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WO2006013367A2 - Proteines chimeres leurs utilisations - Google Patents

Proteines chimeres leurs utilisations Download PDF

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Publication number
WO2006013367A2
WO2006013367A2 PCT/GB2005/003053 GB2005003053W WO2006013367A2 WO 2006013367 A2 WO2006013367 A2 WO 2006013367A2 GB 2005003053 W GB2005003053 W GB 2005003053W WO 2006013367 A2 WO2006013367 A2 WO 2006013367A2
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Prior art keywords
virus
protein
chimeric protein
hiv
amino acid
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WO2006013367A3 (fr
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Rodney Stuart Daniels
Kathryn Marie Copeland
Alexander James Elliot
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Medical Research Council
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Medical Research Council
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Priority claimed from GB0417390A external-priority patent/GB0417390D0/en
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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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—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

Definitions

  • the present invention relates to chimeric proteins which exhibit structural and functional properties of the native parent proteins.
  • the present invention relates to chimeric proteins comprising an amino acid sequence of a viral protein.
  • the present invention further relates to expression of the chimeric proteins, including in recombinant viruses, as well as uses of the chimeric proteins and recombinant viruses expressing them.
  • Env glycoproteins Attachment and entry of human immunodeficiency virus type-1 (HIV-I) to a host cell is mediated by the envelope (Env) glycoproteins.
  • Env monomers are synthesised as 160 kDa glycoproteins (gpl ⁇ O) in the endoplasmic reticulum, where oligimerisation takes place with trimers being the predominant form.
  • the gpl60 precursor is transported to the golgi where it is proteolytically processed by furin and furin-like enzymes into gpl20 and gp41 subunits, which are held together non-covalently.
  • the processed Env glycoproteins are then presented on the surface of the infected cell and subsequently, through budding, form the envelopes of progeny virions.
  • HIV-I attachment to target cells is mediated by the binding of gpl20 to CD4, the primary HIV-I receptor. This binding exposes a site on gpl20 that enables interactions with secondary co-receptors [1] .
  • the two major HIV-I co-receptors are the chemokine receptors CCR5 and CXCR4; CCR5 is generally considered to be the co- receptor utilised by HIV-I virions in early, asymptomatic stages of infection whereas viruses utilising CXCR4 usually emerge during late stages of disease. Binding of gpl20 to the appropriate co-receptor triggers a conformational change in gp41.
  • gp41 contains a short N- terminal hydrophobic sequence of amino acids, the fusion peptide, which mediates fusion of viral and host cell membranes, enabling entry of the virus into the host cell.
  • the conformational change in g ⁇ 41, induced by the gpl20-co-receptor interaction, is necessary in order for this fusion to take place.
  • Both the gpl20 and gp41 subunits act as major targets for the host immune system.
  • HIV glycoproteins It is generally accepted that knowledge of the structures of HIV glycoproteins will yield insights into the functions of the proteins, which will assist in the development of vaccines and drugs for anti-HIV therapies.
  • an obstacle to the development of treatments for HIV is the difficulty in obtaining the required information about the native structure of HIV glycoproteins.
  • Glycosidases have been used to remove oligosaccharides after glycoprotein synthesis [1, 2] in attempts to overcome the former problem. Domains from other proteins have been introduced into the proteins in attempts to improve stability [4, 5] , but with limited success.
  • Retroviral vectors expressing herpes simplex virus thymidine kinase under the control of an HIV-2 LTR have been developed to allow selective killing of HIV-infected cells [6] .
  • use of retroviral vectors that integrate stably into the host genome in a random manner have resulted in detrimental outcomes, including problems of insertional mutagenesis.
  • Recombinant influenza virus has been used to display epitopes from foreign antigens in which sequences encoding the epitopes were substituted in the haemagglutinin (HA) gene.
  • Influenza virus haemagglutinin (HA) is a trimeric membrane glycoprotein with receptor- binding and membrane fusion functions [7] .
  • HA mediates fusion between the viral and endosomal membranes allowing the release of ribonucleoprotein into the infected cell. Since the initial crystallisation of influenza HA [7] , its structure and function have been extensively studied and well characterised [8] . HA is initially synthesised as a trimeric precursor (HAO) containing three identical protein chains each of which is proteolytically processed into two subunits, HAl and HA2 , that are held together covalently by a single disulphide bond.
  • HEO trimeric precursor
  • the HAl subunit forms a globular head and contains the receptor-binding sites and the majority of antigenic sites.
  • the HA2 subunit anchors the structure to the membrane, provides stability to the trimeric structure and contains an N- terminal fusion peptide that has a region of homology with the gp41 fusion peptide.
  • Acidic conditions trigger influenza HA to undergo a conformational change that allows fusion of neighbouring membranes.
  • incorporation of sequences encoding epitopes of foreign antigens into the HA gene results in the expression of the epitopes on the surface of the virus.
  • Recombinant influenza virus displaying foreign epitopes has been used in cancer therapy and as vaccines. Such viruses displaying HIV glycoprotein epitopes have been used as vaccines in mice.
  • the development of a reverse genetics system that allows efficient recovery of recombinant influenza viruses opens up the use of such viruses for gene therapy [11] .
  • the system is based on the early human type A HlNl influenza virus, WSN [11, 12] . It has been shown recently that stable recombinant influenza viruses carrying two foreign genes, by replacing the haemagglutinin (HA) and neuraminidase (NA) genes, can be produced [13] .
  • HA haemagglutinin
  • NA neuraminidase
  • the present invention aims to address the difficulties of obtaining information about the native structure and function of a protein for which obtaining such information is difficult, for example due to instability of the protein, for example glycoproteins.
  • the present invention is particularly applicable to proteins which are associated with a disease state or condition, in particular, a virus-induced disease.
  • the present invention aims to address the need to be able to produce stable proteins, such as glycoproteins, for example viral glycoproteins such as HIV glycoproteins, retaining the structure and function of the native protein.
  • the present invention further aims to address the need to develop therapeutic applications using these proteins, in particular to develop effective therapeutic strategies for diseases such as virus-induced diseases, for example, AIDS caused by HIV.
  • the invention lies in providing a chimeric protein which has structural properties of the native proteins of which it is comprised, such that functional properties of the native proteins are retained.
  • the invention further lies in providing a chimeric protein having a stable structure.
  • the invention lies in providing a chimeric protein which has a stable structure having structural and functional properties of the native proteins.
  • the invention relates to a chimeric protein comprising influenza haemagglutinin (HA) amino acid sequence.
  • the invention relates to a chimeric protein comprising amino acid sequence of the HAl subunit and the HA2 subunit of HA.
  • the invention similarly relates to a chimeric protein comprising a heterologous protein amino acid sequence.
  • the invention relates to a chimeric protein comprising amino acid sequence of a protein associated with a disease, such as a virus- induced disease, for example a viral glycoprotein amino acid sequence, such as an HIV glycoprotein amino acid sequence.
  • the invention is concerned with a chimeric protein comprising HAl and HA2 subunit sequence which has the stable structure of native HA.
  • the invention is further concerned with disulphide bonds between cysteine residues of the chimeric protein.
  • the invention is concerned with disulphide bonds between cysteine residues of HA protein in the chimeric protein.
  • the invention further lies in the expression of chimeric proteins of the invention.
  • the invention further lies in providing recombinant virus expressing chimeric proteins of the invention.
  • the invention is particularly concerned with recombinant virus expressing chimeric protein that has a stable structure of the native proteins and functional properties of the native proteins, for example, a chimeric protein comprising HIV glycoprotein that has a stable structure and function of the native glycoprotein.
