WO1996010631A1 - Acide nucleique codant des proteines matrices mutees utiles pour l'attenuation ou l'activation du virus grippal a - Google Patents

Acide nucleique codant des proteines matrices mutees utiles pour l'attenuation ou l'activation du virus grippal a Download PDF

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Publication number
WO1996010631A1
WO1996010631A1 PCT/US1995/012357 US9512357W WO9610631A1 WO 1996010631 A1 WO1996010631 A1 WO 1996010631A1 US 9512357 W US9512357 W US 9512357W WO 9610631 A1 WO9610631 A1 WO 9610631A1
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virus
enhancing
attenuated
lav
attenuating
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PCT/US1995/012357
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English (en)
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Yoshihiro Kawaoka
Maria R. Castrucci
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St. Jude Children's Research Hospital
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Priority to AU37278/95A priority Critical patent/AU3727895A/en
Publication of WO1996010631A1 publication Critical patent/WO1996010631A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza 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
    • 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
    • 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
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention in the fields of virology, molecular biology and vaccines, relates to mutated nucleic acid and encoded mutated matrix proteins, useful for providing attenuated or enhanced influenza A virus (IAV) by the replacement or addition, in an LAV, of a matrix protein encoding negative strand RNA (nsRNA), with either (1) an attenuating nsRNA, encoding an attenuating matrix protein (AMP); or (2) an enhancing nsRNA, encoding an enhanced matrix protein (EMP), to provide, respectively, attenuated IAVs or enhanced IAVs useful for the production of attenuated or enhanced viral cultures.
  • nsRNA negative strand RNA
  • AMP attenuating matrix protein
  • EMP enhanced matrix protein
  • Vaccines are preparations administered to animals, including humans, to effect prophylactic or therapeutic treatment of disease states through induction of specific immunity.
  • Prophylactic vaccines are given to healthy individuals with the intention of preparing or priming the immune system for more effective defenses against particular infections in the future.
  • the immune system of a vaccinated individual can usually mount an effective secondary immune response and can more rapidly recognize and eliminate the respective pathogens.
  • Therapeutic vaccines are also given to diseased individuals with the intent of stimulating or modulating the immune system which of itself has either failed to mount an immune response or has mounted an ineffective immune response.
  • the causative pathogens or toxins e.g.
  • influenza, polio, and rabies viruses; pneumococcus bacteria; diphtheria and tetanus toxins can be effectively targeted and neutralized in the extracellular fluid by the mechanisms of humoral immunity through antibodies that bind to the pathogens or toxins and thereby lead to their inactivation or destruction (see, e.g. , Plotkin et al. , Vaccines, Saunders, Philadelphia, 1988). In these cases, prophylactic or therapeutic vaccination with preparations that elicit a humoral immune response is generally sufficient for protection or treatment.
  • live, attenuated vaccines have been used to induce immunity against viral infections such as polio. These preparations contain live virions which cause mild, subclinical infections of the vaccinated individuals. In the course of such infections, viral vectors will enter certain host cells and code for the synthesis of virus-specific proteins (Zweerink et al. , Eur. J. Immunol. 7:630, 1977). These endogenously produced antigenic proteins will be processed into smaller 8-9 amino acid peptides and presented as MHC Class I antigens on the cell's surface, thereby eliciting cell-mediated immune responses.
  • Attenuation of viruses involves mutation or modification of one or more of the genes encoding the non-essential viral proteins.
  • the mutation or modification results in sufficiently reduced infectivity and/or replication ability of the virus to induce an immune response, but not to cause pathologies related to viral infection.
  • Influenza infection in a host consists of five interrelated steps: entry into the host, primary replication, spread within the host, secondary replication and termination of replication owing to effective host defense mechanisms, including immunity. Successful completion of the first four steps results in disease, whereas the failure of a step results in either limited or nonproductive infection, or even total failure of infection.
  • Influenza A virus has eight negative-sense RNA (nsRNA) segments which encode at least 10 polypeptides, including RNA-directed RNA polymerase proteins (PB2, PB1 and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (HA), the matrix proteins (M, and M 2 ) and non- structural proteins (NS1 and NS2) (Krug et al., "Expression and Replication of the Influenza Virus Genome, " In The Influenza Viruses, R. M. Krug (ed.),
  • Influenza A viruses are enclosed by lipid envelopes, derived from the plasma membrane of the host cell.
  • the HA and NA molecules are embedded in the envelope by sequences of hydrophobic amino acids (Air et al., Structure, Function, and Genetics (5:341-356 (1989); Wharton et al ,
  • the viral proteins from different viruses can be recombined or
  • reassorted by co-infection in host cells and selection of particular reassortant viruses by rescue from the infected cells. Accordingly, reassortment of new strains of pathogenic viruses with non-pathogenic viruses can provide attenuated new strains which can be used for vaccination.
  • An influenza virus particle binds to cells via interaction between the receptor binding site of the HA and the terminal sialic acid of the cell surface receptor. After binding, the attached virion undergoes endocytosis.
  • the low pH of the endocytotic vesicle triggers a conformational change in the cleavage-activated HA, initiating fusion of the viral and vesicular membranes. Fusion releases the contents of the virion (ribonucleoprotein complex; RNP) into the cytoplasm of the cell (uncoating).
  • RNP ribonucleoprotein complex
  • M2 proteins Prior to fusion, M2 proteins, by ion channeling, introduce protons into the inside of the virion, exposing the core to low pH.
  • Virion morphogenesis and budding Ml protein transported into the nucleus is associated with the migration of RNP out of the nucleus for assembly into progeny viral particles in the cytoplasm. Few details of the assembly process are known. Presumably, RNP in association with the Ml protein buds outward through the cell membrane. Interactions between Ml and the cytoplasmic domains of HA, NA, or M2 have been proposed as signals for budding, but direct evidence for this relationship is lacking. Because influenza virus particles are formed at the cell surface, morphogenesis is regarded as part of the budding process. A virus could initiate morphogenesis but not complete budding. Thus, morphogenesis is defined as "the generation of virus particles", and budding as "the generation and release of virus particles”.
  • This method involves (i) preparation of RNA, containing exactly the same 5' and 3' sequences as viral RNA, from cloned influenza virus genes with RNA polymerase, (ii) encapsulation of the RNA with influenza virus NP and polymerase proteins, (iii) transfection of the encapsulated RNA, and (iv) infection with a helper influenza virus to rescue the transfected RNA Luytjes et al., Cell 59:1107-1113 (1989); Enami et al., J. Virol. 65:2711- 2713 (1991).
  • the matrix (M) gene of IAVs encodes two matrix proteins (MPs), Ml and M2 (Lamb, R. A. "Genes and Proteins of the Influenza Viruses, " p. 1-87.
  • Ml in virions when first treated with a protease or a detergent (Murti et al., Virology 186:294-299 (1992)). Recent cryoelectron microscopy studies suggested that the Ml can modify the lipid bilayer, causing thickening of the viral envelope (Fujiyoshi et al., EMBO J. 5:318-326 (1994)). Ml appears to escort RNP from the nucleus to the cytoplasm (Helenius, A., Cell 69:577-
  • the Ml proteins of influenza A and B viruses have a "zinc- finger" motif (Wakefield et al, Nucl. Acids Res. 77:8569-8580 (1989)), and purified virus contains zinc; however, the amount of zinc is not correlated with its RNA binding activity, implying that zinc binding is not important for this activity.
  • WSNMl arephos ⁇ horylated (Gregoriadese ⁇ * ⁇ /., Virus Res. 76:27-42 (1990)), the role of this property in viral replication is unknown.
  • the influenza B Ml protein is also phosphorylated, suggesting its role in viral replication.
  • This integral membrane protein (Zebedee et al., J. Virol. 62:2762-2772 (1988)) is a homotetramer (Holsinger et al, Virology 755:32-43
  • the M2 protein comprises 97 amino acids, 24 as the ecto-, 19 as the transmembrane, and 54 as the cytoplasmic domain.
  • M2 proteins are palmitoylated at Cys-50 (Sugrue et al, Virology 179:51-56 (1990)).
  • the M2 protein is also phosphorylated at Thr-65 (Hay et al, EMBO J. 4:3021-3024 (1985)).
  • M2 proteins have been proposed to function as an ion channel that permits protons to enter the virion during uncoating and that modulates the pH of intracellular compartments, an essential function for prevention of, the acid- induced conformational change of the intracellularly cleaved HA in the trans ⁇ Golgi network (Hay et al. , EMBO J. 4:3021-3024 (1985)).
  • the activity of the M2 ion channel is blocked by the anti-influenza drug amantadine hydrochloride.
  • the functional role of the M2 cytoplasmic region, the longest among the influenza viral membrane proteins, is unknown.
  • the present invention is directed to overcoming one or more deficiencies of the related arts.
  • the present invention provides attenuated or enhanced influenza viruses (IAV's) using RNA or DNA encoding mutated matrix proteins (MMPs).
  • IAV's attenuated or enhanced influenza viruses
  • MMPs mutated matrix proteins
  • Replacing MP nsRNA with nsRNA encoding attenuating MP (AMP) or enhancing MP (EMP) gives attenuated or enhanced IAVs, respectively.
  • methods of making and using AMP or EMP DNA or RNA for attenuating or enhancing IAV's are also provided.
  • the present invention provides mutant nucleic acid, derived from, or corresponding to, influenza A virus (LAV) matrix genes, encoding mutant matrix protein (MMP).
  • the mutant nucleic acid is provided as either (i) attenuating nucleic acid encoding an attenuating matrix protein (AMP); or (ii) enhancing nucleic acid encoding an enhanced matrix protein (EMP), which
  • MMP is capable of either attenuating or enhancing, respectively, an influenza A virus (LAV) having at least one AMP or EMP encoding nucleic acid.
  • the nucleic acid can be DNA or RNA, such as negative strand RNA (nsRNA).
  • nsRNA negative strand RNA
  • An AMP of the present invention has at least one attenuating mutation which confers attenuating activity, as described herein and/or as known in the art.
  • An EMP of the present invention has at least one enhancing mutation which confers growth enhancing activity, as described herein and/or as known in the art.
  • the present invention also provides attenuated IAVs, and isolated forms thereof, which comprise an pathogenic nsRNA encoding at least one neuraminidase (NA) and hemagglutinin (HA) from at least one pathogenic IAV; and at least one attenuating nsRNA encoding an AMP having at least one attenuating mutation.
  • NA neuraminidase
  • HA hemagglutinin
  • Attenuated IAVs of the present invention are suitable for use as live, attenuated flu vaccines.
  • An attenuated IAV of the present invention is preferably capable of inducing an immune response in an animal to at least one pathogenic IAV strain, which response causes a subclinical LAV infection in the animal, as compared to a clinical LAV infection when a native or non- attenuated pathogenic IAV infection occurs.
  • the at least one pathogenic NA and HA, encoded by the pathogenic nsRNA in an attenuated IAV of the present invention is derived from at least one pathogenic AV strain.
  • An AMP, used in an attenuated virus of the invention can comprise at least one selected from an Ml AMP and an M2 AMP, wherein the at least one attenuating mutation can inhibit the level of at least one of transcription, replication, translation or virion incorporation of an attenuated IAV of the present invention, when the LAV infects a host cell.
  • the attenuating mutation can encode at least one amino acid modification selected from the group consisting of a substitution, a deletion and an insertion.
  • An attenuated virus according to the present invention can further comprise a selection marker selected from the group consisting of a drug resistance marker, a temperature sensitive marker, and an antigenic marker.
