virus (EMCV)).
  • EMCV encephalomyocarditis virus
  • minireplicon constructs can be generated to contain a reporter gene.
  • An example of a minireplicon, CAT-hMPV is shown in Figure 13.
  • a cDNA encoding the reporter protein chloramphenicol acetyltransferase (CAT) can be cloned in negative-sense orientation between the 5' and 3' noncoding viral sequences.
  • a T7 RNA polymerase promoter sequence and a recognition sequence for a restriction enzyme can flank the construct.
  • In vitro transcription will yield virus-like RNA that will form reconstituted RNP complexes when mixed with purified polymerase proteins.
  • the RNPs can be transfected into eucaryotic cells, for example, with helper virus.
  • the rescue can be entirely plasmid driven, i.e., the minireplicon can be co-transfected with a T7 polymerase plasmid, and the N, P, L, and M2.1 genes expressed from pCITE-2a/3a.
  • the polymerase components used to rescue hMPV can be those of RSV, APV, PIV, MPV, or any combination thereof (see above).
  • Virus can be detected using any of a number of assays capable of detecting CAT activity.
  • Rescue of hMPV using a minireplicon system can also be performed by superinfecting the minireplicon-transfected cells with hMPV polymerase components (NL/1/00 and NL/1/99) or polymerase components from MPV, APV, RSV, PIV, or any combination thereof.
  • hMPV polymerase components NL/1/00 and NL/1/99
  • polymerase components from MPV, APV, RSV, PIV, or any combination thereof.
  • a cDNA of the leader region and the adjoining gene can be modified by mutagenesis using synthetic oligonucleotides.
  • a cDNA of the downstream end of another hMPV gene e.g., the L gene, and adjoining trailer region, can be modified to contain an adjacent T7 RNA polymerase promoter.
  • the leader and trailer fragments can be cloned into an expression vector, e.g., pUC19, on either side of an insert of the CAT gene.
  • cDNAs encoding additional hMPV viral analogs can be constructed in the same way. Construct structures can be confirmed using sequencing. Examples of the leader and trailer sequences that can be used for the generation of the minireplicon construct are shown in Figure 14. For comparison, an alignment of APV, RSV and PIV3 leader and trailer sequences are also shown in Figure 14.
  • Full length cDNAs that express the genes of the hMPV virus can be constructed so that infectious viruses can be produced.
  • a cDNA encoding all of the genes or all of the essential genes of hMPV can be constructed; the genome can then be expressed to produce infectious viruses.
  • Genetic alterations, such as mutations and non-native sequences, can be introduced into the cDNA by recombinant DNA technology.
  • the genome of this RNA virus was cloned. For the NL/1/00 isolate of hMPV, eight PCR fragments varying in length from 1-3 kb were generated ( Figure 16).
  • PCR fragments were sequenced and analyzed for sequence errors by comparison to the hMPV sequence deposited in Genbank. Two silent mutations (nucleotide 5780 ile:ile in the SH gene, nucleotide 12219 cys:cys in the L gene) were not corrected. Another change in the L gene at nucleotide 8352 (trp:leu) was not changed since this mutation was observed in two independently generated PCR fragments (C and H), as well as in the hMPV NL/1/99 sequence. Similarly, a 5 nucleotide insertion at nucleotide 4715 in the F-M2 intergenic region was not corrected. Both of these changes may be reflected in the wild type sequence of hMPV.
  • nucleotide 1242 In contrast, at nucleotide 1242, a single A residue was removed in the N-P intergenic region; at nucleotide 3367, a se ⁇ pro was corrected in the F gene; at nucleotide 6296, an asp:val was changed in the G gene; and at nucleotide 7332 a stop codon was changed to a glu in the L gene.
  • Restriction maps of different isolates of hMPV are shown in Figure 17.
  • the restriction sites can be used to assemble the full-length construct.
  • the eight corrected PCR fragments were then assembled in sequence, taking advantage of unique restriction enzyme sites (Figure 18).
  • a genetic marker was introduced at nucleotide 75 generating an AfIII restriction enzyme site without altering the amino acid sequence.
  • a unique restriction enzyme site, Xhol was added at the 3' end of the hMPV sequence.
  • a phage T7 polymerase promoter followed by two G residues was also added to the 3' end of the hMPV sequence.
  • a Hepatitis delta ribozyme sequence and BssHII restriction enzyme site were added.