  • the invention lies in providing chimeric proteins and recombinant viruses expressing the chimeric proteins for use in medical treatment or therapy.
  • the invention particularly relates to recombinant viruses for use as vaccines or in gene therapy for the treatment of a mammal.
  • the invention is concerned with recombinant viruses for use as vaccines or for use in gene therapy as an anti-viral therapeutic strategy, for example, an anti-HIV therapeutic strategy for the treatment of HIV-infected individuals.
  • the present invention provides a chimeric protein comprising (a) an amino acid sequence of the HAl subunit of influenza virus haemagglutinin (HA) protein comprising cysteine residues at positions 30, 68 and 293 as numbered from the N- terminal amino acid of the HAl subunit from influenza virus X31, or corresponding positions in the HAl subunit from a different influenza virus and (b) an amino acid sequence of the HA2 subunit of influenza virus HA protein comprising cysteine residues at positions 153, 160 and 164 as numbered from the N-terminal amino acid of the HA2 subunit from influenza virus X31, or corresponding positions in the HA2 subunit from a different influenza virus and (c) an amino acid sequence of a heterologous protein positioned between the cysteine residues at positions 68 and 293, or the corresponding positions, of (a) and having a disulphide bond between the cysteine residues at position 30 of HAl and position 153 of HA2,
  • the amino acid sequence of the HA2 subunit further comprises the membrane anchor region from position 186 to 207, inclusive.
  • the chimeric protein comprises in the direction from the N-terminus to the C-terminus, (i) an amino acid sequence of the HAl subunit from and including position 1 to and including position 68 as numbered from the N-terminal amino acid of the HAl subunit from influenza virus X31, or corresponding positions in the HAl subunit from a different influenza virus,- (ii) an amino acid sequence of a heterologous protein; (iii) an amino acid sequence of the HAl subunit from and including position 293 to and including position 345 as numbered from the N-terminal amino acid of the HAl subunit from influenza virus X31, or corresponding positions in the HAl subunit from a different influenza virus and (iv) the amino acid sequence of the HA2 subunit from and including position 1 to and including position 237 as numbered from the N-terminal amino acid of the HA2 subunit from influenza virus X31, or corresponding positions in the HA2 subunit from a different influenza virus .
  • the chimeric protein further comprises an amino acid linker sequence at either or both of the junctions between the heterologous protein sequence and the HAl subunit sequence.
  • the amino acid linker sequence is between sequences (i) and (ii) and/or (ii) and (iii) .
  • the linker sequence is Gly-Gly-Gly-Ser.
  • the chimeric protein comprises influenza HA protein from influenza virus X31.
  • the heterologous protein component of the chimeric protein may be from a protein associated with a disease.
  • the heterologous protein component is from a protein associated with a virus- induced disease, for example, from a viral protein.
  • the heterologous protein component may be from a protein that has a trimeric quaternary structure or is functional in a trimeric structure.
  • the heterologous protein component may be from a receptor-binding protein.
  • the amino acid sequence of the heterologous protein is from a protein from a virus belonging to one of the following virus families: Orthomyxoviridae, for example, haemagglutinin glycoprotein of influenza virus
  • Retroviridae for example, surface glycoprotein SU of Rous Sarcoma virus (RSV) , Feline Leukemia virus (FeLV) , Human T-cell Leukemia viruses 1 and 2 (HTLV-1/2) , Bovine Leukemia virus (BLV) , Human Immunodeficiency viruses
  • RSV Rous Sarcoma virus
  • FeLV Feline Leukemia virus
  • HTLV-1/2 Human T-cell Leukemia viruses 1 and 2
  • BLV Bovine Leukemia virus
  • Human Immunodeficiency viruses for example, surface glycoprotein SU of Rous Sarcoma virus (RSV) , Feline Leukemia virus (FeLV) , Human T-cell Leukemia viruses 1 and 2 (HTLV-1/2) , Bovine Leukemia virus (BLV) , Human Immunodeficiency viruses
  • HIV-1/2 HIV-1/2
  • SIV Simian Immunodeficiency virus
  • EIAV Infectious Anemia virus
  • FV Feline Imunodeficiency virus
  • CAEV Caprine Arthritis Encephalitis virus
  • Filoviridae for example, surface glycoprotein GPl of
  • E2 protein of alphaviruses such as Semliki forest virus, Sindbis virus, Ross River, Eastern/Western Equine Encephalitis, and rubiviruses such as Rubella
  • Coronaviridae for example, Sl protein of Severe Acquired
  • SARS Respiratory Syndrome
  • Reoviridae for example, VP2 protein of orbiviruses such as Bluetongue virus, African horse sickness virus
  • Rhabdoviridae for example, G proteins of vesiculoviruses such as Vesicular Stomatitis virus (VSV) and Lyssaviruses such as Rabies
  • VSV Vesicular Stomatitis virus
  • Rabies Rabies
  • Flaviviridae for example, E (E2) proteins of Tick Borne Encephalitis (TBE) , Dengue, Hepatitis C virus (HCV) , West
  • amino acid sequence from a protein from a virus of the family Arenaviridae such as GPl protein of Lassa, Venezuelan hemorrhagic fever, Lymphocytic choriomeningitis may comprise the heterologous protein component .
  • the amino acid sequence of a heterologous protein is from an HIV glycopotein, preferably an HIV-I Env glycoprotein.
  • the HIV-I Env glycoprotein is HIV-I gpl20.
  • the HIV-I gpl20 is from HIV-I strain pNL43 or HIV-I strain JRFL.
  • the amino acid sequence of HIV-I gpl20 comprises cysteine residues at positions 119, 126, 131, 157, 194, 203, 216, 226, 237, 245, 294, 329, 376, 383, 416 and 443 as numbered from the N-terminal amino acid of gpl20 from HIV-I strain pNL43, or corresponding positions in other strains of HIV.
  • the amino acid sequence of HIV-I gpl20 comprises the amino acid from and including position 89 to the amino acid at and including position 495 as numbered from the N-terminal amino acid of gpl20 from HIV-I strain pNL43, or corresponding positions in other strains of HIV.
  • the present invention provides a nucleic acid encoding a chimeric protein of the invention.
  • the present invention provides an expression vector comprising a nucleic acid of the invention.
  • the expression vector may be a mammalian expression vector for expression of the chimeric protein in a mammalian cell, preferably for constitutive expression in a mammalian cell.
  • the expression vector may be a viral expression vector for expression of the chimeric protein by a virus infecting a susceptible host cell.
  • the present invention provides a host cell comprising a nucleic acid of the invention or an expression vector of the invention and a method of producing a host cell of the invention.
  • the host cell may constitutively express the chimeric protein.
  • Another aspect of the invention is a method of producing a chimeric protein of the invention.
  • Yet another aspect of the present invention provides a method of producing a recombinant virus capable of expressing a chimeric protein of the invention, comprising contacting a susceptible cell with an expression vector of the invention and optionally- isolating the virus from the cell.
  • the virus is a vaccinia virus. More preferably, the virus is an influenza virus .
  • the present invention provides a virus obtained by the method of the invention for producing a recombinant virus.
  • a virus of the invention is further capable of expressing a conditionally lethal toxic protein.
  • the conditionally lethal toxic protein may be herpes simplex virus thymidine kinase or an apoptosis inducer such as a caspase .
  • the present invention provides a target cell infected with a virus of the invention.
  • a virus and/or a chimeric protein of the invention may be used as a treatment or therapy for a mammal, preferably a human.
  • a virus and/or a chimeric protein of the invention may be used as a vaccine for a mammal, preferably as a vaccine for a human.