  • the present invention also provides a vaccine composition comprising an attenuated IAV of the present invention, and a pharmaceutically acceptable carrier or diluent.
  • the vaccine composition can further comprise an adjuvant which enhances an IAV immune response to the pathogenic virus in an animal administered the vaccine composition.
  • the present invention also provides a method for obtaining an attenuated IAV capable of being used as a vaccine for at least one pathogenic
  • LAV strain comprising isolating an attenuated IAV which comprises (1) a pathogenic nsRNA encoding at least one NA and HA from at least one pathogenic IAV; and (2) at least one attenuating nsRNA encoding an
  • Such a method according to the present invention can further comprise, prior to the isolating step, a further step of reassorting, in a host, (i) a helper virus having (1) the attenuating nsRNA and (2) sensitivity to at least one selection marker; with (ii) at least one pathogenic nsRNA encoding (1) at least one neuraminidase (NA) and hemagglutinin (HA) from the at least one pathogenic IAV strain; and (2) resistance to the selection marker, to provide the attenuated influenza A virus.
  • the host may be selected from a prokaryotic and a eukaryotic cell, with mammalian cells preferred.
  • the selection marker can be selected from a group consisting of a drug resistance marker, a temperature sensitive marker and an antigenic marker.
  • a method for obtaining an attenuated LAV of the present invention may further comprise removing the resistance to the selection marker from the attenuated LAV to provide a sensitive attenuated IAV lacking resistance to the selection marker.
  • the method optionally further comprises selecting the attenuated LAV using an antibody binding an epitope specific for said atenuated AV.
  • the antibody is selected from polyclonal, monoclonal or a fragment thereof.
  • the present invention further provides a method for vaccinating an animal against an IAV strain, comprising administering to the animal an LAV immune response effective amount of a vaccine composition comprising an attenuated LAV of the present invention.
  • the present invention also provides a method for eliciting an immune response to an IAV in an animal which is prophylactic or therapeutic for an LAV infection, the method comprising administering to an animal a vaccine composition comprising an attenuated influenza A virus of the present invention, which is protective for the animal against a clinical LAV pathology caused by infection of at least one IAV strain.
  • An enhancing nucleic acid of the present invention can comprise nucleic acid encoding at least one Ml EMP or M2 EMP, wherein at least one enhancing mutation in an enhancing nucleic acid can stimulate the level of at least one of transcription, replication, translation or virion incorporation of an enhanced IAV of the present invention, e.g. , when the IAV infects a host cell.
  • the enhancing mutation can encode at least one amino acid modification selected from the group consisting of a substitution, a deletion and an insertion.
  • An enhanced LAV of the present invention can further comprise a selection marker selected from the group consisting of a drug resistance marker, a temperature sensitive marker and an antigenic marker.
  • the present invention also provides a method for obtaining an enhanced IAV, the method comprising isolating an enhanced IAV which comprises at least one enhancing nsRNA encoding an EMP having at least one enhancing mutation which is capable of enhancing the growth of the enhanced
  • Such a method according to the present invention can further comprise, prior to the isolating step, a step of reassorting, in a host, (i) a helper virus having (1) the enhancing nsRNA and (2) sensitivity to at least one selection marker; with (ii) at least one nsRNA encoding (1) at least one neuraminidase (NA) and hemagglutinin (HA) from the at least one LAV strain; and (2) selection resistant nsRNA encoding at least one protein conferring resistance to the selection marker to provide the enhanced LAV.
  • a helper virus having (1) the enhancing nsRNA and (2) sensitivity to at least one selection marker
  • at least one nsRNA encoding (1) at least one neuraminidase (NA) and hemagglutinin (HA) from the at least one LAV strain
  • selection resistant nsRNA encoding at least one protein conferring resistance to the selection marker to provide the enhanced LAV.
  • the method optionally further comprises selecting the enhanced IAV using an antibody binding an epitope specific for said enhanced LAV.
  • the antibody is selected from polyclonal, monoclonal or a fragment thereof.
  • the NA and HA in such a method can be derived from at least one IAV strain.
  • the enhancing nsRNA can encode at least one of an Ml EMP and an M2 EMP.
  • the enhancing mutation can encode at least one animo acid modification selected from the group consisting of a substitution, a deletion and an insertion.
  • the enhanced IAV can be selected from any known or discovered IAV strain.
  • the host may be selected from a prokaryotic and a eukaryotic cell.
  • the selection marker can be selected from a group consisting of a drug resistance marker, a temperature sensitive marker and an antigenic marker.
  • a method for obtaining an enhanced IAV of the present invention may further comprise removing the resistance to the selection marker from the enhanced LAV to provide a sensitive enhanced IAV lacking resistance to the selection marker.
  • Fig. 1 Amino acid sequence of Ml consensus sequence (SEQ ID NO:l).
  • Fig. 2 Amino acid sequence of M2 consensus sequence (SEQ ID NO:2).
  • Fig. 3 Amino acid and nucleotide sequences of M2 mutants (SEQ ID NOS.4-11). The C-terminal portion of the M2 protein is shown. Mutated nucleotides to introduce a stop codon are underlined. Plus (+) and minus (-) signs signify that viruses with the indicated M2 protein were not generated. Fig. 4. Nucleotide sequence of an M gene of one strain of IAV (SEQ ID NOS.4-11). The C-terminal portion of the M2 protein is shown. Mutated nucleotides to introduce a stop codon are underlined. Plus (+) and minus (-) signs signify that viruses with the indicated M2 protein were not generated. Fig. 4. Nucleotide sequence of an M gene of one strain of IAV (SEQ ID NOS.4-11). The C-terminal portion of the M2 protein is shown. Mutated nucleotides to introduce a stop codon are underlined. Plus (+) and minus (-) signs signify that viruses with the
  • IAVs can be specifically mutated as mutant nucleic acids, which encode mutant matrix proteins (MMPs).
  • MMP can be either an attenuating matrix protein (AMP) or an enhancing matrix protein (EMP).
  • AMP attenuating matrix protein
  • EMP enhancing matrix protein
  • An AMP or EMP is sufficient in itself to either attenuate or enhance, respectively, the growth of an LAV containing the mutant nucleic acid, as a negative strand RNA
  • the present invention relates to mutant nucleic acid (e.g. , cDNA or nsRNA) as attenuating nucleic acid, encoding AMPs, or enhancing nucleic acid, encoding EMPs, and to compositions, vaccines, mutant LAV and methods of making and using thereof.
  • mutant nucleic acid e.g. , cDNA or nsRNA
  • the present invention also relates to attenuated IAVs comprising both (i) pathogenic nsRNA encoding at least one NA and HA and (ii) attenuating nsRNA encoding an AMP having at least one attenuating mutation, and to methods of making and using thereof.
  • the now discovered attenuated IAVs and methods provide a utility for attenuating newly found pathogenic strains of LAV, e.g. , as attenuated IAVs, to be used as live, attenuated vaccines.
  • an attenuated IAV of the present invention induces an immune response in an animal infected with the attenuated virus, but the infection is subclinical, such that the infection is suitable for vaccination purposes.
  • the present invention also relates to enhanced IAVs comprising enhancing nsRNA encoding at least one EMP having at least one enhancing mutation, and to methods of making and using thereof.
  • the present invention also provides high titers of IAV using specific matrix gene enhancing mutations, encoding EMPs, which mutations facilitate inactivated vaccine production by providing a high growth master strain, superior to those presently used.
  • the term "virulence” is intended to mean the capacity of a virus, compared to other closely related viruses, to produce disease in a host (Tyler et al, "Pathogenesis of Viral Infections” In Virology, Fields et al., (eds.) Raven Press, Ltd., New York (1990), pp. 191-
  • Attenuated IAVs of the present invention have reduced virulence to the extent that they produce subclinical infections while still eliciting an
  • High yield property The term "high yielding" as used in the literature can be ambiguous, referring to either high HA or high infectivity titers assayed in either eggs or tissue culture.
  • "enhancing" viruses are defined as those capable of producing high infectivity titers in in vitro replication systems (e.g. , tissue culture).
  • a non- limiting example of such a culture is one using mammalian cells, e.g. , Madin- Darby canine kidney (MDCK) cells.
  • MDCK Madin- Darby canine kidney
  • Different influenza viruses show different infectivity titers. For example, the WSN and A/Puerto Rico/8/34
  • H1N1(PR8) strains both of which are lethal to mice, produce high infectivity titers (approximately 10 9 plaque-forming units (PFUs)/ml) in MDCK cells in contrast to ordinary viruses (e.g. , A/Aichi/2/68 (H3N2)(Aichi); approximately 10 7 PFUs/ml).
  • ordinary viruses e.g. , A/Aichi/2/68 (H3N2)(Aichi); approximately 10 7 PFUs/ml.
  • Influenza virus particles are known to be pleomorphic (Hoyle, L., "Morphology and Physical Structure” In The Influenza Viruses, Springer- Verlag, New York (1968), pp. 49-68); however, clinical isolates of early passages in eggs or tissue cultures contain more filamentous than spherical particles, whereas laboratory strains passaged extensively in such cultures contain spherical virions predominantly (Hoyle, L., "Morphology and Physical Structure” In The Influenza Viruses, Springer- Verlag, New York (1968), pp. 49-68).
  • the filamentous virions possess many of the serologic, hemagglutinating and enzymatic characteristics of the spherical particles.
  • the M gene is now discovered to be a major determinant of this morphologic difference.
  • the morphologic change and general reduction of virulence in many viruses during passage in vitro suggest an association of the shape of influenza virus with virulence.
  • viruses produce higher infectivity titers but become less virulent after repeated passaging in vitro, whereas the high-yield phenotype correlates with increased virulence (Smeenk et al, J. Virol. 65:530-534 (1994)) and a filamentous virion shape with high infectivity titers. This seeming contradiction originates from the polygenic nature of viral replicative efficiency. During adaptation to in vitro passage, multiple viral genes mutate, affecting the growth of the virus in ways that obscure the direct relationship between high infectivity titers and virulence.
  • mutations in the M gene as at least one nsRNA encoding an Ml or an M2 matrix protein (MP), provide attenuation or enhanced growth of influenza A virus, such that live IAV reassortant and/or reverse genetics produced viruses and vaccines, having attenuation or growth enhancement can be provided according to the present invention.
  • Ml or an M2 matrix protein MP
  • MMP Matrix .Protein
  • AMP Attenuating Matrix Protein
  • EMP Enhanced Matrix Protein
  • An MMP as an AMP or EMP, according to the present invention, can refer to any AMP or EMP, or a subset thereof, which is capable of replacing a matrix protein (MP), or nsRNA encoding therefor, in an IAV to provide an attenuated IAV (using an AMP or encoding nsRNA) or an enhanced IAV
  • AMP or EMP has at least one amino acid substitution, deletion or insertion of a wild type MP form or variant which provides attenuating (AMP) or enhancing (EMP) activity.
  • MMPs, as AMPs or EMPs are incorporated into a virus by use of mutant nucleic acid encoding therefore, e.g. , nsRNA.
  • nsRNA can be provided using genetic engineering to manipulate the corresponding cDNA.
  • the cDNA can thus be mutated to provide mutant cDNA-encoding attenuating or enhancing mutations, as part of an attenuating or enhancing nucleic acid.