  • Helper plasmids encoding the hMPV L, N, P and M2.1 proteins in a pCITE plasmid were also generated. Once the full-length hMPV cDNA is generated, virus recovery by reverse genetics can be performed in Vero cells using fowl-pox T7 or MVA-T7 as a source of T7 RNA polymerase, or a cell line or a plasmid expressing T7 RNA polymerase.
  • RNA polymerase derived from the bacteriophage T7 A unidirectional or bi-directional pol I-pol II transcription system can be used to express viral RNA molecule intracellularly. This systems proved to be very efficient for the generation of influenza virus from cloned cDNA (Hoffmann et. al., PNAS, 97 6108-61 13 (2000).
  • RNA polymerase I transcripts do not contain cap structures at their 5 '-end and do not have poly A tails at the 3 '-end.
  • systems employing the cellular transcription machinery are designed to express proteins from a pol II promoter and viral (-)vRNA or (+) cRNA which do not have a cap structure or a polyA tail from a pol I promoter.
  • To provide virus-like primary transcripts which do not contain additional non viral sequences is critical because the terminal structures are crucial for viral replication and transcription.
  • a minigenome system may be designed to compare the replication efficiency of each.
  • Replication efficiency can be measured by the transcription of a reporter molecule expressed by the minigenome, e.g., a CAT gene.
  • plasmids expressing the L, N, P, and M2.1 genes of hMPV, under the control of a pol II promoter are cotransfected into a host cell together with a CAT-minigenome-plasmid.
  • the relative efficiency of replication is measured by determining the relative level of expression of the CAT reporter molecule.
  • RNA pol I can be used to synthesize positive strand copies of the hMPV viral genome (cRNA).
  • the viral cDNA is inserted between an RNA pol I promoter and a terminator sequence.
  • the whole pol I transcription unit is inserted in the positive-sense orientation between an RNA pol II promoter and a polyadenylation site.
  • Two types of positive-sense RNAs are synthesized. From the pol II promoter, an mRNA with a 5 '-cap structure is transcribed. From the pol I promoter full-length, positive-sense hMPV cRNA with a triphosphate group at the 5'end is transcribed by cellular RNA polymerase I.
  • a cloning vector can be used for the insertion of arbitrary cDNA fragments, e.g., pHWl 1 (Hoffmann & Webster, J. Gen Virol. 2000 Dec 81(Pt 12):2843-7).
  • This plasmid contains the pol II promoter (immediate early promoter of the human cytomegalovirus) and the human pol I promoter that are upstream of a pol I terminator sequence and a poly(A) site.
  • the viral polymerase proteins are provided by plasmid vectors with a pol II promoter, such as the immediate early promoter of human cytomegalovirus.
  • plasmids contain the cDNAs representing four gene segments of hMPV, i.e., the L-gene, the N-gene, the P-gene, and the M2.1 gene.
  • Those four plasmids (1 - 5 ⁇ g) are cotransfected with the pol I/pol II plasmid (1-5 ⁇ g) representing the full length genome of hMPV into 10 6 -10 7 293T cells, COS-7 or Vero cells.
  • 293T cells can be cocultured with cells permissive for MPV, such as Vero or tMK cells. The addition of trypsin to the cell culture medium results in the generation of infectious virus particles.
  • the coculturing of primate cells with MDCK cells was employed for the efficient rescue of influenza A virus (Hoffmann et. al., PNAS, 97 6108-61 13 (2000).
  • the supernatant after different times after transfection i.e., 3d to 1Od
  • the coexpression of all viral structural proteins i.e., M, M2.2, SH, F, and G
  • M, M2.2, SH, F, and G may improve the efficiency of virus recovery.
  • a successful system was developed to rescue recombinant hMPV.
  • expression plasmids encoding various polymerase proteins, were co-transfected with the cloned hMPV to be rescued into appropriate host cells. Upon collection and treatment, the cells and supernatant were then used to inoculate Vero cells. Infectious rescued virus was detected using immunostaining methods.