  • a virus and/or a chimeric protein of the invention may be used for the preparation of a vaccine.
  • a vaccine comprising a virus and/or a chimeric protein of the invention is also provided by the present invention.
  • a virus and/or a chimeric protein of the invention may be used for the preparation of a medicament for the treatment of a mammal .
  • a virus of the invention which expresses a chimeric protein of the invention comprising an amino acid sequence of a protein associated with a disease state and/or a chimeric protein comprising an amino acid sequence of a protein associated with a disease state may be used as a vaccine for individuals suffering from the disease or those susceptible to developing the disease, or for the preparation of a vaccine or for the preparation of a medicament for the treatment of an individual suffering from the disease.
  • the disease is a virus-induced disease.
  • a virus of the invention which expresses a chimeric protein of the invention comprising an amino acid sequence of an HIV Env glycoprotein and/or a chimeric protein comprising an amino acid sequence of an HIV Env glycoprotein may be used as an anti-HIV vaccine for HIV-infected individuals or those susceptible to infection, or for the preparation of a vaccine or for the preparation of a medicament for the treatment of an HIV-infected individual.
  • a virus of the invention may be used in gene therapy for a mammal, preferably a human.
  • a virus of the invention which expresses a chimeric protein of the invention comprising an amino acid sequence of a protein associated with a disease state and/or a chimeric protein comprising an amino acid sequence of a protein associated with a disease state may be used in the treatment of an individual suffering from the disease.
  • Such a virus may also be used for the preparation of a medicament for the treatment of an individual suffering from the disease.
  • the disease is a virus-induced disease.
  • a virus of the invention which expresses a chimeric protein of the invention comprising an amino acid sequence of an HIV-I Env glycoprotein and/or a chimeric protein comprising an amino acid sequence of an HIV-I Env glycoprotein may be used in the treatment of an HIV- infected individual.
  • a virus may be used as part of a therapeutic strategy to clear HIV from an infected individual .
  • Such a virus may also be used for the preparation of a medicament for the treatment of an HIV- infected individual .
  • a virus and/or a chimeric protein of the invention may be used as a treatment or therapy either alone or in combination with other medicaments and/or treatments.
  • compositions comprising a virus and/or a chimeric protein of the invention for the treatment or therapy of a mammal, preferably a human.
  • Figure 1 shows a schematic representation of the assembly of the EnvHA chimeric protein of an embodiment of the present invention.
  • the influenza X31 (H3) HA, mRNA sense, with 5' and 3' non-coding regions is shown as is the disulphide-bonded structure of the encoded HA in the processed form.
  • HA1/HA2 subunits are covalently linked by a single disulphide bond between residue 30 of HAl and residue 153 of HA2.
  • the positions of the signal peptide , fusion peptide, and membrane anchor, are shown (residue numbering is for influenza virus X31 and includes the signal peptide and fusion peptides in HAl and HA2 respectively) as is the bromelain (Br) cleavage site in HA2.
  • B The HIV-I Env is presented in the processed form of gpl20/gp41 with its disulphide bonding pattern. The two subunits are held together non covalently. All numbering refers to the HIV-I pNL43 Env sequence [14] and the positions of the signal peptide, fusion peptide, and membrane anchor, are shown.
  • Figure 2 shows expression of EnvHA chimeric proteins of embodiments of the present invention.
  • Cell lysates were prepared from recombinant vaccinia virus-infected CV-I cells expressing the proteins indicated and separated by SDS-PAGE under reducing conditions. Lysates were probed using (a) anti-Env sheep polyclonal serum (ARP401) and (b) anti-HA rabbit polyclonal serum (X31) . Neither serum reacted with lysates from parental vaccinia (vRB12) infected and uninfected (NEG) cells. The locations of specific glycoprotein species are indicated.
  • ARP401 anti-Env sheep polyclonal serum
  • X31 anti-HA rabbit polyclonal serum
  • Figure 3 shows the oligomeric forms of EnvHA chimeric proteins of embodiments of the present invention expressed in CV-I cells.
  • Cell lysates were prepared in the absence of ⁇ -mercaptoethanol, separated by SDS-PAGE and probed using anti-Env (ARP401) and anti-HA rabbit polyclonal sera.
  • Results for EnvHA constructs (R3 and Yl) , wt Env (JRFL and NL43) and HA (X31) are shown. With both sera, monomeric EnvHA can be seen to run at approx 150 kDa, and two higher molecular weight bands corresponding to dimeric and trimeric forms are observed. Similar forms are observed for Env and HA with their respective anti-sera.
  • Figure 4 shows detection of EnvHA chimeric protein cell surface expression by immunofluorescence.
  • CV-I cells infected with recombinant vaccinia viruses were probed with anti-JRFL, anti-NL43 and anti-HA antibodies to detect surface expression of EnvHA protein.
  • Figure 5 shows the effect of trypsin on EnvHA (R3) , wt NL43 Env and HA (X31) proteins.
  • CV-I cells expressing the proteins indicated (R3, NL43, X31) were incubated with increasing concentrations of trypsin at 37 0 C for 30 min.
  • Cell lysates were prepared and separated by SDS-PAGE under reducing conditions then probed using (a) anti-Env monoclonal antibody (EVA3012) to detect wt EnvHAO (150 kDa; gpl20-HA0) and (b) anti-HA polyclonal serum (X31) to detect EnvHAO, HAO, HAl and HA2.
  • EVA3012 anti-Env monoclonal antibody
  • X31 anti-HA polyclonal serum
  • FIG. 6 shows the identification of the 28kDa protein as HA2.
  • CV-I cells expressing the proteins indicated R3, Yl, X31
  • Cell lysates were prepared and separated by SDS-PAGE under reducing conditions. Lysates were probed with R185 polyclonal serum to detect HA2.
  • Cross-reactive bands were present in parental vaccinia (vRB12) infected and uninfected (NEG) cells but HA2 was absent.
  • Figure 7 shows an assay of membrane fusion under conditions optimal for X31 HA.
  • ghost cell lines and BHK cells were infected with recombinant vaccinia viruses to express the proteins indicated (R3, Yl, JRFL, NL43, X31) for 16 h, then treated with 5 ⁇ g/ml trypsin and briefly exposed to low pH cell buffer (pH 5) .
  • Membrane fusion was then monitored as described in materials and methods. Uninfected cell lines (NEG) and those infected with wt vaccinia (vRB12) were treated similarly.
  • the present invention provides a chimeric protein comprising amino acid sequences of both the HAl subunit and the HA2 of influenza virus HA protein and of a heterologous protein.
  • the HAl subunit amino acid sequence comprises cysteine residues at positions 30, 68 and 293 as numbered from the N-terminal amino acid of the HAl subunit from influenza virus X31, or the corresponding positions in the HAl subunit from other strains of influenza virus.
  • the amino acid sequence of the HA2 subunit comprises cysteine residues at positions 153, 160 and 164 as numbered from the N-terminal amino acid of the HA2 subunit from influenza virus X31, or the corresponding positions in the HA2 subunit from other strains of influenza virus.
  • the amino acid sequence of the heterologous protein is positioned between the cysteine residues at positions 68 and 293 of the HAl subunit.
  • the chimeric protein has a disulphide bond between the cysteine residues at position 30 of HAl and position 153 of HA2 , between the cysteine residues at positions 68 and 293 of HAl, and between the cysteine residues at positions 160 and 164 of HA2.
  • the chimeric protein has the structural properties of the native HA protein such that the structure and function of the native heterologous protein are retained.
  • Influenza virus HA is a trimeric membrane glycoprotein.