  • any subset of an AMP or an EMP, e.g. , as a peptide fragment of an AMP, can be prepared by recombinant DNA methods discussed in more detail below, and/or by any other method capable of producing an attenuating or enhancing nucleic acid encoding, respectively, an AMP or EMP having a conformation similar to an attenuating (AMP) or enhancing (EMP) portion of an MP and having attenuating or enhancing activity, according to suitable screening assays, e.g. , as described herein and/or as known in the art.
  • an MMP of the present invention alternatively includes polypeptides having attenuating or enhancing activity as comprising a portion of an MP amino acid sequence which substantially corresponds to at least one 10-252 or 43 to 252 amino acid fragment and/or consensus sequence of a known wild type MP variant or group of MP variants.
  • an MMP can have homology of at least 80% to a known MP, such as 80-99% homology, or any range or value therein, while providing attenuating or enhancing activity.
  • An AMP or EMP, or encoding nucleic acid, of the present invention is not naturally occurring or is naturally occurring but is in a purified or in an isolated form which does not occur in nature.
  • an MMP of me present invention substantially corresponds to a MP domain of an MP or a group of MPs, as a consensus sequence, such as a consensus sequence of 2 or more naturally occurring wild type variants of M2 or Ml proteins.
  • Percent homology may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol Biol 48:443 (1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482 (1981). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e. , nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • the AMP of the present invention is a mutant form of at least one Ml or M2 MP.
  • Table 1 presents alternative variants of Ml matrix protein, which can be used or mutated to provide AMPs or EMPs, or nucleic acid encoding therefor, according to present invention. See, e.g. , Ito et al., J. Virol. 65:5491-5498 (1991), which is entirely incorporated herein by reference to alternative MP sequences.
  • An MMP of the present invention also includes mutants of MP variants, wherein at least one amino acid residue in the polypeptide has been replaced, inserted or deleted by at least one different amino acid, which mutation either confers attenuation by the resulting AMP, or confers enhancement by the resulting EMP, on an IAV strain containing the AMP or
  • an amino acid or nucleic acid sequence of an MMP of the present invention is said to "substantially correspond" to an MP amino acid or nucleic acid sequence respectively, if the sequence of amino acids or nucleic acid in both molecules provides polypeptides having biological activity that is substantially similar, qualitatively or quantitatively, to the corresponding fragment of at least one MP domain, but where the MMP sequence also has attenuating or enhancing activity.
  • Such "substantially corresponding" MMP sequences include conservative amino acid or nucleotide substitutions, or degenerate nucleotide codon substitutions wherein individual amino acid or nucleotide substitutions are well known in the art.
  • MMPs of the present invention include a finite set of substantially corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.
  • substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein.
  • For a detailed description of protein chemistry and structure see Schulz, G.E. et al., Principles of Protein Structure, Springer- Verlag, New York, 1978, and Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference.
  • substitutions of an AMP or EMP of the present invention include, alternatively or in addition to attenuating or enhancing mutations, substitutions of at least one amino acid residue which has been replaced, inserted or deleted by at least one different amino acid.
  • substitutions preferably are made in accordance with the following list as presented in Table 3, which substitutions can be determined by routine experimentation to provide modified structural and functional properties of a synthesized polypeptide molecule, while maintaining MP, attenuating and/or enhancing biological activity, as determined by suitable activity assays.
  • MMP, AMP, MMP or “substantially corresponding to” includes such substitutions.
  • alternative substitutions can be made by routine experimentation, to provide alternative MMPs of the present invention, e.g. , by making one or more conservative substitutions of MP fragments which provide MP activity.
  • substitutions of MMPs of the present invention are those in which at least one amino acid residue in the protein molecule has been removed and a different residue inserted in its place according to the following Table 4.
  • the types of substitutions which can be made in the protein or peptide molecule of the present invention can be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al , infra. Based on such an analysis, alternative conservative substitutions are defined herein as exchanges within one of the following five groups: Table 4
  • Conservative amino acid substitutions included in the term “substantially corresponding” or “corresponding”, according to the present invention, e.g. , as presented herein, are well known in the art and would be expected to maintain biological and structural properties of the polypeptide after amino acid substitution. Most deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule. "Characteristics" is defined in a non-inclusive manner to define both changes in secondary structure, e.g. ⁇ -helix or /3-sheet, as well as changes in physiological activity, e.g. in receptor binding assays.
  • Amino acid sequence insertions as included in an MMP can also include amino and/or carboxyl-terminal fusions of from one residue to polypeptides of essentially unrestricted length, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions can range generally from about 1 to 10 residues, more preferably 1 to 5.
  • An example of a terminal insertion includes a fusion of a signal sequence, whether heterologous or homologous to the host cell, to an MMP to facilitate secretion from recombinant bacterial hosts.
  • One additional group of variants according to the present invention is that in which at least one amino acid residue in the peptide molecule, and preferably, only one, has been removed and a different residue inserted in its place.
  • Most deletions, insertions and substitutions of MMPs according to the present invention are those which maintain or improve the attenuating or growth enhancing characteristics of the MMP.
  • substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.
  • an MMP made by site-specific mutagenesis of an MP-encoding nucleic acid and expression of an MMP in cell culture or, alternatively, by chemical synthesis can be tested for attenuating or enhancing activity (e.g. , as is known or as described herein).
  • the activity of the cell lysate or purified MMP can be screened in a suitable screening assay for the desired characteristic, e.g. , attenuating or enhancing activity in any of the several assays.
  • Modifications of protein or peptide properties can also be assayed by methods well known to the ordinarily skilled artisan.
  • salts of the MMPs of the present invention As used herein, the term “salts" refers to both salts of carboxyl groups and to acid addition salts of amino groups of the protein or peptide molecule.
  • Amino acid sequence variations in an MMP of the present invention can be prepared by mutations in the DNA. Such MMPs include, for example, deletions, insertions or substitutions of nucleotides coding for different amino acid residues within the amino acid sequence.
  • deletion, insertion, and substitution can also be made to arrive at the final construct, provided that the final construct encodes an MMP having some attenuating or enhancing activity. Preferably improved attenuating or enhancing activity is found over that of the corresponding MP.
  • mutations that will be made in nucleic acid encoding an MMP must not place the sequence out of reading frame and preferably will not create complementary domains that could produce secondary mRNA structures (see, e.g. , EP Patent Application
  • Mutant nucleic acid as attenuating or enhancing nucleic acid of the present invention, can be prepared by site-directed mutagenesis of nucleotides in the DNA or nsRNA encoding an MP, thereby producing mutant nucleic acid encoding an AMP or EMP, and thereafter reverse transcribing the EMP or AMP encoding DNA to produce nsRNA.
  • MMPs typically exhibit the same qualitative biological activity as the naturally occurring MP (see, e.g. , Ausubel, infra; Sambrook, infra), except for the additional attenuating or enhancing activity produced by the at least one mutation. Knowledge of the three-dimensional structures of proteins is crucial in understanding how they function.
  • the three-dimensional structures of hundreds of proteins are currently available in the protein structure databases (in contrast to several hundred thousand known protein and peptide sequences in sequence databases, e.g. , Genbank, Chemical Abstracts REGISTRY, etc.). Analysis of these structures shows that they fall into recognizable classes or motifs. It is possible to model the three-dimensional structure of protein based on homology to a related protein of known structure. Examples are known where two proteins that have relatively low sequence homology, but are found to have almost identical three dimensional structure. Such homologous variants of MPs or MMPs are also included in AMPs or EMPs of the present invention.
  • Attenuating or enhancing nucleic acid can be recombinantly or synthetically produced, or optionally purified, to provide commercially useful amounts of attenuating or enhancing nucleic acid for use in diagnostic or research applications, according to known method steps (see, e.g.,
  • any known and/or suitable screening assay can be used, as is known in the art.
  • virus replication can be used to screen both attenuation or enhancement.
  • Other activities suitable, alone or in any combination, for screening include, but are not limited to, quantitative and/or qualitative measurement of transcription, replication, translation, virion incorporation, virulence, viral yield, and/or morphogenesis, using such methods as reverse genetics, reassortment, complementation, and infection. See, e.g., The Influenza Viruses, R.M. Krug (ed.), Plenum Press, New York, (1989).
  • Attenuated IAVs of the present invention comprise at least one pathogenic nsRNA and at least one attenuated nsRNA.
  • the pathogenic nsRNA encodes at least one neuraminidase (NA) and at least one hemagglutinin (HA) from at least one pathogenic IAV strain.
  • NA neuraminidase
  • HA hemagglutinin
  • the inclusion of at least one NA and HA encoding negative strand RNA provides a host immune response specificity of the resulting attenuated virus for the at least one pathogenic IAV strain from which the NA an HA encoding RNA are derived.
  • At least one attenuating nsRNA which codes for an AMP having at least one mutation which attenuates the resulting IAV, provides a attenuated IAV having utility as a vaccine against at least one pathogenic IAV strain.
  • one or more sources of NA or HA encoding nsRNA, or cDNA encoding therefore, can be used to provide attenuated IAVs which can be used to vaccinate against one or more pathogenic strains of LAV.
  • At least one, such as 1-20 NA and/or HA encoding nsRNA can be included such that the attenuated live virus can be used as a vaccine against at least one or more pathogenic IAV strains, such as 1-2, 2-4, 5-8, 9-20 or 1-20 strains.
  • An attenuated LAV according to the present invention is attenuated to the degree such that, while the attenuated virus is capable of inducing a pathogenic IAV strain specific immune response in an animal, the immune response involves a subclinical IAV infection in the animal.
  • Such an attenuated virus or vaccine composition of the present invention can be used for prophylactic or therapeutic treatment, as described herein.
  • An attenuated IAV of the present invention can contain an attenuating nsRNA encoding for an Ml AMP.
  • the attenuated virus contains an attenuating nsRNA encoding an attenuating Ml matrix protein
  • the attenuating mutation comprise at least one mutation in an Ml domain selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a lipid binding domain, a zinc finger domain, a phosphorylation site, a morphology domain, a transcription inhibiting domain and an RNA binding domain, which domains are known in the art.
  • Such domains include 1-252 amino acids, or any range or value therein, corresponding to positions 1-252 of an Ml amino acid sequence.
  • An attenuated or enhanced virus according to the present invention can contain at least one mutation in at least one matrix protein wherein the mutation encodes at least one amino acid modification selected from the group consisting of a substitution, a deletion or an insertion.
  • the attenuating or enhancing mutation causes at least part of the attenuation or enhancement of the virus, and the amino acid modification affects the functioning of the virus in an animal host, such that an IAV, as either inhibiting an IAV with an attenuating mutation, or stimulating the growth of an IAV with an enhancing mutation.
  • the attenuated IAV induce a suitable immune response in the infected animal, which immunizes the animal while only producing a subclinical influenza A virus infection in the animal.
  • the growth of the virus is enhanced in a statistically significant manner, over an LAV not having the enhancing nucleic acid and/or EMP.
  • an Ml or M2 AMP having one or more mutations in an Ml or M2 matrix protein can provide attenuating or enhancing activity on an influenza A virus.
  • the mutation can be in any coding or non-coding region which is sufficient to encode an effect in the transcription, replication, translation or virion incorporation of a mutated IAV of the present invention, when the LAV infects a host cell, or other mutation in the matrix amino acid sequence which confers the attenuating or enhancing activity of a IAV containing the attenuating or enhancing nucleic acid.
  • an Ml attenuating mutation can comprise at least one mutation in an Ml MP domain selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a zinc finger domain, a phosphorylation domain, a transcription inhibiting domain, a morphology changing domain and an RNA binding domain.