  • the expression plasmids and the cloned hMPV to be rescued were mixed in 100 ⁇ l optiMEM (per well) in the following amounts: 0.4 ⁇ g of plasmid encoding the hMPV P gene (in pCITE 2a/3a, designated clone #41-6), 0.4 ⁇ g of plasmid encoding the hMPV N gene (in pCITE 2a/3a, designated clone #35-11), 0.3 ⁇ g of plasmid encoding the hMPV M2 gene (in pCITE 2a/3a, designated clone #25-6), 0.2 ⁇ g of plasmid encoding the L gene (in pCITE 2a/3a, designated clone #2), and 4 ⁇ g of hMPV plasmid clone #2 which has the leader and trailer like APV or clone #10 which has hMPV leader and trailer sequences.
  • the expression plasmids used have the wild type sequence restored in the second amino acid position.
  • the transfection reagent Lipofectamine 2000 (8 ⁇ l) was mixed into 100 ⁇ l of optiMEM and then added to the plasmid mixture. This combined mixture was applied to the 293T cells. Six days after transfection, the cells and supernatant were collected, frozen, thawed, and used to inoculate Vero cells. Nine days post inoculation, the infected cells were fixed in methanol, immunostained with a guinea pig polyclonal antibody followed by anti-guinea pig HRP and the DAKO AEC substrate. Plaque formation demonstrated that the rescued virus was infectious.
  • Animal hosts can be infected in order to characterize the virulence of MPV strains. For example, different hosts can be used in order to determine how infectious each strain is in an organism.
  • Balb/c mice, cotton rats, and Syrian Golden hamsters were infected with hMPV using a dose of 1.3 x 10 6 pfu/animal.
  • the animals were inoculated intranasally with 1.3 x 10 pfu of hMPV in a 0.1 ml volume.
  • the tissue samples were quantified by plaque assays that were immunostained on Day 9 with the hMPV guinea pig antiserum.
  • Four days post-infection the animals were sacrificed, and the nasal turbinates and lungs were isolated and quantified for hMPV titers by plaque assays that were immunostained (Table 9).
  • the primary infection was allowed to progress for fifty-four days when the guinea pigs were inoculated with the homologous and heterologous subtypes (10e4 TCID50/ml), i.e., two guinea pigs had a primary infection with NL/1/00 and a secondary infection with NL/1/99 in order to achieve a heterologous infection, three guinea pigs had a primary infection with NL/1/00 and a secondary infection with NL/1/00 to achieve a homologous infection, two guinea pigs had a primary infection with NL/1/99 and a secondary infection with NL/1/00 to achieve a heterologous infection and three guinea pigs had a primary infection with NL/1/99 and a secondary infection with NL/1/99 to achieve a homologous infection.
  • the homologous and heterologous subtypes 10e4 TCID50/ml
  • Throat and nose swabs were collected for 12 days (primary infection) or 8 days (secondary infection) post infection, and were tested for the presence of the virus by RT-PCR assays.
  • the results ( Figure 19) of the RT-PCR assays showed that guinea pigs inoculated with virus isolate NL/1/00 showed infection of the upper respiratory tract on days 1 through 10 post infection.
  • Guinea pigs inoculated with 99-1 showed infection of the upper respiratory tract day 1 to 5 post infection. Infection of guinea pigs with NL/ 1/99 appeared to be less severe than infection with NL/1/00.
  • Virus isolates NL/1/00 (subtype A) and NL/1/99 (subtype B) (Ie5 TCID50) was used to inoculate two cynomologous macaques per subtype (intratracheal, nose and eyes).
  • the macaques were inoculated for the second time with NL/1/00.
  • Throat swabs were collected for 14 days (primary infection) or 8 days (secondary infection) post infection, and were tested for presence of the virus by RT-PCR assays.
  • Cynomologous macaques inoculated with virus isolate NL/1/00 showed infection of the upper respiratory tract day 1 to 10 post infection.
  • Sera were collected from the macaques that received NL/ 1/00 during six months after the primary infection (re-infection occurred at day 240 for monkey 3 and day 239 for monkey 6). Sera were used to test for the presence of IgG antibodies against either NL/ 1/00 or APV, and for the presence of IgA and IgM antibodies against 00-1.
  • IgA and IgM antibodies showed the raise in IgM antibodies after the first infection, and the absence of it after the reinfection. IgA antibodies were only detected after the re-infection, showing the immediacy of the immune response after a first infection. Sera raised against hMPV in macaques that were tested in an APV inhibition ELISA showed a similar response as to the hMPV IgG ELISA.