  • HA is initially synthesized as a trimeric precursor (HAO) containing three identical protein chains. Each of the chains is proteolytically processed into two subunits, HAl and HA2, that are held together covalently by a single disulphide bond between the cysteine residue at position 39 of HAl and the cysteine residue at position 153 of HA2.
  • the HAl subunit forms a globular head and contains the receptor-binding sites and the majority of antigenic sites.
  • the HA2 subunit stably anchors the HA protein to the membrane, helping to stabilize the HA trimers.
  • Incorporating part of influenza virus HA into the chimeric protein results in a chimeric protein which is expressed at the cell surface and which has a stable structure having structural properties of the native HA. This helps to ensure that the structural and functional properties of the native heterologous protein are retained in the chimeric protein.
  • use of influenza virus HA in the chimeric protein allows the expression of a protein with a stable structure and the function of the native protein. Without being bound by any particular theory, it is believed that the incorporation of amino acid sequence of the HAl subunit and of the HA2 subunit stabilizes the chimeric protein. The presence of disulphide bonds in the HA protein similarly serves to stabilize the structure of the chimeric protein.
  • the disulphide bond between the HAl subunit (at cysteine residue 30) and the HA2 subunit (at cysteine residue 153) has a stabilizing effect on the structure.
  • the presence of HA2 subunit and disulphide bonds helps to ensure the expression of a heterologous protein having its native structure. Ensuring the correct, native structure of the heterologous protein in turn ensures the protein retains its native function.
  • the amino acid sequence of the HA2 subunit further comprises the membrane anchor region from position 186 to 207, inclusive. This serves to hold the chimeric protein on the cell surface and allows membrane fusion functional studies to be performed.
  • the chimeric protein comprises in the direction from the N-terminus to the C-terminus, (i) an amino acid sequence of the HAl subunit from and including position 1 to and including position 68 as numbered from the N-terminal amino acid of the HAl subunit from influenza virus X31, or corresponding positions; (ii) an amino acid sequence of a heterologous protein; (iii) an amino acid sequence of the HAl subunit from and including position 293 to and including position 345 as numbered from the N-terminal amino acid of the HAl subunit from influenza virus X31, or corresponding positions and (iv) an amino acid sequence of the HA2 subunit from and including position 1 to and including position 237 as numbered from the N-terminal amino acid of the
  • a preferred chimeric protein comprises the complete HAl subunit sequence without residues 69 to 292, inclusive. It also has the complete HA2 subunit sequence. Therefore, such a chimeric protein can be expressed by a recombinant influenza virus infecting a susceptible host cell as the complete HA2 subunit sequence has the components required for this.
  • the complete HA2 subunit has the membrane anchor which serves to hold the chimeric protein on the virus surface and facilitate membrane fusion, the cytoplasmic tail which is important for trafficking the chimeric protein and assisting virus assembly, and the fusion peptide important for infectivity of the virus.
  • the chimeric protein further comprises an amino acid linker sequence at either or both of the junctions between the heterologous protein sequence and the HAl subunit sequence.
  • the amino acid linker sequence is between sequences (i) and (ii) and/or (ii) and (iii) .
  • the linker sequence preferably comprises between one to four amino acids, most preferably four amino acids.
  • the linker sequence is the sequence Gly-Gly-Gly-Ser.
  • the linker sequences may be based on the HAl subunit sequence directly C-terminal to the cysteine residue at position 68, and/or directly N-terminal to the cysteine residue at position 293, as numbered form the N-terminal amino acid of the HAl subunit from influenza virus X31, or corresponding positions in the HAl subunit from a different influenza virus. Still further, the linker sequences may be based on the sequence of the heterologous protein. However, the linker sequence is not limited to any particular amino acid sequence as long as the resultant chimeric protein has the structural properties of the native HA protein such that the structure and function of the native heterologous protein are retained.
  • linkers at one or both of the junctions should not have an adverse effect on the structure or function of the chimeric protein.
  • the linker sequence above may have the addition, deletion or substitution of one or more amino acids. It is preferable that the amino acid sequence comprises mainly residues with small side- chains. It is also preferable that the amino acids have hydrophilic side-chains.
  • a chimeric protein of the invention may have additional amino acid sequences derived from HAl and/or the heterologous protein providing the chimeric protein includes the specific cysteine residues described or their equivalents in other influenza HA proteins such that disulphide bonds are present between these residues as specified.
  • the chimeric protein comprises influenza HA protein from influenza virus X31.
  • the HA protein may be that from any influenza virus.
  • HA protein may be from Hl viruses PR8 or WSN, or from more recent strains such as New Caledonian.
  • the HA used is preferably one for which nucleic acid has been isolated and the sequence determined. The skilled person is able to determine the corresponding amino acid positions in HA from other influenza viruses relative to those of X31 HA, for example by sequence homology comparisons.
  • the term "corresponding position" of an amino acid is defined as an amino acid position in HA from influenza virus other than X31 which is equivalent to a given amino acid position in influenza virus X31.
  • an amino acid at an equivalent position in HA protein from a different influenza virus has the same structural and/or functional role as the amino acid at the given position in X31 HA protein.
  • the chimeric protein also has a heterologous protein component.
  • Heterologous protein is defined as any protein which does not derive from the same source as the HA protein.
  • the heterologous protein is one that is inherently unstable, such that standard techniques for expressing proteins are not effective in producing the protein in a structurally and functionally correct form.
  • the heterologous protein component is from a protein associated with a disease.
  • a disease may be any disease for which it is desirable to be able to develop therapeutic strategies, resulting from a greater understanding of a protein associated with that disease.
  • a disease may be an infectious disease or a cancer.
  • the disease may be induced by a virus such as HIV, or a virus-induced disease, for example, a cancer induced by a virus, more particularly, a cancer induced by Rous Sarcoma virus (RSV) , Feline
  • RSV Rous Sarcoma virus
  • the heterologous protein component is from a protein associated with a virus-induced disease, that is, from a viral protein.
  • the heterologous protein component (amino acid sequence (c) and (ii) in certain embodiments) may be HA derived from a different influenza virus than the source of the HA components (amino acid sequences (a) , (b) , (i) , (iii) and (iv) in certain embodiments) of the chimeric protein.
  • the technique described in the present invention may be used to produce an attenuated virus strain from a highly pathogenic one which contains a polybasic amino acid sequence at its HA1/HA2 processing site.
  • the heterologous protein component is preferably from a protein that has a trimeric quaternary structure.
  • a protein with a trimeric structure consists of three polypeptide chains, which may be identical or different to one another, linked together to form a trimer.
  • a trimeric protein will generally only be functionally active when it is in trimer form.
  • the heterologous protein component may be from any protein that is functional in a trimeric structure, even if it is able to form other quaternary structures .
  • the heterologous protein component is from a protein associated with a disease, preferably from a viral glycoprotein, and preferably one that has a trimeric structure or is active in trimeric form.
  • the heterologous protein component may be from a receptor-binding protein.
  • receptor-binding protein is defined as any protein which is capable of interacting with one or more molecules, for example, protein, lipid, carbohydrate or combinations thereof, present on the surface of a cell, preferably a mammalian cell, more preferably a human cell.
  • a receptor-binding protein is capable of targeting the chimeric protein to a specific cell type that has on its surface the corresponding "receptor" molecule (s) .
  • the heterologous protein component is from a protein associated with a disease, preferably from a viral glycoprotein, and preferably one that is a receptor-binding protein.
  • a protein also has a trimeric structure or is active in trimeric form.