  • an Ml attenuating mutation can comprise at least one mutation in an M2 MP domain selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a domain having at least one cysteine involved in oligomerization, a phosphorylation domain, a transmembrane domain, and a C-terminus domain.
  • an Ml enhancing mutation can comprise at least one mutation in an Ml MP domain selected from the group consisting of a 5 ' non-coding domain, a 3 ' non-coding domain, a high yielding domain, a phosphorylation domain, a transcription stimulating domain, a morphology changing domain and an RNA binding domain.
  • an M2 enhancing mutation can comprise at least one mutation in an M2 MP domain selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a morphology domain, a phosphorylation domain, and a transcription stimulating domain.
  • AMPs generated according to the present invention can contain at least one change from Ser or Thr to Ala, Gly or Pro. Phosphorylation of these mutant Ml proteins can be performed as known or described in Gregoriades et al, J. Virol - ⁇ 9:229-235 (1984) and Gregoriades et al, Virus Res. 16:27-42 (1990). Substitution mutations of Ml where at least one Ser and Thr replaced with Ala, Gly or Pro does not contain phosphorylated residues, showing which residues are phosphorylated. Replacement of phosphorylated residues with other amino acids, preferably Ala, can generate AMPs according to the present invention.
  • a non-limiting example of a group of attenuating mutations which can provide an Ml AMP of the present invention can include at least one substitution of a Ser or Thr in the phosphorylation domain of amino acids 94-129 of a IAV Ml MP with an animo acid selected from the group consisting of Ala, Gly or Pro, with Ala preferred.
  • Further non-limiting examples of such substitutions include at least one of Alal l ⁇ , Alall ⁇ , Alal20 and Ala 128, corresponding to substitutions of serine residues (or AlalO ⁇ or Alal26, corresponding to substitutions at Thr residues, or substitutions are made for one or more negative variance or consensus sequence sera.
  • EMPs are generated according to the present invention that contain at least one change from Ala, Gly or Pro to Ser or Thr. Phosphorylation of these mutant Ml proteins is performed. Replacement of Ala, Gly or Pro with phosphorylated residues, such as Ser or Thr, can generate EMPs according to the present invention.
  • Such phosphorylation domains can comprise at least two amino acids corresponding to positions 94-129 of said naturally occurring Ml MP.
  • An enhancing nucleic acid can include a substitution of a Ser or Thr for at least one selected from the group consisting of Alall6, Glyll ⁇ , Alall ⁇ , Gly 118, Alal20, Glyl20, Alal2 ⁇ or Glyl2 ⁇ .
  • AMPs generated according to the present invention can contain at least one change from Ser or Thr to Ala, Gly or Pro. Phosphorylation of these mutant M2 proteins can be -34-
  • a non-limiting example of a group of attenuating mutations which can provide an M2 AMP of the present invention can include at least one substitution of a Ser or Thr in the phosphorylation domain of amino acids
  • EMPs can be generated according to the present invention that contain at least one change from Ser or Thr to Ala, Gly or Ser. Replacement of Ser or Thr with phosphorylated residues with other amino acids, such as Ala, Gly or Pro, can generate EMPs according to the present invention.
  • a non-limiting example of such enhancing mutations can include mutations of at least one amino acid corresponding to positions 64-65 of an M2 MP.
  • An enhancing nucleic acid can include, e.g. , a substitution of at least one of Ser or Thr for at least one selected from the group consisting of
  • Attenuated Ml Zinc Finger Mutations which can provide Ml AMPs of the present invention include zinc-finger substitutions of a least one His or Cys residue encompassing positions corresponding to one or more of 148 to 162 of a naturally occurring Ml matrix protein.
  • Such attenuating substitutions can include, but are not limited to, substituting a Cys by an amino acid selected from the group consisting Ser,
  • substitutions include at least one of Serl4 ⁇ , Serl51, Tyrl59 and Tyrl62, e.g. , for at least one of Cysl4 ⁇ , Cysl51, Hisl59 and
  • Enhanced Ml Zinc Finger Mutations include zinc-finger substitutions of a least one His or Cys residue encompassing positions corresponding to one or more of 148 to 162 of a naturally occurring Ml matrix protein.
  • Such enhancing substitutions can include, but are not limited to, substituting a Cys for an amino acid selected from the group consisting Ser, Gly and Ala with Ser preferred; or substitution of a His residue for an amino acid selected from the group consisting of Tyr, Trp and Phe, with Tyr preferred.
  • Further non-limiting examples of such substitutions, e.g. , of a zinc-finger domain, of an Ml MP include at least one of Alal49, Alal55, Serl57, Serl61, Serl4S, Serl51, Tyrl59 and Tyrl62, e.g. , by at least one of Cys or His.
  • One or more of such zinc-finger substitutions can enhance an Ml matrix protein to provide an EMP according to the present invention.
  • Zinc blot assays (Schiff et al, Proc. Natl. Acad. Sci. USA 55:4195- 4199 (1988)) are used according to the present invention to confirm that the mutant Ml proteins have lost the zinc-binding activity, as preliminary screening before determination of attenuation or enhancement.
  • This assay involves fractionation of the viral proteins by SDS-PAGE, transfer of the proteins to nitrocellulose, and blotting with "Zn.
  • Ml Attenuating Morphology Domain Mutations include, but are not limited to, morphology changing domain substitution of at least one of Asn207, Ser207, His222 and
  • an attenuating mutation can comprise at least one substitution of at least one of Asn207 and His222 by at least one of Gln207, Ser207 or His207 and one of His222, Asn222 and Gln222.
  • an Ml protein enhancing substitutions which can provide an Ml EMP of the present invention include, but are not limited to, morphology changing domain substitutions of at least one of an Ser207, His222 and/or Pro222 for a corresponding amino acid selected from the group consisting of Asn207, Gly207, Arg222 and His222.
  • Non-limiting examples of an M2 protein attenuating substitution which can provide an M2 AMP of the present invention include, but are not limited to, morphology changing domain substitution of at least one of Asn82 by Ser82.
  • an attenuating mutation can comprise at least one substitution of at least one of Asn207 and His222 by at least one of Gln ⁇ 2 by Gln ⁇ 2 or Ser ⁇ 2.
  • an Ml protein enhancing substitutions which can provide an Ml EMP of the present invention include, but are not limited to, morphology changing domain substitutions of at least one of an Asn207 and/or His222 for a corresponding amino acid selected from at least one of the group consisting of Ser207, Gln207, Arg222 and Pro222.
  • an attenuating mutation can comprise at least one substimtion of at least one of Asn207 and His222 by at least one of Gln207 or His207 and one of Pro222,
  • an M2 protein enhancing substitutions which can provide an M2 EMP of the present invention include, but are not limited to, morphology changing domain substitution of an Asn for SerS2.
  • an attenuating mutation can comprise at least one substitution of Ser82 by Asn, Gin or Pro.
  • Attenuating substitutions to provide an Ml AMP include substitutions in a lipid bonding domain, as amino acids
  • Non-limiting examples of such substitutions include at least one of a Gly for Pro69 and a Pro for at least one of Glul41 and Thrl40.
  • Non-limiting examples of such substitutions include Gly for at least one of Pro69, Glnl41 and Thrl40.
  • a further example of a Ml domain which can be mutated to provide an Ml AMP of the present invention include substimtions in an RNA binding domain which can include, as a non-limiting example, amino acids 90-108 and/or 128-164 of a native Ml amino acid sequence.
  • Non-limiting examples of such substitutions include Ala for at least one of Arg95, Lys98, ArglOl, Lysl02, Lysl04, Argl05, Argl34, Argl60, Hisl62 and Argl63.
  • a domain having a cysteine involved in oligomerization of an M2 MP can be modified by an amino acid substimtion in an M2 MP sequence to provide an M2 AMP of the present invention.
  • Such domains include, as non-limiting examples, sequences corresponding to amino acids Cysl7, Cysl9 and Cys50 of an M2 amino sequence.
  • Such a attenuating mutations can include, but are not limited to, a replacement of Cys with a Ser, Gly or Ala, such a replacement of at least one of Cysl7, Cysl9 and Cys50 by an Ala, Gly or Ser, with Ser preferred.
  • Additional, M2 AMPs of the present invention can be provided by deleting 1-54 amino acids from the C-terminus.
  • Such deletions include, but are not limited to, a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 amino acid deletions.
  • Such deletions correspond to positions 44-97 of any M2 matrix protein.
  • an attenuated IAV of the present invention contains a mutation in either the Ml or M2, 3' or 5', non-coding domain, as an M-gene domain it is preferred that the mutation inhibit the level of at least one of transcription, replication, translation or virion incorporation of said attenuated
  • IAV in a host cell capable of being infected by said attenuated influenza A virus, such that the resulting virus is attenuated.
  • Attenuating or enhancing mutations in a 3' non-coding region or domain of an M-gene nsRNA can also provide an Ml or M2 AMP or EMP of the present invention.
  • Such modifications include, but are not limited to at least one 3' mutation selected from the group consisting of 1U by G, A or C; 2C by G, A or U; 3G by U, C or A; 4U by G, A or C; 5U by G, A or C; 6U by G, A or C; 7U by G, A or C; 8C by G, U or A; 9G by A, U or C; 10U by G, A or C; 11C by G, A or U; 12C by G, A or C; 13C by G, A or U; 14A by U, G or A and 15C by G, U or A; according to SEQ ID NO:3.
  • Preferred attenuating 3' non-coding region substimtion are at least one selected from the group consisting of 1A, 2G, 2A, 2U, 3A, 5G, 5C, 10A and 11G.
  • Preferred enhancing substimtions are at least one selected from the group 1G, 1C, 4G, 4A, 6G, 6C, 8A and 14U. . See e.g. , Piccone et al. , Virus Res. 25:99-112 (1993), which is entirely incorporated herein by reference. Additional mutations in the 5 ' non-coding regions or domains of an Ml or M2 nsRNA can also provide an Ml or M2 AMP or EMP of the present invention.
  • Such modifications include, but are not limited to 5' mutations at least one selected from the group consisting of 1015G by A, U or C; 1016G by A, U or C; 1017A by G, U or C; 1018A by G, C or U; 1019C by A, G or U; 1020A by G, U or C; 1021A by G, C or U; 1022A by G, C or U; 1023G by A, C or U; 1024A by G, C or U; 1025U by A, G or C; 1026G by
  • Preferred attenuating 5' non-coding region substimtions include, but are not limited to, at least one selected from the group consisting of 1017C, 1017U, 1017A, 1019U, 1020U, 1021U, 1023C, 1023U, 1023A, 1024C,
  • Preferred enhancing substimtions include, but are not limited to, at least one selected from the group consisting of 1015A, 1016C, 1018C, 1022C, 1022G, 1024G, 1025A, 1025G, 1026U and 1026A. See e.g. , Piccone et al , J. Virol 66:443 l-433 ⁇ (1992).
  • modifications in the sequence of the transmembrane domain corresponding to amino acids 25-43 of an M2 MP can include, but are not limited to, substimtion of a Leu, He or Val for at least one selected from Ala, Gin, Glu, Asn, Asp, Lys, Leu, Pre and Arg.
  • Preferred substimtions include, but are not limited to at least one selected from the group consisting of Leu26, Val27, Val28, Ile32, Ile33, Ile35, Leu36, Leu38, Leu40, Ile42 and Leu43.