  • Virus cross-neutralization assays were preformed on sera collected from hMPV infected cynomologous macaques. The sera were taken from day 0 to day 229 post primary infection and showed only low virus neutralization titers against NL/1/00 (0-80), the sera taken after the secondary infection showed high neutralisation titers against NL/1/00, i.e., greater than 1280. Only sera taken after the secondary infection showed neutralization titers against 99-1 (80-640), and none of the sera were able to neutralize the APV C virus. There was no cross reaction between APV-C and hMPV in virus cross-neutralization assays, however, there was a cross reaction between NL/1/00 and NL/1/99 after a boost of the antibody response.
  • INFECTION OF HUMANS The sera of patients ranging in ages under six months or greater than twenty years of age were previously tested using IFA and virus neutralization assays against 00-1. These sera were tested for the presence of IgG, IgM and IgA antibodies in an ELISA against NL/1/00. The samples were also tested for their ability to in inhibit the APV ELISA.
  • FIG. 21 A map of the M2 gene of hMPV strain hMPV/NL/1/00 is shown in Figure 21.
  • a Bsp El site is constructed at nucleotide position
  • restriction sites are constructed by site-specific mutagenesis. Restriction digestion of the recombinant genome at the two Bsp El sites using the restriction endonuclease Bsp El and subsequent ligation results in a deletion of the sequence between nucleotide position 4741 and nucleotide position 5444.
  • Nhe I sites are introduced at nucleotide positions 4744 and 5241.
  • the restriction sites are constructed by site-specific mutagenesis. Restriction digestion of the recombinant genome at the two Nhe I sites using the restriction endonuclease Nhe I and subsequent ligation results in a deletion of the sequence between nucleotide position 4744 and nucleotide position 5241.
  • Swa I sites are introduced at nucleotide positions 5311 and 5453.
  • the restriction sites are constructed by site-specific mutagenesis. Restriction digestion of the recombinant genome at the two Swa I sites using the restriction endonuclease Swa I and subsequent ligation results in a deletion of the sequence between nucleotide position 5311 and nucleotide position 5453.
  • primers used to introduce the restriction enzyme sites were used:
  • primer set For putting BspEI into hMPV/NL/1/00 to make M2 deletion from 4741 to 5444: primer set
  • the primer sets for cloning the hMPV/NL/1/00 virus with the SH deletion are as follows:
  • This process allows recovery of recombinant hMPV using plasmids only, in the absence of helper viruses.
  • the recovery of hMPV is carried out using SF Vero cells, which are propagated in the absence of animal and human derived products.
  • This process allows recovery of recombinant hMPV with similar efficiency to previous methods using helper viruses (recombinant vaccinia or fowl-pox viruses expressing T7 polymerase). Because no helper viruses are needed in the recovery process, the vaccine viruses are free of contaminating agents, simplifying downstream vaccine production.
  • the cells used for vaccine virus recovery are grown in media containing no animal or human derived products. This eliminates concerns about transmissible spongiform encephalopathies (e.g. BSE), for product end users.
  • BSE transmissible spongiform encephalopathies
  • This method enables generation of a recombinant vaccine seed that is completely free of animal or human derived components.
  • the seed is also free of contaminating helper viruses.
  • Plasmid-based expression systems for rescue of viruses from cDNA are described, e.g., in RA Lerch et al., Wyeth Vaccines, Pearl River NY, USA (Abstract 206 from XII International Conference on Negative Strand Viruses, June 14 th -19 th 2003, Pisa Italy) and G. Neumann et. al., J. Virol., 76, pp 406-410. (b) Methods and Results
  • hMPV N plasmids (4 ⁇ g; marker: kanamycin resistancy), hMPV P plasmids (4 ⁇ g; marker: kanamycin resistancy), hMPV L plasmids (2 ⁇ g; marker: kanamycin resistancy), cDNA encoding hMPV antigenomic cDNA (5 ⁇ g; marker: kanamycin resistancy) and pADT7(N)DpT7 encoding T7 RNA polymerase (5 ⁇ g; marker: blasticidin) are introduced into SF Vero cells using electroporation in serum-free medium.
  • hMPV virus For the rescue of hMPV virus, 4 expression plasmids are used. They are for the genes N, P, L and also M2 of hMPV. In particular the following plasmids are used: 4 ug hMPV N pCITE plasmid,
  • the viral genome to be rescued is in a full length plasmid with a T7 promoter.
