US20090041725A1 - Replication-Deficient RNA Viruses as Vaccines - Google Patents

Replication-Deficient RNA Viruses as Vaccines Download PDF

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US20090041725A1
US20090041725A1 US11/836,322 US83632207A US2009041725A1 US 20090041725 A1 US20090041725 A1 US 20090041725A1 US 83632207 A US83632207 A US 83632207A US 2009041725 A1 US2009041725 A1 US 2009041725A1
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virus
egfp
cell
sevv
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Wolfgang J. Neubert
Sascha Bossow
Sabine Schlecht
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MAX-PLANK DER WISSENSCHAFTEN EV Gesell zur Forderung
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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    • 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
    • C07K14/08—RNA viruses
    • C07K14/115—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18811—Sendai virus
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a replication-defective and transcription-competent negative-strand RNA virus, which can be used for the expression of transgenes and in particular for the area of vaccine development.
  • Another target group are the elderly, whose immune system is no longer so efficient, so that it can be overloaded by vaccination, and increased multiplication of the vaccine virus may lead to vaccination lesions. There is therefore the problem of making the immunologically excellent live vaccination even safer for application in certain target groups, as well as increasing the safety profile for general use.
  • EP-A-0 702 085 describes the production of recombinant, infectious, replicating unsegmented negative-strand RNA viruses from cloned cDNA.
  • EP-A-0 863 202 describes a recombinant Sendai virus, in whose genome a heterologous gene is inserted or a gene is deleted or inactivated, but whose genome replication is still intact.
  • Negative-strand RNA viruses are especially suitable as the backbone of vaccines, as their multiplication in the cytoplasm takes place at the RNA level and genes within the viral genome can simply be exchanged.
  • RNA viruses with surface proteins of various virus types and using them as vaccines in animal experiments (Schmidt et al., J. Virol. 75 (2001), 4594-4603 and WO 01/42445).
  • this bovine parainfluenza virus with human PIV 3 and RSV surface antigens should already be sufficiently attenuated for application in humans. Reversions to the wild type should not be expected, as complete genes were exchanged for viral surface proteins. Clinical testing of the vaccine has already begun.
  • virus mutants described are, however, replication-competent, virus multiplication will undoubtedly occur in the vaccinee, the intensity of which is attenuated by modification of the virus, but is not excluded completely.
  • the intensity of the viremia that is to be expected and therefore the side-effects suffered by the vaccinee then depend on individual factors.
  • an attempt is to be made to substantially reduce the risks of live vaccination, and especially the risks for vaccinees in certain target groups.
  • a fundamental difficulty in this approach is that the viral RNA polymerase performs two functions: synthesis of viral mRNA and multiplication of the viral genomes. This coupling must be removed in the new vaccine, as the vaccine must now only be capable of synthesis of viral mRNA.
  • Another problem is that the recombinant virus must perform efficient synthesis of viral mRNA, if it is to be suitable at all as live vaccine. There are thus two basically contradictory requirements, which mean that considerable difficulties are to be expected in the production of safe, but efficient live vaccines based on negative-strand RNA viruses.
  • Shoji et al. describe the production and characterization of a P gene-deficient rabies virus.
  • the virus was produced by means of P-protein-expressing helper cells. Without de novo synthesis of P-protein, the viruses are only capable of primary transcription. The slight viral gene expression is manifested in a very weak signal for N-protein in immunofluorescence and only in very few cells, and convincing proof of this slight viral gene expression will only be provided by PCR analysis.
  • Use of this mutant virus in a challenge test in the mouse model should show protection, but there is no control experiment with transcription-inactive virus and the time interval for the viral challenge is too short. The duration of supposed protection is not being investigated. The use of such mutant viruses for the development of an attenuated rabies vaccine therefore seems not to be very promising.
  • decoupling of the replication and transcription functions can be achieved by partially removing the constituents of polymerase that are essential for the genome replication function. This may involve one of the viral proteins N, P and L, or a special functional domain of such a protein.
  • One object of the present invention is thus a recombinant negative-strand RNA virus, which is replication-deficient and transcription-competent.
  • the virus according to the invention contains a viral genome with a mutation in at least one of the genes N, L and P, with the mutation leading to loss of genome replication without loss of secondary transcription capacity.
  • the virus according to the invention is a prerequisite for the production of live vaccines, especially for the production of live vaccines with an enhanced safety profile, which is especially suitable for use in high-risk patients with a weak or damaged immune system.
  • the invention also relates to a nucleocapsid of the recombinant virus, comprising the viral negative-strand RNA, complexed with the proteins N, L and P plus the negative-strand RNA of the recombinant virus in isolated form.
  • the invention also relates to a cDNA, which codes for a negative-strand RNA according to the invention, in particular a viral RNA and/or an RNA complementary to it.
  • the invention further relates to a cell line for multiplication of the recombinant negative-strand RNA virus according to the invention.
  • the recombinant negative-strand RNA virus according to the invention can be obtained by mutation of a starting virus in at least one of the genes N, L and P.
  • the starting virus can be a natural negative-strand RNA virus, especially from the families Paramyxoviridae or Rhabdoviridae or recombinant variants thereof.