  • Enhanced nsRNA, EMPs and Enhanced IAVs are examples of modifications in the sequence of the transmembrane domain corresponding to amino acids 25-43 of an M2 MP.
  • Enhanced IAVs of the present invention comprise at least one enhancing nsRNA.
  • the at least one enhanced nsRNA which codes for an
  • EMP having at least one mutation which attenuates the resulting virus can provide an enhanced LAV having utility, e.g. , as a master strain for culturing
  • An enhanced LAV of the present invention can contain an enhanced nsRNA encoding for an EMP selected from an Ml EMP or an M2 EMP.
  • the enhanced virus contains an enhancing nsRNA encoding an Ml EMP
  • the enhancing mutation comprises at least one mutation in a domain an Ml matrix protein or encoding nsRNA selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a zinc finger, a lipid binding domain, a phosphorylation site, a transcription inhibiting domain, a morphology domain, and an RNA binding domain, which domains are known in the art.
  • Such domains include 1-100 amino acids of
  • an enhancing nsRNA of the present invention encodes an M2 EMP
  • the enhancing mutation is preferably selected from at least one mutation in an M2 domain selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a domain containing at least one cystine residue involved in oligomerization; a transmembrane domain and a phosphorylation domain.
  • Such domains can include 1-60 amino acids corresponding to positions 1-97 of an M2 amino acid sequence.
  • a suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding (or which is complementary to a sequence encoding) an MP fragment of a matrix gene is identified as above, isolated or synthesized, and hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from cells having matrix genes and which are capable of expressing an MP.
  • Single stranded oligonucleotide probes complementary to an attenuating or enhancing activity encoding sequence can be synthesized using method steps (see, e.g. , Ausubel, infra;
  • Such a labeled, detectable probe can be used by known procedures or as a basis for synthesizing PCR probes for amplifying a cDNA generated from an isolated RNA encoding a target nucleic acid or amino acid sequence.
  • transformants can be selected by use of selection media appropriate to the vector or, virus or MP used, RNA analysis or by the use of antibodies specific for a target protein as an MP or MMP used in a method according to the present invention.
  • a target, detectably labeled probe of this sort can be oligonucleotide that is complementary to a polynucleotide encoding a target protein, as an
  • a synthetic oligonucleotide can be used as a target probe which is preferably at least about 10 nucleotides in length (such as 10, 15, 16, 17, l ⁇ , 19, 20, 21, 22, 23, 24, 25, 26, 27, 2 ⁇ , 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more, or any combination or range therein, in increments of 1 nucleotide), in order to be specific for a target a nucleic acid to be detected, amplified or expressed.
  • the probe can correspond to such lengths of a DNA or RNA encoding an MP, such as a sequence encoding a peptide corresponding to a portion of SEQ ID NO:l or SEQ ID NO:2, wherein the probe sequence is selected according to the host cell containing the DNA, e.g. , as presented in Table A1.4 of Ausubel, infra.
  • MMP encoding mutant nucleic acids of the present invention can include 30-756, such as 30-300, 40- 200, 30-100, 101-200, and 201-300 nucleotides, or any range or value therein, substantially complementary to a portion of nucleotides encoding SEQ ID NO:l or SEQ ID NO:2, or complementary to SEQ NO:3 or 4, wherein the codons can be substimted by codons encoding the same or conservatively substimted amino acids, as well known in the art.
  • Culturing of the host or rescue virus and introduction of complementary nsRNA into an IAV can be induced by methods known per se.
  • a nucleic acid sequence encoding an MMP of the present invention can be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed, e.g., by Ausubel, infra, and are well known in the art.
  • the cultured and/or amplified cDNA is then incorporated into IAVs, according to known techniques, e.g.
  • RNA containing exactly the same 5' and 3' sequences as viral RNA, from cloned influenza virus genes (e.g. , cDNA) with RNA polymerase, (ii) encapsulation of the RNA with influenza virus NP and polymerase proteins, (iii) transfection of the encapsidated RNA, and (iv) infection with a helper influenza virus to rescue the transfected RNA
  • cloned influenza virus genes e.g. , cDNA
  • Host cells comprising a nucleic acid which encodes an MMP of the present invention may be grown under conditions that provide expression of a desired MMP or mutant nsRNA in recoverable or commercially useful amounts. See, e.g., Ausubel, infra, at ⁇ 1 and 13; Palese, U.S. Patent No. 5,166,057, which are entirely incorporated herein by reference.
  • a mutant nucleic acid can also be recombinantly expressed in a host cell after the nucleic acid is incorporated into a plasmid or viral vector capable -44-
  • any of a wide variety of vectors can be employed for this purpose. See, e.g. , Ausubel, infra, ⁇ 1.5, 1.10, 7.1, 7.3, ⁇ .l, 9.6, 9.7, 13.4, 16.2, 16.6, and 16.8-16.11.
  • Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector can be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • a substimtion in a matrix protein to induce attenuation involves a replacement of an alanine for a charged amino acid in an attenuating matrix protein, which mutation results in a cold sensitive mutant.
  • the mutation in a matrix protein involves a substimtion of a conserved amino acid for a similar amino acid, as described herein.
  • the deletion is selected from the group consisting of 1-54 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, or 54, or any range or value therein, from the C-terminal or N-terminal ends of at least one of an Ml or M2 MP.
  • the insertion is selected from the group consisting of 1-54 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, or 54, or any range or value therein, from the C-terminal or
  • any influenza A virus strain can be used to obtain an attenuated influenza A virus which is capable of inducing an immune response in an animal, but which responds involves a subclinical infection.
  • the present invention also provides attenuated viruses wherein the mutant negative strand RNA and pathogenic negative strand RNA are derived from influenza A virus strains which have the same or similar host strains, such as a host strains including the same order genus or species.
  • the attenuated virus contains mutant negative strand
  • RNA and pathogenic negative strand RNA which are derived from influenza A viruses both capable of infecting human hosts or primate hosts.
  • An attenuated virus according to the present invention can further comprise a selection marker used to select for a reassortant or recombinant influenza A virus containing a mutant negative strand RNA according to the present invention.
  • selection marker can be selected from the group consisting of a drug resistance marker, a temperature sensitive marker, and an antigenic marker.
  • the selection marker is a drug resistance marker
  • the drug resistance can be used in any form which provides the ability to select an attenuated influenza A virus containing both the pathogenic negative strand RNA and the mutant negative strand RNA.
  • a drug resistance marker useful in the present invention is a drug selected from the group consisting of an amantadine-like compound, amantadine, rimantadine or other tricyclic symmetric amine.
  • an amantadine-like compound such a compound can be selected from the group consisting of amantadine, rimantidine, or other tricyclic symmetric amine analog.
  • the drug is amantadine.
  • rescued IAVs can be further selected using an MMP antibody, e.g.
  • a method for obtaining an attenuated influenza A virus which is capable of being used as a vaccine for at least one pathogenic influenza A virus strain.
  • the method can comprise isolating an attenuated influenza A virus of the present invention, as described herein.
  • the method may further comprise, prior to the isolating step, reassorting in a host a helper virus having said at least one mutant negative strand RNA and sensitivity to at least one selection marker, with at least one of said pathogenic negative strand RNA encoding (1) at least one NA and HA from at least one pathogenic influenza virus strain and selection RNA encoding at least one selection protecting conferring resistance to the selection marker.
  • helper virus containing the mutant negative strand RNA which helper virus also has sensitivity to a selection marker, the selected reassortant having resistance to the selection marker, are also discovered to be obtainable as the reassortant attenuated virus containing both the pathogenic negative strand RNA and the mutant negative strand RNA.
  • Such attenuated influenza A viruses are found to be useful as vaccines for inducing a suitable immune response against the at least one pathogenic influenza A virus strain from which the at least one NA and HA encoding pathogenic negative strand RNA is derived. Additionally, such attenuated viruses induce a suitable immune response while at the same time inducing a subclinical influenza A viral infection.
  • attenuated vaccines of the present invention are useful for vaccine compositions and methods for prophylactic and therapeutic vaccine treatment of animals, preferably primates and humans.
  • RNA can be derived from at least one pathogenic influenza A virus strain which is not limited to any particular strains. Currently, any known or discovered influenza A virus strain can be used as a source of the at least one NA and HA encoding pathogenic negative strand RNA. According for methods for generating attenuated viruses of the present invention, any host cell which is suitable for replicating and/or reassorting attenuated viruses of the present invention may be used. Such host cells may be selected from prokaryotic or eukaryotic cells. It is preferred that the eukaryotic cells be selected from the group consisting of a mammalian cell, an insect cell, a yeast cell and a bird cell, with mammalian cells preferred. Non-limiting examples of cell lines suitable for methods of the present invention, include MDCK, MDBK, VERO and CV-1 cells, readily available from commercial stores (e.g., ATCC, Rockville, MD).
  • the present invention also provides methods for generating reassortant viruses wherein the resistance to the selection marker is removed from the attenuated virus to provide an attenuated virus having sensitivity to the selection marker or attenuated virus which lacks resistance to the selection marker. For example, if amantadine resistance is used as a selection marker for obtaining reassortant attenuated viruses of the present invention, which are to be used as vaccines for human treatment, then it is preferred that such resistance to amantadine be removed.
  • Such removal can be used by either removing the selection marker by using culture systems which, while allowing rescue of mutant M gene, do not support replication of amantadine resistant viruses.
  • mutant viruses with changes in the M gene generated by reverse genetics exert host range alteration; that is, the mutants do not grow in certain culture systems (e.g. , MDBK cells or eggs) which support the replication of the parent virus.
  • the host range mutants can be used as helper viruses in such nonpermissive culture. Unlike the first strategy, this system allows the generation of viruses with temperature-sensitive attenuating mutations.
  • a temperature sensitive helper virus can be used as a selection system.
  • Helper viruses can be eliminated during the reassortant process by use of the appropriate temperature selection.
  • a non- limiting example of such a helper virus is tS51 virus containing a temperature sensitive mutation in the Ml protein.
  • the invention relates to the making and using of an anti-MP antibody (e.g., an M2 antibody) or an anti-MMP antibody (e.g. , an AMP or EMP antibody).
  • An AMP or EMP antibody binds specifically to an epitope of either an AMP or an EMP, respectively.
  • the AMP or EMP antibody binds an epitope that distinguishes an AMP or EMP from an non-attenuating or non-enhancing MP, respectively.
  • an AMP or EMP antibody binds an epitope within or resulting from the attenuating or enhancing amino acid mutation in the AMP or EMP, respectively.
  • An MP antibody binds to an epitope specific for an MP.
  • Anti-MP or anti-MMP antibodies are useful for the analysis, isolation or purification of: (a) MPs; (b) MMPs; or (c) MP- or MMP-containing IAVs.
  • An MP or MMP of the present invention can be used as an antigen to provide antibodies or hybridomas.
  • MP or MMP antibodies of the present invention include monoclonal and polyclonal antibodies, fragments, and single chain antibodies. Such antibodies are provided by known method steps, such as hybridoma or recombinant technology. Antibody fragments which contain the idiotype of an MP or MMP antibody molecule can be generated by known techniques. See, e.g., Kohler and Milstein, Nature 256:495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Assoc. and Wiley Interscience, N.Y., (19 ⁇ 7,
  • Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD, or any subclass thereof.
  • a monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites.
  • a hybridoma producing a MAb of the present invention can be cultivated in vitro, in situ or in vivo. Preferred methods of antibody production include hybridoma or recombinant techniques, which provide high titers of antibody.