  • the amino acid sequence of the heterologous protein is from a protein from a virus belonging to one of the following virus families : Orthomyxoviridae, for example, haemagglutinin glycoprotein of influenza virus
  • Retroviridae for example, surface glycoprotein SU of Rous Sarcoma virus (RSV) , Feline Leukemia virus (FeLV) , Human T-cell Leukemia viruses 1 and 2 (HTLV-1/2) , Bovine Leukemia virus (BLV) , Human Immunodeficiency viruses (HIV-1/2) , Simian Immunodeficiency virus (SIV) , Equine Infectious Anemia virus (EIAV) , Feline Imunodeficiency virus (FIV) , Caprine Arthritis Encephalitis virus (CAEV) , Visna/Maedi
  • RSV Rous Sarcoma virus
  • FeLV Feline Leukemia virus
  • HTLV-1/2 Human T-cell Leukemia viruses 1 and 2
  • BLV Bovine Leukemia virus
  • HV-1/2 Human Immunodeficiency viruses
  • SIV Simian Immunodeficiency virus
  • EIAV Equine Infectious Anemia virus
  • FV Feline
  • Filoviridae for example, surface glycoprotein GPl of Ebola, Marburg
  • E2 protein of alphaviruses such as Semliki forest virus, Sindbis virus, Ross River, Eastern/Western Equine Encephalitis, and rubiviruses such as Rubella
  • Coronaviridae for example, Sl protein of Severe Acquired Respiratory Syndrome (SARS) Reoviridae, for example, VP2 protein of orbiviruses such as Bluetongue virus, African horse sickness virus
  • Rhabdoviridae for example, G proteins of vesiculoviruses such as Vesicular Stomatitis virus (VSV) and Lyssaviruses such as Rabies . These proteins are all trimeric glycoproteins.
  • VSV Vesicular Stomatitis virus
  • Rabies Lyssaviruses
  • Flaviviridae for example, E (E2) proteins of Tick Borne Encephalitis (TBE) , Dengue, Hepatitis C virus (HCV) , West Nile, Yellow fever. These proteins are dimeric in their native state but at low pH dissociation occurs and trimers form to allow fusion.
  • TBE Tick Borne Encephalitis
  • HCV Hepatitis C virus
  • amino acid sequence from a protein from a virus of the family Arenaviridae such as GPl protein of Lassa, Venezuelan hemorrhagic fever, Lymphocytic choriomeningitis may comprise the heterologous protein component.
  • These proteins are tetrameric in their native state but may adopt a trimeric structure when in the chimeric protein format due to the influence of the HA protein components, retaining at least their antigenic properties .
  • the amino acid sequence of a heterologous protein is from an HIV Env glycoprotein.
  • the heterologous protein amino acid sequence is from an HIV-I Env glycoprotein.
  • the HIV-I Env glycoprotein is HIV-I gpl20.
  • the HIV-I gpl20 is from HIV-I strain pNL43 or HIV-I strain JRFL.
  • the present invention provides the use of an Env glycoprotein amino acid sequence from any strain, isolate, clone or sub ⁇ clone of HIV.
  • HIV is defined as a human immunodeficiency virus, such as HIV-I or HIV-2.
  • HIV also includes a virus equivalent to the human virus, but having a different host species, for example, SIV.
  • the amino acid sequence of HIV-I gpl20 comprises cysteine residues at positions 119, 126, 131, 157, 194, 203, 216, 226, 237, 245, 294, 329, 376, 383, 416 and 443 as numbered from the N-terminal amino acid of gpl20 from HIV-I strain pNL43, or corresponding positions in other strains of HIV [15] , where disulphide bonds are present between pairs of cysteine residues as found in native HIV-I gpl20.
  • disulphide bonds are present between pairs of cysteine residues as found in native HIV-I gpl20.
  • only some of these cysteine residues and their corresponding disulphide bonds may be present or further cysteine residues and their corresponding disulphide bonds may be present.
  • the amino acid sequence of HIV gpl20 comprises the amino acid from and including position 89 to the amino acid at and including position 495 as numbered from the N-terminal amino acid of gpl20 from HIV-I strain pNL43, or corresponding positions in other strains of HIV.
  • Such a sequence is chosen to optimise joining of the HAl subunit sequence and heterologous protein sequence at comparable positions in each of the proteins (in the "stalk" regions) .
  • the skilled person is able to determine the corresponding amino acid positions in gpl20 from other HIV strains relative to those of gpl20 from HIV-I strain pNL43, for example by sequence homology comparisons.
  • corresponding position of an amino acid is defined as an amino acid position in gpl20 from HIV other than strain pNL43 which is equivalent to a given amino acid position in HIV-I pNL43.
  • an amino acid at an equivalent position has the same structural and/or functional role as the amino acid at the given position in HIV-I pNL43.
  • the HIV gpl20 amino acid sequence comprises one or more antigenic epitopes.
  • the present invention provides a nucleic acid encoding the chimeric protein of the invention.
  • the nucleic acid may be DNA or RNA.
  • a nucleic acid, or fragments thereof, of the present invention may be obtained by standard methods known in the art.
  • a nucleic acid may be generated by chemical synthesis or preferably, by polymerase chain reaction (PCR) using specific primers. The generation of a nucleic acid of the present invention is described under Materials and Methods in the Examples.
  • the present invention provides an expression vector comprising a nucleic acid of the invention.
  • the type of vector used is not limited to any particular type.
  • expression vector is defined as a vector having the elements necessary for transcription of nucleic acid which are operably linked to that nucleic acid, for example, a nucleic acid of the invention.
  • Nucleic acid is "operably linked” when it is placed in a functional relationship with another nucleic acid sequence.
  • a promoter is operably linked to a nucleic acid if it effects transcription of that nucleic acid in a host cell.
  • Such elements include a promoter, terminator and polyadenylation signal .
  • the expression vector may be a mammalian expression vector for expression of the chimeric protein in a mammalian cell.
  • Suitable mammalian expression vectors include the pEE14tpa and pcDNA3.1 vectors.
  • the expression vector may be a viral expression vector for expression of the chimeric protein by a virus infecting a susceptible host cell .
  • Suitable viral expression vectors include, but are not limited to, the vaccinia virus shuttle vector pRB21 [16] and recombinant influenza viruses generated using the vector pHH21 [11] .
  • the present invention provides a host cell comprising a nucleic acid of the invention or an expression vector of the invention.
  • the invention also includes a method of producing a host cell of the present invention, which may be produced by introducing a nucleic acid or expression vector of the present invention into a suitable cell using techniques known in the art.
  • a suitable host cell may be any cell which can express the introduced nucleic acid or expression vector.
  • Suitable host cells including bacteria, in particular E. coli, yeast, fungi and higher eukaryotic cells are well known in the art.
  • a suitable host cell for a mammalian expression vector includes mammalian cells such as Chinese Hamster Ovary (CHO) cells.
  • Suitable host cells for viral expression vectors include mammalian cells such as CV-I, BHK, NS20, 293T and MDCK.
  • CV-I cells are used with vaccinia expression vectors and 293T and MDCK cells are used with influenza expression vectors.
  • suitable cells for use with influenza virus expression vectors of the invention which express a chimeric protein comprising an HIV Env protein include Jurkat, CEM C8166, HVS T and PMBC cells .
  • the invention also provides a method of producing a chimeric protein of the invention, comprising culturing a host cell of the invention under conditions allowing expression of the encoded chimeric protein and optionally recovering the chimeric protein from the host cell culture.
  • the host cell is a CHO cell. Suitable conditions for the expression of proteins in host cells are known in the art.
  • a further aspect of the present invention provides a method of producing a recombinant virus capable of expressing a chimeric protein of the invention, comprising contacting a susceptible cell with an expression vector of the invention and optionally isolating the virus from the cell.