  • T7 DNA-dependent RNA polymerase transcribes a full length viral RNA genome using this full length plasmid. After the viral genome is made, the viral polymerase complex will transcribe the viral genome and generate viral messenger RNAs and virus is subsequently recovered.
  • the pulse for the electroporation is 220V and 950 microfarads.
  • 5.5 X 10 6 SF Vero cells are used per electroporation.
  • the electroporated cells are allowed to recover at 33°C in the presence of OptiC (a custom formulation from GIBCO Invitrogen Corporation) overnight. Recovered cells are washed twice with 1 mL of PBS containing calcium and magnesium and overlayed with 2 mL of OptiC. Electroporated cells are further incubated at 33 0 C for 5-7 days. At the end of the incubation period, cells are scraped into the media and total cell lysate is analyzed for presence of hMPV. Virus recovery is confirmed by immunostaining of plaque assays using hMPV specific polyclonal antibodies.
  • FIG. 24 The cells (Vero cells) were infected at a MOI of 1.
  • viruses When viruses are inoculated into an animal, an array of antibodies against the virus are produced. Some of these antibodies can bind virus particles and neutralize the infectivity of the viruses. In this example, a microneutralization assay was used to analyze the remaining infectivity of the viruses after the viruses were incubated with dilutions of serum containing antibodies.
  • a 96-well plate is divided (i) into rows A (dilution 1 :32); B (1 :64); C (1 :128); D (1 :256); E (1 :512); F (1 : 1024); G (1 :2048); and H ( No Antibody) and (ii) into columns 1 to 12 for the different samples (first sample: columns 1 to 3; second sample: columns 4 to 6; third sample: columns 7 to 9; and fourth sample: columns 10 to 12).
  • 230 ⁇ l of sample dilution are added to row A.
  • 115 ⁇ l of Opti-MEM are added to rows B-H.
  • Microneutralization assay was performed as follows: sera were serially diluted. Each test sample and each control was diluted by 1 :32 by adding 22.5 ⁇ l of sera to 697.5 ⁇ l of Opti-MEM Medium (Ix). Serum and medium were mixed gently by inversion three times and place on ice. Each dilution of serum was incubated with the virus hMPV/GFP2. Cells were washed with phosphate buffered saline (“PBS").
  • PBS phosphate buffered saline
  • Vero cells from ATCC are maintained in MEM (JRH Biosciences) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, nonessential amino acids, and 100 units/ml penicillin G, 100 ⁇ g/ml streptomycin sulfate.
  • the virus/sera mixtures were added to cells and incubated for one hour at 35°C. All of the medium, which contained the virus, were removed, and cells were washed with PBS. Opti- MEM medium was added to the cells and the cell cultures were incubated for three days.
  • Opti-MEM I Reduced-Serum Medium (GIBCO 31985-070) contains, among others, HEPES buffer, 2400 mg/L sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, growth factors, and phenol red reduced to 1.1 mg/L.
  • the remaining infectivity of the viruses was measured by quantifying eGFP green foci on the images captured with fluorescence microscope.
  • results demonstrate that the microneutralization assay using hMPV/GFP2 provides reliable and reproducible results.
  • the use of hMPV-GFP in the microneutralization assay facilitates the high throughput screening of different vaccines and antibodies in animal model systems such as ferrets and monkeys. This technique also provides efficient means for diagnosing and monitoring infections in humans.
  • results demonstrate a linear correlation between plaque reduction and microneutralization using hMPV/GFP2.
  • Table 10 Titers of ferret sera using hMPV/GFP2 microneutralization assay and plaque reduction assay. Complement from Guinea pig (add 100 ⁇ l in 20 ml of Opti-MEM) was used for plaque reduction assay. NT50 is 1 /dilution that confers 50% neutralization of input virus. The numbers in the table indicate the titers of sera.
  • Table 11 Titers of Monkey sera using hMPV/GFP2 microneutralization assay and plaque reduction assa .