  • an antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody.
  • Epitopes or "antigenic determinants” usually consist of surface groupings of molecules such as amino acids or sugar side chains and can have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • Antibodies directed against an MP or MMP can be used to detect an MP, MMP or an enhanced or attenuated IAV, using known techniques, based on the teaching and guidance presented herein.
  • the antibodies distinguish either: (a) human IAV proteins from non-human LAV proteins; or (b) MMPs from MPs.
  • these antibodies distinguish a host cell LAV MP from a helper cell IAV MP, when helper cells are used that express non-human LAV proteins.
  • Host cell-specific anti-LAV antibodies therefore select reassortants having proteins of the host cell-specific virus.
  • antibodies specific for a host cell MP can be used to select attenuated or enhanced IAVs from other IAVs having MPs derived from the helper virus.
  • antibodies which distinguish an MMP from an MP can be used to select an attenuated or enhanced IAV of the present invention.
  • the antibodies bind an epitope found in an MMP (e. g. , in an AMP or EMP), but not found in an MP.
  • Such antibodies bind an MMP in an attenuated or enhanced LAV to select rescued assortants (alternatively or in addition to amantidine selection).
  • One screening method of the invention is an immunoassay employing an enzyme immunoassay (El A).
  • El A enzyme immunoassay
  • These assays are based on the use of specific antibodies (monoclonal or polyclonal) to an MP or an MMP, including those present in an LAV.
  • samples containing attenuated or enhanced IAVs can be used in addition to those containing an MP or an MMP.
  • An MP- or MMP-specific antibody can be detectably labeled by linking it to an enzyme for use in an EIA. This enzyme, when later exposed to an appropriate substrate, will produce a product detectable by spectrophotometric, fluorometric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuc lease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Calorimetric methods also can be used which employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection can also be accomplished using any of a variety of other immunoassays.
  • immunoassays for example, by radioactively labeling the antibodies, it is possible to detect or measure an MP, MMP, or attenuated or enhanced LAV, through the use of a radioi munoassay (RIA).
  • RIAs are described in Laboratory Techniques and Biochemistry in Molecular Biology, Work et al ,
  • the radioactive isotope can be detected by such means such as the use of a gamma coimter, a scintillation counter or by autoradiography. See, e.g., Harlow and Lane, infra, Colligan, infra.
  • binding activity of a given sample of anti-MP or anti-MMP antibody can be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • an anti-MP or anti-MMP antibody with a fluorescent compound.
  • fluorescent labeled antibody When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can be then be detected by its fluorescence.
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 1S2 EU or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as ethylene-di-amine tetraacetic acid (EDTA).
  • EDTA ethylene-di-amine tetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescent compound can be used to label the antibody of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting luminescence qualitatively or quantitatively.
  • Non-limiting examples of bioluminescent compounds for purposes of labeling include luciferin, luciferase and aequorin.
  • An antibody molecule of the present invention can be adapted for utilization in a immunometric assay, also known as a "two-site", “forward” or “sandwich” assay.
  • a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid- phase antibody, antigen, and labeled antibody. See, e.g., Harlow and Lane, infra, Colligan, infra.
  • An antigenic MP or MMP peptide can be modified or administered with an adjuvant in order to increase the peptide antigenicity.
  • Methods of increasing the antigenicity of a peptide are well-known in the art. Such procedures include coupling the antigen with a heterologous protein (such as globulin or ⁇ -galactosidase) or through the inclusion of an adjuvant during immunization.
  • a heterologous protein such as globulin or ⁇ -galactosidase
  • the specific concentrations of detectably labeled antibody, MP, MMP, or IAV, the temperature and time of incubation, as well as other assay conditions, can be varied depending on various factors including the concentration of an MP or MMP in the sample, the nature of the sample, and the like.
  • the binding activity of a given lot of anti-MP or anti-MMP antibody can be determined according to well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • the assay of the present invention is also ideally suited for the preparation of a kit.
  • kit can comprise a carrier being compartmentalized to receive in close confinement therewith one or more containers, such as vials, tubes and the like, each container comprising the separate elements of the immunoassay.
  • containers such as vials, tubes and the like
  • each container comprising the separate elements of the immunoassay.
  • containers such as vials, tubes and the like
  • each container comprising the separate elements of the immunoassay.
  • Further containers can contain standard solutions comprising serial dilutions of the MP or MMP to be detected.
  • the standard solutions of an MP or MMP can be used to prepare a standard curve with the concentration of MP or MMP plotted on the abscissa and the detection signal on the ordinate.
  • results obtained from a sample containing an MP or an MMP can be interpolated from such a plot to give me concentration of the MP or the MMP. See, e.g. , Harlow and Lane, infra; Colligan, infra.
  • compositions of the present invention suitable for inoculation or for parenteral administration, include attenuated virus containing sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which can also contain auxiliary agents or excipients which are known in the art. See, e.g. , Berkow et al , eds., The Merck Manual, 15th edition, Merck and Co., Rah way, N.J., 1987; Goodman et al , eds.,
  • compositions may also include other immunomodulators, such as cytokines which accentuate an immune response to a viral infection.
  • Heterogeneity in the vaccine may be provided by mixing specific species for at least one influenza A virus strain.
  • the vaccine preparation may contain one or more specific attenuated viruses of the present invention, which viruses contain at least one NA or HA encoding RNA and at least one mutant matrix encoding RNA.
  • viruses contain at least one NA or HA encoding RNA and at least one mutant matrix encoding RNA.
  • vaccines can be provided for variations in a single strain of influenza A virus or for more than one strain of influenza A virus, using techniques known in the art.
  • a pharmaceutical composition according to the present invention may further or additionally comprise at least one viral chemotherapeutic compound selected from gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon- ⁇ , interferon-/3, interferon- ⁇ , thiosemicarbarzones, methisazone, rifampin, ribvirin, a pyrimidine analog, a purine analog, foscamet, phosphonoacetic acid, acyclovir, dideoxynucleosides, or ganciclovir. See, e.g. , Katzung, infra, and the references cited therein on pages 798-800 and 680-6 ⁇ l, respectively, which references are herein entirely incorporated by reference.
  • at least one viral chemotherapeutic compound selected from gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon- ⁇ , interferon-/3, interfer
  • the administration of the vaccine may be for either a "prophylactic” or "therapeutic" purpose.
  • the compound (s) are provided in advance of any symptom of influenza A viral infection.
  • the prophylactic administration of the compound(s) serves to prevent or attenuate any subsequent infection.
  • the attenuated viral vaccine is provided upon the detection of a symptom of actual infection.
  • the therapeutic administration of the compound(s) serves to attenuate any actual infection. See, e.g., Berker, infra, Goodman, infra, Avery, infra and Katzung, infra, which are entirely incorporated herein by reference, including all references cited therein.
  • An attenuated vaccine or vaccine composition of the present invention may, thus, be provided either prior to the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection.
  • a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient.
  • Such an agent is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant.
  • a vaccine or composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
  • the "protection” provided need not be absolute, i.e. , the disease need not be totally prevented or eradicated, provided that there is a statistically significant improvement relative to a control population. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of the disease.
  • the vaccine of the present invention when it is provided to an individual, it may be in a composition which may contain salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition.
  • Adjuvants are substances that can be used to specifically augment a specific immune response. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the animal being immunized. Adjuvants can be loosely divided into several groups based upon their composition.
  • These groups include oil adjuvants, mineral salts (for example, AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH 4 (SO 4 ), silica, kaolin, and carbon), polynucleotides (for example, poly IC and poly AU acids), and certain natural substances (for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, or Bordetella pertussis, and members of the genus Brucella.
  • substances particularly useful as adjuvants are the saponins such as, for example, Quil A. (Superfos A S, Denmark). Examples of materials suitable for use in vaccine compositions are provided in Remington 's Pharmaceutical Sciences (Osol, A, Ed, Mack Publishing Co, Easton, PA, pp. 1324-1341 (19 ⁇ 0), which reference is incorporated herein by reference).
  • a vaccine of the present invention may confer resistance to one or more strains of an IAV by either passive immunization or active immunization.
  • active immunization a live attenuated vaccine or composition is administered prophylactically, according to a method of the present invention.
  • passive immunization the vaccine is provided to a host (i.e. a human or mammal) volunteer, and the elicited antisera is recovered and directly provided to a recipient suspected of having an infection caused by an IAV strain.
  • the vaccine is provided to a female (at or prior to pregnancy or parturition), under conditions of time and amount sufficient to cause the production of antisera which serve to protect both the female and the fetus or newborn (via passive incorporation of the antibodies across the placenta).
  • the present invention thus concerns and provides a means for preventing or attenuating infection by an IAV strain, or by organisms which have antigens that can be recognized and bound by antisera to the polysaccharide and/or protein of the conjugated vaccine.
  • a vaccine is said to prevent or attenuate a disease if its administration to an individual results either in the total or partial attenuation (i.e. suppression) of a symptom or condition of the disease, or in the total or partial immunity of the individual to the disease.
  • At least one attenuated IAV of the present invention may be administered by any means that achieve the intended purpose, using a pharmaceutical composition as previously described.
  • administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes.
  • Parenteral administration can be by bolus injection or by gradual perfusion over time.
  • a preferred mode of using a pharmaceutical composition of the present invention is by subcutaneous, intramuscular or intravenous application. See, e.g. , Berker, infra, Goodman, infra, Aveiy, infra and Katzung, infra, which are entirely incorporated herein by reference, including all references cited therein.
  • a typical regimen for preventing, suppressing, or treating a disease or condition which can be alleviated by a cellular immune response by active specific cellular immunotherapy comprises administration of an effective amount of a vaccine composition as described above, administered as a single treatment, or repeated as enhancing or booster dosages, over a period up to and including between one week and about 24 months.
  • an "effective amount" of a vaccine composition is one which is sufficient to achieve a desired biological effect. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the ranges of effective doses provided below are not intended to limit the invention and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. See, e.g., Berkow et al , eds., The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992;
  • the dosage for a human adult will be from about lO O 7 plaque forming units (PFU)/kg or colony forming units (CFU)/kg.
  • PFU plaque forming units
  • CFU colony forming units
  • the dosage should be a safe and effective amount as determined by conventional methods, using existing vaccines as a starting point.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art.
  • non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and i ⁇ jectable organic esters such as ethyl oleate.
  • Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption.
  • Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form.
  • Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water.
  • inert diluents such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents. See, e.g. , Berker, infra, Goodman, infra, Avery, infra and Katzung, infra, which are entirely incorporated herein by reference, included all references cited therein.
  • the recipients of the vaccines of the present invention may be any vertebrate animal which can acquire specific immunity via a cellular or humoral immune response, where said response is mediated by an MHC class I (cellular response) or a class LI (humoral response) protein.
  • MHC proteins have been identified in mammals, birds, bony fish, frogs and toads.
  • the preferred recipients are mammals of the Orders Primata (including humans, apes and monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora (including cats, and dogs).
  • the preferred recipients are turkeys, chickens and other members of the same order.
  • the most preferred recipients are humans.
  • Influenza A/equin 63 H3N ⁇
  • HlNl A/Puerto Rico/ ⁇ /34
  • PR ⁇ A/duck/Oklahoma/4/77
  • Madin-Darby bovine kidney (MDBK) cell line was cultured in Eagle's minimal essential medium (MEM) containing 10% fetal calf serum.