  • the virus is a vaccinia virus, such as vaccinia virus strain vRB12. More preferably, the virus is an influenza virus, such as the human HlNl influenza virus WSN.
  • a susceptible cell is a suitable host cell infected with wt virus.
  • a chimeric protein of the invention which comprises an HIV Env glycoprotein
  • specific mutations are made in the HAl and/or HA2 domains, using techniques familiar to those skilled in the art, based on studies of influenza HA fusion mutants [17] , in order to adjust the pH at which fusion occurs to ensure optimum infectivity of the viruses expressing the chimeric protein.
  • the CD4 receptor the presence of which is required for a cell to be infected by a virus expressing a chimeric protein comprising an HIV Env protein, is commonly found internalized in early endosomes in the absence of specific internalization stimulators.
  • the present invention provides a virus obtained by the method of the invention for producing a recombinant virus.
  • a virus of the invention is further capable of expressing a conditionally lethal toxic protein.
  • the conditionally lethal toxic protein may be herpes simplex virus thymidine kinase (HSVTK) . This protein becomes toxic to a cell infected with recombinant virus expressing HSVTK when the drug ganciclovir is administered.
  • HSVTK herpes simplex virus thymidine kinase
  • expression of HSVTK in a cell is also dependent on the target cell being infected with HIV.
  • expression of the HSVTK gene under control of a modified HIV-2 LTR makes expression of HSVTK conditional on the presence of HIV, in particular the presence of HIV-I, HIV-2 or SIV Tat proteins.
  • recombinant virus expressing HIV gpl20 and HSVTK under the control of HIV-2 LTR will selectively kill only CD4-expressing cells which are infected with HIV.
  • conditionally lethal toxic protein may be an apoptosis inducer.
  • Apoptosis is a normal physiological response that can occur in mammalian cells in response to different stimuli such as T-lymphocyte signalling or DNA damage.
  • the cell Upon stimulation, the cell enters a regulated suicide program; an efficient method of cell death where the cell contents are not spilled, such that inflammatory responses do not occur.
  • a family of proteases called cysteine-aspartic acid proteases (caspases) , play a major role in apoptosis. There are at least eleven human caspases of which there are two main classes: initiators and effectors/executors.
  • Initiators such as caspases-2, -8, -9 and -10 can activate themselves whereas effector/executor caspases-3, -6, and -7 require proteolytic processing by initiator caspases. They are all synthesized as two inactive heterologous subunits that require processing at a specific Asp site in the linker between the subunits in order to become active. Activation of caspases creates hierarchical caspase cascades that lead to proteolytic cleavage of cellular factors, DNA degradation and ultimately cell death. Thus, the use of a recombinant virus expressing a caspase allows targeted killing of a cell infected by the virus.
  • a reverse caspase-3 capable of autocatalytic processing has been created [18] .
  • expression of revCasp3 under control of a modified HIV-2 LTR makes expression of revCasp3 conditional on the presence of HIV, in particular the presence of HIV-I, HIV-2 or SIV Tat proteins.
  • recombinant virus expressing HIV gpl20 and revCasp3 under the control of HIV-2 LTR will selectively kill only CD4-expressing cells which are infected with HIV.
  • conditionally lethal toxic protein may be any of those developed or being developed for gene therapy purposes.
  • An alternative method of conferring conditional toxicity may result from including sequences corresponding to the HIV Rev Response Element (RRE) in the toxic gene segment .
  • the RRE would serve to retain the toxic gene mRNA in the cell nucleus, thereby preventing production of the toxic protein [19] .
  • This block would be overcome in cells expressing HIV Rev protein.
  • recombinant viruses expressing HIV gpl20 and toxic genes (HSVTK or revCasp3) containing the RRE will selectively kill only CD4- expressing cells which are infected with HIV.
  • the present invention provides a target cell infected with a virus of the invention.
  • Preferred target cells for infection by vaccinia virus include CV-I cells.
  • 293T and MDCK cells are preferably used for infection with influenza virus.
  • preferred cells for use with recombinant influenza virus of the invention expressing a chimeric protein comprising an HIV Env protein include Jurkat, CEM C8166, HVS T and PMBC cells. These cells express CD4 which is necessary for their recognition and infection by recombinant viruses expressing HIV Env protein.
  • target cells such as 293T and MDCK, which support efficient influenza replication, are genetically modified to express human CD4 and/or chemokine receptors using techniques known to those skilled in the art.
  • a virus and/or a chimeric protein of the invention may be used as a treatment or therapy for a mammal, preferably a human.
  • a virus and/or a chimeric protein of the invention may be used as a vaccine for a mammal, preferably as a vaccine for a human.
  • a virus and/or a chimeric protein of the invention may be used for the preparation of a vaccine.
  • a vaccine comprising a virus and/or a chimeric protein of the invention is also provided by the present invention.
  • a virus and/or a chimeric protein of the invention may be used for the preparation of a medicament for the treatment of a mammal .
  • the virus and/or chimeric protein may be used to treat individuals suffering from the disease or at risk from suffering from the disease, and in therapeutic strategies against that disease.
  • diseases or conditions that may be targeted using a virus and/or chimeric protein of the invention include infectious diseases and cancer.
  • the disease may be induced by a virus such as HIV, or may be a virus-induced disease, for example, a cancer induced by a virus.
  • a virus and/or a chimeric protein of the invention is used in combination with a suitable carrier as a vaccine.
  • Virus and/or a chimeric protein at the desired degree of purity and in a sufficient amount to induce antibody formation is mixed with a physiologically acceptable carrier.
  • a physiologically acceptable carrier is non-toxic to a recipient at the dosage and concentration employed in the vaccine.
  • the vaccine is formulated for injection, usually- intramuscular or subcutaneous injection.
  • Suitable carriers for injection include sterile water.
  • Preferred suitable carriers include physiological saline solutions, such as normal saline or buffered salt solutions such as phosphate-buffered saline or Ringer's solution.
  • the vaccine generally contains an adjuvant, for example an adjuvant which can enhance cellular or local immunity.
  • Additional exipients that can be present in a vaccine include low molecular weight polypeptides, proteins, amino acids, carbohydrates including glucose or dextrans, chelating agents such as EDTA, stabilizing agents and anti-microbial agents.
  • a recombinant virus and/or a chimeric protein of the invention used as a vaccine, or a vaccine prepared using recombinant virus and/or a chimeric protein of the invention may be administered to uninfected individuals or infected individuals to enhance immune response to the heterologous protein comprised in the chimeric protein. Administration may be combined with administration of other vaccines, medicaments or compositions, or in combination with other treatments.
  • a preferred recombinant virus for use as a vaccine expresses a chimeric protein of the invention comprising an HIV Env glycoprotein, preferably a gpl20 protein.
  • a preferred chimeric protein for use as a vaccine comprises an HIV Env glycoprotein, preferably a gpl20 protein.
  • the gpl20 has more than one antigenic epitope.
  • Such a recombinant virus and/or chimeric protein, or vaccine prepared using such a virus and/or chimeric protein is administered to an HIV- infected individual or an uninfected individual at risk of becoming infected.
  • multiple recombinant viruses each expressing a chimeric protein comprising gpl20 from a different HIV strain, clone or isolate and/or multiple chimeric proteins comprising gpl20 from different HIV strains are used as a vaccine or to prepare a vaccine.
  • Multiple recombinant viruses and/or chimeric proteins may be administered simultaneously or used in the preparation of a single vaccine.
  • multiple recombinant viruses and/or chimeric proteins may be administered sequentially or used individually in the preparation of separate vaccines.
  • a virus of the invention may be used in gene therapy for a mammal .