  • Table 12 Linear Correlation between plaque reduction assay and microneutralization assay usin hMPV/GFP2
  • SEQ ID NO: 1 isolate NL/ 1/99 HMPV cDNA sequence (vaiant Bl)
  • SEQ ID NO:2 isolate NL/1/00 HMPV cDNA sequence (variant Al)
  • SEQ ID NO:4 isolate NL/1/94 HMPV cDNA sequence (variant B2)
  • SEQ ID NO:8 leader sequence of aMPV-A
  • SEQ ID NO: 17 F protein sequence for HMPV isolate NL/1/00
  • SEQ ID NO: 18 F protein sequence for HMPV isolate NL/17/00
  • SEQ ID NO: 19 F protein sequence for HMPV isolate NL/1/99
  • SEQ ID NO:20 F protein sequence for HMPV isolate NL/1/94
  • SEQ ID NO:26 G protein sequence for HMPV isolate NL/17/00
  • SEQ ID NO:28 G protein sequence for HMPV isolate NL/1/94
  • SEQ ID NO:33 L protein sequence for HMPV isolate NL/1/00
  • SEQ ID NO:34 L protein sequence for HMPV isolate NL/17/00
  • SEQ ID NO:35 L protein sequence for HMPV isolate NL/1/99
  • SEQ ID NO:36 L protein sequence for HMPV isolate NL/1/94

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Abstract

L'invention concerne un virus isolé ARN négatif monobrin de mammifère, le métapneumovirus (MPV), dans la sous-famille des Pneumoviridae, de la famille des Paramyxoviridae avec une ou plusieurs modifications génétiques. La présente invention concerne également les composants mutants, à savoir les acides nucléiques et les protéines, de ces MPV mammifères mutants. Ces MPVm mutants peuvent être atténués. Ces MPVm mutants peuvent coder pour des séquences non natives. L'invention concerne en outre des formulations de vaccin comportant le MPVm, incluant des formes recombinantes et chimériques desdits virus. Les préparations de vaccin de l'invention englobent des vaccins multivalents, incluant des préparations de vaccins bivalents et trivalents. De plus, l'invention concerne un complexe d'ARN polymérase viral chimérique et des analyses utilisant ces complexes d'ARN polymérase chimérique. Les complexes d'ARN polymérase chimérique de l'invention sont composés de différents composants d'ARN polymérase provenant de différents virus de la famille des Paramyxoviridae.
PCT/EP2007/009942 2007-11-16 2007-11-16 Souches de métapneumovirus atténués vivants et leur utilisation dans des formulations de vaccin et souches de métapneumovirus chimériques WO2009062532A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018039221A1 (fr) * 2016-08-23 2018-03-01 Xiaoyong Bao Hmpv recombinant vivant atténué possédant des mutations au niveau des motifs pdz de la protéine m2-2, vaccin contenant ce dernier et utilisation de ce dernier
WO2020120910A1 (fr) * 2018-12-14 2020-06-18 Universite Claude Bernard Lyon 1 Production de vaccins viraux sur une lignee cellulaire aviaire

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002057302A2 (fr) * 2001-01-19 2002-07-25 Vironovative B.V. Virus provoquant des maladies respiratoires chez des mammiferes y etant sensibles
WO2006099360A2 (fr) * 2005-03-10 2006-09-21 Medimmune Vaccines, Inc. Souches de metapneumovirus et leur utilisation dans des formulations de vaccin et comme vecteurs pour l'expression de sequences antigeniques et methodes de propagation de virus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002057302A2 (fr) * 2001-01-19 2002-07-25 Vironovative B.V. Virus provoquant des maladies respiratoires chez des mammiferes y etant sensibles
WO2006099360A2 (fr) * 2005-03-10 2006-09-21 Medimmune Vaccines, Inc. Souches de metapneumovirus et leur utilisation dans des formulations de vaccin et comme vecteurs pour l'expression de sequences antigeniques et methodes de propagation de virus

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018039221A1 (fr) * 2016-08-23 2018-03-01 Xiaoyong Bao Hmpv recombinant vivant atténué possédant des mutations au niveau des motifs pdz de la protéine m2-2, vaccin contenant ce dernier et utilisation de ce dernier
US11478517B2 (en) * 2016-08-23 2022-10-25 Board Of Regents, The University Of Texas System Live attenuated recombinant HMPV with mutations in PDZ motifs of M2-2 protein, vaccine containing and use thereof
WO2020120910A1 (fr) * 2018-12-14 2020-06-18 Universite Claude Bernard Lyon 1 Production de vaccins viraux sur une lignee cellulaire aviaire
FR3089789A1 (fr) * 2018-12-14 2020-06-19 Universite Claude Bernard Lyon 1 Production de vaccins viraux sur une lignee cellulaire aviaire

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