  • Madin- Darby canine kidney (MDCK) cells were cultured in the same conditions as MDBK cells, except that 5% calf serum was used.
  • a plasmid (pPR ⁇ M-10) containing the PR ⁇ M gene was constructed as described by Huddleston and Brownlee (Huddleston, J.A. et al , Nucl. Acids Res. 70:1029-1037 (1982)).
  • a second plasmid (pUCT3PRM), containing the PR8 M gene flanked by the Ksp632I site and T3 RNA polymerase promoter sequence, was made by cloning the polymerase chain reaction (Saiki, R.K. et al. , Science 25P:4 ⁇ 7-491 (19 ⁇ )) product made with pPR ⁇ M-10 as a template and with primers 5 '- ATCGATGAATTCTCTTCGAGCGAAAGCAGGTAGATATTG-3' (SEQ ID NO: 12) and 5'-GAGGACAAGCTTATTAACCCTCACTAAAAG- TAGAAACAAC ⁇ GAGTTTTTTACT-3' (SEQ ID NO: 13).
  • pUCT3PRM contains a T3 RNA polymerase promoter upstream and a Ksp632I site downstream of the M gene, so that viral sense RNA transcripts are generated when digested with Ksp632I, filled-in with Klenow fragment, and transcribed with T3 RNA polymerase (Enami, M. et al. , Proc. Natl Acad. Sci. USA 57:3802-3805 (1990)).
  • pUCT3COOH- 1, pUCT3COOH-5, and pUCT3COOH-10 were constructed by replacing M gene nucleotides, which convert the M2 carboxyl-terminal Glu, amino acid residue 93, and 89 codon to a stop codon, respectively, using oligonucleotide- directed mutagenesis (Kunkel, T.A. et al. , Methods in Enzymology 154:367-
  • Nucleoprotein (NP) and polymerase (P) proteins were purified from A/duck/Oklahoma/4/77 by glycerol and glycerol-cesium chloride (CsCl) gradients as previously described (Parvin, J.D. et al , J. Virol. 65:5142-5152 (19 ⁇ 9)).
  • RNP M ribonucleoprotein
  • M RNP complex was then transfected into 70-90% confluent MDBK cells infected 1 hr before transfection with Eq/MIA at a multiplicity of infection of 1.
  • Alternative Selection 1 Amantidine selection was carried out as follows. Eighteen hours after transfection, MDCK cells were infected with transfectants in supernatant fluid in the presence of amantadine (l ⁇ /ml). Three days later, viruses in the supernatant were plaque-purified in MDCK cells three times in the presence of amantadine (l ⁇ /ml), and then inoculated into embryonated eggs.
  • Alternative Selection 2 Alternatively, amantidine and antibody selection of transfectant viruses (containing the M gene encoding the amantadine resistant M2 protein) was carried out as follows. Eighteen hours after transfection, MDCK cells were infected with transfectants in supernatant fluid in the presence of amantadine (1 ⁇ g/ml). Three days later, viruses were further identified with the use of a monoclonal antibody specific for human
  • IAV M2 IAV M2. This antibody reacts with the M2 protein encoded by the transfected M gene, but not with the M2 protein produced by the helper virus. Appropriate numbers of plaques were picked, according to the ratio of transfectant and helper viruses identified with the monoclonal antibody specific for the M2 protein. Pure populations of the transfectant viruses were obtained by plaque purifying three times in the presence of amantadine (1 ⁇ g/ml).
  • the M gene of the viruses was sequenced as disclosed (Katz, J.M. et al , J. Gen. Virol 73:1159-1165 (1992)), to confirm the origin of the gene and the intended mutations and to ensure no unwanted mutations as described.
  • HA titration and hemagglutination inhibition (HI) tests were performed with receptor-destroying enzyme (RDE)-treated antisera in microliter plates (Palmer, D.F. et al , Immunol Ser. 6:51-52 (1975)).
  • RDE receptor-destroying enzyme
  • ferrets were infected with the wild type virus (10 7 PFU) 3 weeks after initial infection, and virus titers in nasal wash samples were examined as described above.
  • amantadine resistance conferred by the M2 protein of PR ⁇ strain a naturally amantadine-resistant virus
  • PCR amplification and partial sequencing of the M gene of viruses derived from individual plaques grown in the presence of amantadine showed that approximately 50% of the plaques represented viruses with the PR ⁇ M gene (designated Eq/MLA-PR ⁇ M). All remaining plaques were considered amantadine-resistant mutants of the helper Eq/MIA virus, and were not examined further.
  • the carboxyl-terminal Glu in the M2 protein is not essential for viral replication.
  • M2 protein M gene product
  • the total number of M2 amino acid residues is identical among all influenza A viruses examined (Hay, A.J. et al , EMBO J. 4:3021-3024 (19 ⁇ 5)), suggesting that the entire M2 protein is important for normal functioning.
  • the COOH-1 mutant produced slightly smaller plaques on MDCK cells than did the transfectant virus with the PR ⁇ M gene (2-mm vs. 3-mm diameter on day 3), suggesting that the former was attenuated. When examined in ferrets, the COOH-1 mutant had a 10-fold lower titer than did the parental
  • a rescue system for the M gene of influenza A viruses whose efficiency is sufficient for generating transfectants that contain a mutation in the M2 or Ml protein.
  • the establishment of an M gene rescue system provides an elegant means to produce live, attenuated influenza vaccines.
  • a master strain used in vaccine production can contain attenuating mutations in genes for internal proteins in addition to HA and NA gene from a currently circulating strain.
  • the present invention provides such attenuating mutations, and screening methods for determining an optimal balance between attenuation and viral replication.
  • Amantadine resistance the selection pressure used to rescue the M gene from cloned cDNA, is not a desirable feature for vaccine strains, because amantadine and its derivative, rimantadine, are the only licensed antiviral drugs against influenza A.
  • an alternative system that would generate viruses with attenuating mutations in the M gene, without introducing amantadine resistance, is also provided by use of alternative selection systems, or by the removal of the amantadine resistance after selection of attenuated viruses of the present invention.
  • this objective is met by production of attenuated viruses with a specific M-gene-determined host range.
  • viruses with mutations in the NA gene have been generated by reverse genetics, and their host range differs from that of the parent virus (Luo, G. et al , Virus Res. 29:141-153 (1993); Castrucci, M.R. et al , J. Virol 67:759-764 (1993)).
  • viruses have similarly different host ranges, useful for selection of attenuated or enhanced viruses are used as helper viruses in the generation of amantadine- sensitive, or other selection sensitive transfectant viruses.
  • Enhancing mutations are provided by the present invention, e.g. , by comparing the M genes of high-yield (WSN) and ordinary (Aichi) viruses
  • Mutations are introduced in the M gene of the PR ⁇ virus, a high-yielding strain (10 9 PFUs/ml in MDCK cells) for which we already have a reverse genetics system. Among 11 positions (41, 204, 205 and 21 ⁇ in the Ml and 2 ⁇ , 31, 54, 56, 57, ⁇ 9 and 93 in the M2) where the Aichi and WSN M gene products differ, only two (41 in the Ml (Table 5) and 31 in the M2 are common to enhancing WSN and PR ⁇ viruses. A change found at residue 31 of the M2 protein is responsible for amantadine resistance, which does not correlate with the enhancing phenotype.
  • the Ala-to-Val change at residue 41 of the Ml is also found in a mouse- adapted A/Port Chalmers/73 (H3N2) virus.
  • H3N2 mouse-adapted A/Port Chalmers/73
  • a change at residue 139 from Thr to Ala in the Ml has been identified in the mouse-adapted A FM/ 1/47 strain and shown to be responsible for enhancing characteristics (Smeenk et al, J. Virol 65:530-534 (1994)).
  • This change has also been detected in another mouse-adapted A/WS/33 strain, NWS.
  • NWS mouse-adapted A/WS/33 strain
  • enhanced viruses are generated containing at least one amino acid change at residue 41 from Val to Ala (41A), at residue 139 from Thr to Ala (139A), or double mutations at residues 41 from Val to Ala and 139 from Thr to Ala (41A139A) in the reverse genetics system described herein.
  • PR ⁇ Ml protein (Table 5).
  • a helper virus a reassortant is used containing only the M gene from an amantadine-sensitive A/equine/Miami/ 1/63 (H3N8) and all the other genes from PR ⁇ .
  • H3N8 amantadine-sensitive A/equine/Miami/ 1/63
  • the entire M gene is optionally sequenced before the rescue experiments are begun, to determine whether undesired or unspecified mutations are provided.
  • the entire M genes of transfectant viruses are also optionally sequenced to ensure that they contain only desired mutations. Mutations may also be introduced into other genes upon rescue of the mutant M, thus affecting the viral phenotype. At least two viruses are generated for each mutant.
  • PR ⁇ strain is mouse-adapted, the effects of mutations in the M gene are determined using this animal model.
  • Ferrets are also optionally used, which are routinely employed in virulence evaluations of human viruses.
  • virus titer in nasal washes for ferrets and trachea and lung for mice
  • MDCK cells three days after infection (highest virus titers were found on day 3 in previous experiments).
  • virus titers in trachea and lung are determined to examine the spread from upper respiratory tract to the lung. The virus titers in these organs of mice correlates with virulence
  • a dose required to kill 50% of mice (MLD*,) and to infect animals (IDso) is also determined.
  • a transfectant virus with the wild-type PR ⁇ M gene is the control.
  • the enhancing phenotype does in fact confer virulence. This results allows prediction of increased virulence on the basis of the amino acid sequence in the Ml or M2 EMP.
  • One or more amino acid changes correlate with virulence. Changes resulting in the enhancing phenotype in vitro but not virulence are useful to introduce into live vaccine strains for culturing. If neither substimtion correlates with virulence, replication in vitro may not relate to virulence, although the likelihood of this result is remote according, to recent findings (Smeenk et al, J. Virol. 65:530-534 (1994)).
  • Ml protein is involved in the uncoating and transport of RNP from the nucleus to cytoplasm. It is also expected to alternatively or additionally contribute to budding. To determine if enhancing viruses have a short replication cycle (thus leading to high titers in a given period of time) or produce more virus particles in a single replication cycle than do low-yield viruses, one-step growth characteristics are tested using the transfectant viruses described herein. The differences in Ml functions -6 ⁇ -
  • influenza A virus In the current view of uncoating, upon endocytosis, the core of influenza A virus is exposed to low pH by the function of the M2 ion channel.
  • NP gene Enami et al, Virology 194:822-827 (1993)).
  • Kinetics of the NP and Ml transport of incoming virus particles has been quantitated by confocal microscopy (Martin et al, J. Virol 65:232-244 (1991)) after synchronizing infection by letting virus bind to cells at 4°C and penetrate at 37°C.
  • Quantitative confocal microscopy is optionally preformed using a suitable imaging system, as is known in the art. MAbs to these proteins and the NP gene for the probe preparation are readily available from research or commercial sources.
  • the enhancing virus' NP and NP gene appear in the nucleus after infection more rapidly than those of the low-yield virus, it is expected that the uncoating step is responsible for the difference in growth rate and that the enhancing virus' Ml dissociates faster from RNP.
  • the molecules in RNP to which the Ml binds RNA, NP, polymerase proteins or NS2
  • RNA binding region Ye et al, J. Virol. 63:3586-3594 (1989)
  • the Ml of enhancing viruses may dissociate more rapidly than the Ml of low-yield viruses because of an altered Ml -RNA association.