  • a virus of the invention which expresses a chimeric protein of the invention comprising an amino acid sequence of an HIV Env glycoprotein and/or a chimeric protein comprising an amino acid sequence of an HIV Env glycoprotein may be used in the treatment of an HIV-infected individual.
  • Such a virus may be used as part of a therapeutic strategy to clear HIV from an infected individual .
  • Such a virus and/or a chimeric protein may also be used for the preparation of a medicament for the treatment of an HIV-infected individual .
  • a virus of the invention may be used as a treatment either alone or in combination with one or more medicaments and/or treatments.
  • medicaments and/or treatments for example, a number of medicaments and therapeutic strategies for the treatment of HIV-infected individuals are known to those skilled in the art.
  • a recombinant virus and/or a chimeric protein of the invention may also be used to provide a composition for the treatment or therapy of a mammal, preferably a human.
  • One or more recombinant viruses and/or one or more chimeric proteins can be formulated in pharmaceutical compositions.
  • These compositions may comprise, in addition to one or more of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient .
  • the precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant or an inert diluent.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • Such compositions and preparations generally contain at least 0.1% by weight of the active ingredient (s) .
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • administration is preferably in a "prophylactically effective amount” or a "therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • a prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
  • the medicaments and pharmaceutical compositions of the invention may be administered by any of several routes, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • the dose of the medicament or pharmaceutical composition is based on well known pharmaceutically acceptable principles. General dosages are based on mg of the active ingredient per kg body weight, for example, 0.01 mg/kg to 100 mg/kg, more preferably 0.5 mg/kg to 10 mg/kg. The dose will depend on the route of administration in addition to the factors described above.
  • the medicaments and pharmaceutical compositions are administered alone or in combination with other medicaments or compositions, or treatments or methods of managing a condition or disease.
  • EnvHA chimeric protein
  • Figure 1 The designed construct maintains disulphide-bonds between positions 68 and 293 of HAl, the eight in the globular head of gpl20, that between residues 30 of HAl and 153 of HA2 , and that between residues 160 and 164 in HA2.
  • Such a structure is expected to possess the stability and therefore the membrane fusion characteristics of native HA and the receptor-binding properties of HIV-I Env.
  • envHA chimeric genes a clone of wt X31- HA was obtained [20] and the env genes of HIV-I strains pNL43 [14] and JRFL [21] were rescued by polymerase chain reaction (PCR) using proviral DNA, cloned and sequenced as described [22] .
  • pNL43 and JRFL were chosen as they are well characterised strains that use CXCR4 and CCR5 chemokine co-receptors respectively [23] .
  • the required fragments of each gene were generated using PCR with native Pfu-polymerase (Stratagene) and a panel of oligonucleotide primers (Table 1) .
  • the fragments were purified from 1.5% ( ⁇ 1 Kb) and 0.5% (>1 Kb) agarose gels using 45 ⁇ m filter units (Millipore #UFC30HVNB) according to manufacturer's instructions and the three required fragments joined together using PCR-splice overlap extension (PCR-SOE) with Pfu-polymerase and primers X31HAFU and X31HARU. All PCRs were performed on a PTC- 100 cycler (MJ Research Inc) using 25 cycles of 96°C/2 min, 50°C/1.5 min, 72°C/lO min (for products up to 1300 bp) or 20 min (for final products of 2360-2600 bp) .
  • PCR-SOE PCR-splice overlap extension
  • the envHA genes were ligated into the vaccinia shuttle vector pRB21 using Pst I and Hind III restriction sites, the resultant ligations used to transform DH5 ⁇ E. coli and resultant clones sequenced prior to being used in the production of recombinant vaccinia (Copenhagen strain) viruses [16, 20] .
  • a number of constructs were made with/without linker sequences (GGGS) at the HAl/gpl20 junctions to allow a degree of flexibility such that the gpl20 globular head might be more easily accommodated on the HA stalk to allow trimer formation (Table 2) .
  • Env, HA and EnvHA chimeric proteins in mammalian cells using recombinant vaccinia virus
  • Recombinant vaccinia viruses were plaque purified twice in CV-I cells before use in experiments [20] .
  • CV-I cells were infected with recombinant vaccinia virus at a moi of 1 and 16 h post-infection cell medium was removed and cells were resuspended in 1 ml of phosphate-buffered saline (PBS) and centrifuged (6K/30 sec) .
  • Cell pellets were lysed with 120 ⁇ l SDS loading buffer (2% SDS, 62.5 mM Tris pH 6.8, 5% ⁇ -mercaptoethanol, 0.1% bromophenol blue) and separated by SDS-PAGE.
  • Western blotting was performed using a 1 : 5000-dilution of anti-Env sheep polyclonal serum (ARP401, UK Centralised Facility for AIDS Reagents; CFAR) , and a 1 : 1000-dilution of anti-X31- HA rabbit polyclonal serum (supplied by D A Steinhauer [35]) , with HRP-labelled donkey antisheep (Sigma, 1:5000- dilution) and HRP-labelled Protein A (Amersham
  • CV-I cells were grown on 13 mm glass coverslips in 24- well tissue culture plates. Cells were infected with recombinant vaccinia virus at a moi of 1 for 16 h and fixed with 4% paraformaldehyde (w/v) in PBS. Surface expressed EnvHA proteins were detected using HIV-I strain-specific anti-Env and anti-HA antibodies. A 1:20 dilution of the monoclonal antibody EVA3012 (CFAR) was used to probe NL43-based constructs, with a fluorescein linked sheep anti-mouse secondary antibody (1:200 dilution, Amersham Biosciences) .
  • EVA3012 CFAR
  • EVA435 1 : 100-dilution, CFAR
  • 1 :200-diluted fluorescein linked donkey anti-rabbit secondary antibody (Amersham Biosciences) were used to probe JRFL-based constructs.
  • cells were permeabilised prior to immunofluorescent staining by treating with PBS/2% Triton X-100 (v/v) for 30min at room temperature. Cells were counterstained with 4 ',6' diamidino-2-phenylindole (DAPI) for 1 min at room temperature to visualize nuclei. Coverslips were mounted on glass slides and analysed on a Nikon Labophot- 2 fluorescent microscope.
  • CV-I cells were infected with recombinant vaccinia virus at a moi of 1.
  • DMEM Dulbecco's modified Eagle medium
  • TPCK-treated trypsin Sigma
  • Cells were then incubated with equivalent concentrations of trypsin inhibitor (0, 2.5, 5, 10, 20 ⁇ g/ml; Sigma) for 10 min at 37°C, washed with PBS and cell lysates prepared.
  • Membrane fusion assays were performed following a method modified from that described previously [26] . Briefly, ghost cells constitutively expressing either the CD4 receptor (g -parental) , or CD4 and CCR5 co-receptor (g- R5) , or CD4 and CXCR4 co-receptor (g-X4) [27] were infected at a moi of 1 with recombinant vaccinia virus . At 16 h post infection, cells were washed with DMEM and incubated with 5 ⁇ g/ml TPCK-treated trypsin for 5 min at 37°C.
  • Cells were then washed and treated for 30 s with cell buffers (20 mM HEPES, 150 mM NaCl, 2 mM CaCl 2 ) that had been adjusted to particular pHs (6.0-5.0 in 0.2 steps) with citrate, as specified in the results section, after which pH was returned to neutral. Cells were incubated in DMEM with 5% fetal calf serum for 1 h at
  • Heterokaryons/syncytia were fixed with PBS/0.25% glutaraldehyde (v/v) , stained with 1% Toluidine Blue (w/v; Sigma) , and photographed using a Nikon Diaphot phase contrast microscope and F-301 camera.