  • the molecular basis for this distinction is optionally determined. Because the Ml dissociates from the RNP upon exposure to low pH (Zhirnov, O.P., Virology 176:274-279 (1990)), a difference in pH threshold may influence the speed of uncoating. To determine the pH threshold of Ml dissociation from RNP, an assay is employed as method steps used by Zirnov (Zhirnov, O.P., Virology 176:274- 279 (1990)).
  • Virus is ultracentrifuged through 15% glycerol in water/25% glycerol in Tris-Mes (morpholinoehanesulfonic acid) buffer, containing 1% Nonidet P-40, protease inhibitors, and 150 mM NaCl, adjusted to a pH ranging from 5 to 8. Viral envelope is disrupted under this condition and the core RNP is then precipitated. Since the dissociation of the Ml from RNP depends upon pH values of buffer, determination of the pH threshold for Ml dissociation is provided by comparing the ratio of Ml to NP in the pellet.
  • Tris-Mes morpholinoehanesulfonic acid
  • RNA binding region Ye et al, J. Virol. 63:3566-3594 (19 ⁇ 9).
  • the mode of Ml binding to vRNA could differ between the high- and low-yield viruses.
  • the differences are expected to lie in Ml binding to other viral proteins in RNP, such as the NP.
  • Other expected mechanisms include, but are not limited to interaction of the Ml -RNP complex with cellular proteins for its transport. Budding
  • Ml is now expected to associate with other viral proteins (i.e. , HA, NA, or M2), in ways that could be essential for budding. It is therefore expected that a difference in budding efficiency results in the enhancing phenotype by examining mutation of an Ml MP to form an Ml EMP of the present invention as an Ml or Ml -RNP complex with cellular membranes.
  • Ml protein from the cDNA is expressed in MDCK cells in the presence of ( 35 S)methionine, using either the vaccinia or Semliki forest virus (SFV) (Liljestrom et al, Biotechnology 9: 1356-1361 (1991)) expression system, both of which are currently in use in my laboratory (P3,P29).
  • the vaccinia system expresses sufficient wild-type Ml proteins to correct the RNP-escorting defect of the ts51 Ml at a nonpermissive temperature (Dr. J. Ye, personal communication). Equivalent or greater amounts of protein are expressed by the SFV system (Liljestrom et al, Biotechnology 9:1356-1361 (1991)).
  • the vaccinia system has proved useful in a variety of smdies, including virus assembly (Chong et al, J. Virol. 67:407-414 (1993); Chong et al., J. Virol. 65:441-447 (1994); Simpson et al, J. Virol 66:790- ⁇ 03 (1992)).
  • the SFV system is also used (only four nonstructural SFV proteins are made).
  • Cell membranes are fractionated according to published methods (Chong et al, J.
  • Virol 65:441-447 (1994) known to give good separation of membranes derived from ER, Golgi apparatus, and the plasma membrane.
  • the fractions containing these membranes are identified by measuring the sucrose density of individual fractions and the presence of the HA (expressed from cDNA) for the plasma membrane, Bip for the ER, or -mannosidase II for Golgi membranes.
  • marker proteins are identified by radioimmunoprecipitation (HA and Bip) or enzyme activity ( ⁇ -mannosidase II (Storrie et al, Methods Enzymol. 752:203-225 (1990))).
  • the distribution of the Ml proteins is then compared between the high- and low-yield viruses expressed from the cDNA among membrane fractions. Because the entire M gene encodes the M2 protein as well, a construct containing only the Ml coding region is used, so that the effect of the M2 protein is eliminated.
  • Experiments expressing other viral proteins are also performed (HA, NA, and M2) in addition to the Ml to examine their effect on the Ml association with membrane.
  • the cDNA clones are provided as described herein and the protein in each fraction will be identified by either radioimmunoprecipitation or Western blotting. MAbs to these proteins are also available from research or commercial sources.
  • the Ml-RNP complex is prepared, e.g. , in pH 7.2 buffer by ultracentrifugation of lysate of cells infected with either high- or low-yield virus labeled with ( 35 S)methionine as described (Deshpande et al, Virology 139:32-42 (19 ⁇ 4); Martin et al, J. Gen.
  • results with the membranes expressing other viral proteins show specific interactions of the Ml-RNP complex with these proteins.
  • the findings of a greater quantity of the Ml-RNP complex in association with the plasma membrane (with or without other viral proteins) of enhancing viruses would show that association of the complex is a major factor in development of the enhancing phenotype. If a difference only with the expression of other viral proteins is shown, then the responsible proteins are identified by expressing them individually.
  • Virion shape is expected to influence influenza pathogenesis
  • amino acid substimtions in the M gene products are provided according to the present invention by determining the morphology of influenza virions, testing the influence of virion shape on virulence, and then showing how the mutations influence virion morphology.
  • A/Korea/426/68 Ml contains Pro
  • the PR ⁇ Ml contains His at position 222 (P14).
  • Electron microscopic analysis showed that A/Korea/426/68 was filamentous but a transfectant virus containing the PR ⁇ M gene was spherical, demonstrating a strong correlation between the Pro-222 in the Ml and the filamentous phenotype.
  • transfectant viruses are generated, each containing a change from Asn-207 to Ser, His-222 to Pro in the Ml, or Ser- ⁇ 2 to Asn in the M2 of PR ⁇ strain by reverse genetics as described earlier.
  • the transfectant viruses evaluated under the electron microscope.
  • the filamentous A/Korea/426/68 containing Pro-222 in the Ml and a spherical transfectant containing the wild type PR ⁇ Ml serve as controls.
  • Amino acid changes associated with a shift in virion shape from spherical to filamentous are expected to show that a single amino acid change in the Ml determines the ultimate morphology of influenza virus. If the transfectant viruses are spherical, a virus is generated containing all three changes. If this triple mutant is filamentous, viruses are generated containing different combination of changes to pinpoint those with the major influence.
  • Changes in the shape of influenza A viruses are expected to affect infectivity, as shown by examining the ratio of PFUs per virion. Electron microscopy is used to count the number of virions as compared to an internal standard (latex beads). PFUs are determined with MDCK cells. If the filamentous virions are more infectious than the spherical ones on the basis of virion number, virion shape promotes the infectivity of influenza viruses in vitro.
  • the MLD JO , ID ⁇ and virus titers are examined in organs in mice and in nasal washes in ferrets as described earlier. If the filamentous viruses are more infectious in animals, evidence of increased virulence (i.e. , reduced
  • MLDso ID 50 and/or increased virus titers in organs and nasal wash
  • ID 50 and/or increased virus titers in organs and nasal wash
  • virion shape can be influenced by interactions of the Ml with itself or other viral proteins, differences are distinguished between filamentous and spherical viruses in interactions of the Ml protein or Ml-RNP complex with the plasma membrane, in the presence or absence of the HA, NA and/or
  • Ml protein is associated with the plasma membrane for filamentous than for spherical viruses, the degree of Ml association with the plasma membrane influences virus morphology. If the difference occurs only when the plasma membrane contains other proteins (HA, NA, or M2), this relationship depends on interaction of the Ml with other viral proteins. Using data obtained by methods described herein, proteins are identified which interact with the Ml and therefore have important roles in determining virion shape.
  • ADDRESSEE STERNE, KESSLER, GOLDSTEIN & FOX
  • Lys Cys lie Tyr Arg Arg Leu Lys Tyr Gly Leu Lys Arg Gly Pro Ser 50 55 60
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

Virus grippaux atténués ou activés (IAVs) obtenus à l'aide de protéines matrices mutées (MMPs) codant de l'ARN ou de l'ADN. Le remplacement de l'ARN à brin négatif (nsARN) d'une protéine mutée par nsARN codant des protéines matrices atténuantes (AMP) ou activantes (EMP) permet d'obtenir des IAVs atténués ou activés. Des procédés de fabrication et d'utilisation d'ADN ou d'ARN de AMP ou de EMP pour atténuer ou activer le IAV sont également décrits.
PCT/US1995/012357 1994-09-30 1995-10-02 Acide nucleique codant des proteines matrices mutees utiles pour l'attenuation ou l'activation du virus grippal a WO1996010631A1 (fr)

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US6579528B1 (en) * 1998-08-13 2003-06-17 The University Of Pittsburgh - Of The Commonwealth System Of Higher Education Cold-adapted equine influenza viruses
WO2008036146A2 (fr) 2006-07-14 2008-03-27 Sanofi Pasteur Biologics Co. Construction de vaccins antiviraux de recombinaison par insertion directe à médiation par transposon de déterminants immunologiques étrangers dans des protéines de virus vecteur
WO2008100290A2 (fr) 2006-09-29 2008-08-21 Sanofi Pasteur Biologics Co Vecteurs rhinoviraux recombinants
US20090074812A1 (en) * 2007-06-18 2009-03-19 Wisconsin Alumni Research Foundation Influenza M2 protein mutant viruses as live influenza attenuated vaccines
US20110076297A1 (en) * 2009-09-30 2011-03-31 Saint Louis University Peptides for Inducing Heterosubtypic Influenza T Cell Responses
US8128938B1 (en) 2004-05-18 2012-03-06 Vical Incorporated Influenza virus vaccine composition and methods of use
US9446116B2 (en) 2006-02-07 2016-09-20 Peptcell Limited Peptide sequences and compositions
US9890363B2 (en) 2015-07-06 2018-02-13 Wisconsin Alumni Research Foundation (Warf) Influenza virus replication for vaccine development
US9950057B2 (en) 2013-07-15 2018-04-24 Wisconsin Alumni Research Foundation (Warf) High titer recombinant influenza viruses with enhanced replication in MDCK or vero cells or eggs
US10130697B2 (en) 2010-03-23 2018-11-20 Wisconsin Alumni Research Foundation (Warf) Vaccines comprising mutant attenuated influenza viruses
US10633422B2 (en) 2015-06-01 2020-04-28 Wisconsin Alumni Research Foundation (Warf) Influenza virus replication by inhibiting microRNA lec7C binding to influenza viral cRNA and mRNA
US10808229B2 (en) 2009-10-26 2020-10-20 Wisconsin Alumni Research Foundation (“WARF”) High titer recombinant influenza viruses with enhanced replication in vero cells
US11046934B2 (en) 2014-06-20 2021-06-29 Wisconsin Alumni Research Foundation (Warf) Mutations that confer genetic stability to additional genes in influenza viruses
US11197925B2 (en) 2016-02-19 2021-12-14 Wisconsin Alumni Research Foundation (Warf) Influenza B virus replication for vaccine development
US11241492B2 (en) 2019-01-23 2022-02-08 Wisconsin Alumni Research Foundation (Warf) Mutations that confer genetic stability to genes in influenza viruses
US11390649B2 (en) 2019-05-01 2022-07-19 Wisconsin Alumni Research Foundation (Warf) Influenza virus replication for vaccine development
US11807872B2 (en) 2019-08-27 2023-11-07 Wisconsin Alumni Research Foundation (Warf) Recombinant influenza viruses with stabilized HA for replication in eggs
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WO2008100290A2 (fr) 2006-09-29 2008-08-21 Sanofi Pasteur Biologics Co Vecteurs rhinoviraux recombinants
US9474798B2 (en) * 2007-06-18 2016-10-25 Wisconsin Alumni Research Foundation Influenza M2 protein mutant viruses as live influenza attenuated vaccines
US10119124B2 (en) 2007-06-18 2018-11-06 Wisconsin Alumni Research Foundation (Warf) Influenza M2 protein mutant viruses as live influenza attenuated vaccines
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