  • the envHA genes were ligated into the vector pEE14tpa to generate pEE14tpa-envHA constructs. These constructs were used to transfect mammalian cells, in particular Chinese Hamster Ovary (CHO) cells. The cells are treated with the drug methyl-sulphoximine (MSX) to allow selection of clones which constitutively express the EnvHA protein. Thus, cell lines are generated which constitutively express EnvHA proteins. Established procedures exist for the purification of HIV envelope and influenza HA proteins from mammalian cells [24, 25] . Generation of recombinant influenza virus expressing EnvHA chimeric proteins
  • the envHA genes are modified using PCR with specific primers to allow incorporation of the genes into the pHH21 vector system [11] by methods familiar to those skilled in the art.
  • the vectors are propagated in E. coli for subsequent generation of recombinant influenza virus.
  • Recombinant influenza viruses bearing a chimeric protein of the invention are produced based on known methods [11, 12] .
  • up to 9 mRNA-sense plasmids encoding influenza proteins
  • 8 vRNA-sense plasmids (carrying the eight influenza genes) to transfect host 293T cells.
  • Suitable transfection reagents include, but are not limited to, Lipofectamine, Effectene, DOTAP and calcium phosphate.
  • the mRNA-sense plasmids provide the viral proteins to allow virus replication and assembly and the vRNA segments provided by the vRNA-sense plasmids are packaged into progeny viruses.
  • Replacement of the HA vRNA plasmid with that of a vector encoding a chimeric protein of the invention means that after one round of infection in the 293T cells, progeny viruses carry the nucleic acid encoding. the chimeric protein and express the chimeric protein on their surfaces.
  • replacement of the HA vRNA plasmid with that of a vector encoding EnvHA means that progeny viruses carry the envHA gene and express EnvHA on their surfaces.
  • these recombinant viruses infect cells expressing CD4 and certain chemokine receptors instead of epithelial cells of the respiratory tract that express suitable sialic acid receptors.
  • wt Envs could fuse all cell types used including Baby Hamster Kidney (BHK) cells which do not express either CD4 or chemokine coreceptors
  • BHK Baby Hamster Kidney
  • wt Envs maintained their co- receptor specificities with JRFL fusing ghost cells expressing CCR5 only whilst NL43 preferentially fused cells expressing CXCR4 although there was some fusion observed on the other ghost cell lines. The latter observation is probably related to the parental and CCR5 expressing ghost cell lines both having low level endogenous expression of CXCR4.
  • EnvHA proteins displayed a mix of the properties of their parental proteins.
  • EnvHA protein In order to use the EnvHA protein for structural studies of gpl20, surface expression is preferable as it infers correct protein folding and can assist protein purification.
  • Initial experiments involving the infection of mammalian cells with recombinant vaccinia viruses produced proteins of the size predicted for EnvHA chimeras.
  • the antigenic characteristics of the Env and HA subunits have been retained as we were able to utilise anti-Env and anti-HA antibodies for the detection of EnvHA, a portion of which was trimeric, in cell lysates and at the cell surface ( Figures 2-6) .
  • Certain highly pathogenic avian influenza viruses have an insertion of a series of basic amino acids adjacent to the trypsin cleavage site thereby increasing the relative size of the "cleavage loop" .
  • the HAs of these viruses can be cleaved by enzymes other than those residing within the tissues of the respiratory and alimentary tracts allowing such viruses to infect other organs within the host resulting in serious systemic disease.
  • Membrane fusion induced by HIV-I Env has been shown to be a staged event with gpl20 CD4 binding inducing conformational changes in gpl20 that create/expose the chemokine co-receptor binding site, with the gpl20/co-receptor interaction driving further conformational changes in the Env gpl20/gp41 trimer to trigger the fusion event .
  • Much effort has been put into mapping the sites on gpl20 responsible for CD4-binding and co-receptor interaction, and whilst the V3-loop plays a major role, other variable domains and amino acid residues in constant regions are involved [1] .
  • EnvHA constructs were designed (Figure 1) to contain all the gpl20 residues identified as being important in these interactions.
  • Figure 1 In the experiments reported here we have not directly monitored co-receptor binding by EnvHAs but results suggest that it is not essential for induction of membrane fusion whereas CD4 binding is.
  • the stability conferred on the EnvHA constructs by the HA1/HA2 stalk region notably with there being a disulphide bond between HAl and HA2 , impedes the conformational change induced by gpl20-CD4 binding thereby preventing the creation/exposure of the co-receptor binding site. If this is so, clearly EnvHA- CD4 binding followed by a low pH trigger is sufficient to induce membrane fusion (Figure 7, Table 3) .
  • HAs of influenza mutants selected in the presence of high concentrations of amantadine contain amino acid substitutions at a number of positions in HAl and/or HA2 that result in fusion occurring at elevated (more neutral) pHs, inferring less structural stability in HA1/HA2 interaction [17] .
  • membrane fusion was monitored against a pH range of 6-5 in 0.2 steps it was found that the pH at which fusion occurred was higher by approximately 0.2 pH units for EnvHAs when compared to wt HA. This shift is comparable to those observed for influenza mutants containing amino acid substitutions in the globular head of HAl [17] .
  • EnvHAs might be expected to be less stable than HA as the globular head of HAl (224 amino acids and 2 N-linked glycosylation sequons) was replaced with gpl20 proteins of approximately twice the size (NL43, 407 amino acids and 23 N-linked glycosylation sequons; JRFL, 401 amino acids and 22 N linked glycosylation sequons) .
  • the membrane fusion induced by influenza HA has been shown to be pH, temperature and time dependent [20, 26] . In the current work all fusion experiments were conducted at 37°C and there were no discernable time differences for heterokaryon formation caused by X31-HA and EnvHAs .
  • recombinant influenza viruses expressing EnvHA chimeric proteins allow the development of strategies for the effective treatment of HIV-I.
  • Such recombinant viruses target CD4- expressing cells.
  • Recombinant viruses additionally carrying a conditionally lethal toxic gene, such as HSVTK [6] or reverse Caspase-3 that induces apoptosis [18] , under the control of an HIV-2 LTR, allow the targeted killing of only HIV-infected CD4-expressing cells such that uninfected CD4 cells remain viable to perform their immune functions.
  • Wilson IA, Skehel JJ, Wiley DC Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature 1981;289: 366-373.
  • Flandorfer A, Garcia-Sastre A, Basler CF, Palese P Chimeric influenza A viruses with a functional influenza
  • Cullen BR Nuclear mRNA export: insights from virology. Trends in Biochemical Sciences 2003,-28: 419-24.
  • Doms RW, Edinger AL, Moore JP Coreceptor use by primate lentiviruses; in Korber B, Kuiken C, Foley B, Hahn B, McCutchan F, Mellors JW and Sodroski J (eds) : Human retroviruses and AIDS. Los Alamos, Theoretical
  • Table 2 Composition of EnvHA chimeric proteins.

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Abstract

L'invention porte sur des protéines chimères comprenant une séquence d'acides aminés d'une protéine virale et présentant des propriétés structurelles et fonctionnelles de protéines parents natives, l'invention portant également sur des acides nucléiques et sur des vecteurs d'expression codant ces protéines et sur des cellules hôtes exprimant ces protéines. L'invention porte également sur l'expression des protéines chimères, incluses dans des virus de recombinaison, ainsi que sur leurs utilisations et sur les virus de recombinaison les exprimant.
PCT/GB2005/003053 2004-08-04 2005-08-02 Proteines chimeres leurs utilisations Ceased WO2006013367A2 (fr)

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