  • paramyxoviruses e.g. Sendai virus, human or bovine parainfluenza virus, e.g. human parainfluenza virus (hPIV) type 1, 2, 3, 4a or 4b, Newcastle disease virus, mumps virus, measles virus or human respiratory syncytial virus (hRSV) or rhabdoviruses, e.g.
  • VSV vesicular stomatitis virus
  • the virus is a Sendai virus, e.g. of the Fushimi strain (ATCC VR105).
  • Fushimi strain ATCC VR105
  • RNA viruses are representatives of the Rhabdoviridae, Filoviridae, Bornaviridae, Arenaviridae or Bunyaviridae, e.g. VSV.
  • the Sendai virus is an enveloped virus with a helical nucleocapsid ( FIG. 1 ).
  • the envelope consists of a lipid membrane, which is derived from the plasma membrane of the host cell from which the virus was released.
  • Transmembrane glycoproteins namely the fusion protein (F) and hemagglutinin-neuramidase (HN)
  • F fusion protein
  • HN hemagglutinin-neuramidase
  • M matrix protein lines the inside of the membrane.
  • the nucleocapsid contained in the envelope consists of single-stranded RNA complexed with nucleoprotein (N), with in each case 6 nucleotides of the RNA bound by one N protein, an RNA-dependent RNA polymerase (L) and the co-factor phosphoprotein (P).
  • N nucleoprotein
  • L RNA-dependent RNA polymerase
  • P co-factor phosphoprotein
  • the negative-strand RNA genome of the Sendai virus contains the genes of the 6 structural proteins in the order: 3′-N-P/C-M-F-HN-L-5′ ( FIG. 2 ).
  • the P/C gene codes for a total of 8 proteins, the structural phosphoprotein and all non-structural proteins known to date.
  • the proteins P, N and L are important for functional transcription and replication (Lamb et al., Paramyxoviridae: The Viruses and their Replication. Fields Virology, 4th edition (2001), Lippincott, Williams & Wilkins, Philadelphia, 1305-1340).
  • the recombinant negative-strand RNA virus according to the invention contains a mutation in at least one of the genes N, L and P.
  • the mutation can be a deletion, substitution and/or insertion in one of the genes N, L or P, which gives rise to a replication deficiency of the virus, but does not disturb the capacity for transcription.
  • the mutation preferably affects a partial sequence of the proteins encoded by the genes N, L and/or P, which is necessary for replication, whereas other partial sequences necessary for transcription remain functional.
  • the recombinant virus has a mutation in gene P, namely in an N-terminal partial sequence of gene P.
  • the mutation preferably affects at least the region of amino acids 33-41 of the protein P, which are important for the capacity for replication. It is further preferred that the C-terminal region (starting from amino acid 320) does not have any mutations impairing the transcription function.
  • the mutation is a mutation in the region of amino acids 2-77 leading to loss of capacity for replication, for example a deletion of (a) the amino acids 2-77 of the protein encoded by gene P or (b) a partial sequence of (a) sufficient for loss of the capacity for replication.
  • Corresponding mutations can also take place in P proteins of other negative-strand RNA viruses, e.g. of other paramyxoviruses, e.g. hPIV3.
  • the recombinant virus according to the invention is replication-deficient and transcription-competent. Loss of the capacity for replication means that in a target cell (a cell which does not produce in trans any of the functions deleted by mutation) no detectable virus genome multiplication is found, and in contrast to a reduced or conditional replication deficiency, also no permissive conditions exist, in which replication can occur.
  • the loss of the capacity for replication can be determined as described in Example 8.
  • the virus according to the invention is capable of transcribing the gene products encoded by it after infection in a target cell, so that expression of the viral proteins including one or more heterologous gene products can take place in the target cell. It is important that the recombinant virus according to the invention should possess the capacity for secondary transcription, i.e.
  • the viral gene products that arise through primary transcription with the protein components originally contained in the nucleocapsid are capable of bringing about and/or supporting a secondary transcription themselves.
  • the extent of the secondary transcription then leads to protein synthesis of preferably at least 1%, at least 2%, at least 3%, at least 4% or at least 5% relative to a corresponding wild-type virus, i.e. a virus without the mutation in at least one of the genes N, L and P.
  • the capacity for secondary transcription can be reduced relative to the corresponding wild-type virus, though preferably at most by a factor of 20, especially preferably at most by a factor of 10.
  • the capacity for secondary transcription can be determined as in Example 7.1 and/or 7.3 by quantitative determination of the expression of a heterologous gene product, e.g. a reporter protein.
  • the recombinant virus according to the invention preferably contains at least one transgene, i.e. at least one sequence coding for a heterologous gene product.
  • the heterologous gene product can be a protein, for example a reporter protein, e.g. a fluorescence protein such as GFP or a derivative thereof, or an antigen, against which an immune response is to be produced, or a therapeutic protein, e.g. a protein for virotherapy or a functional RNA molecule, e.g. an antisense RNA, a ribozyme or an siRNA molecule capable of RNA interference.
  • the heterologous gene product is an antigen, originating from a pathogen, such as a virus, a bacterium, a fungus or a protozoon, a tumor antigen or an autoantigen.
  • the antigen is a viral antigen, derived from a heterologous negative-strand RNA virus, such as a human parainfluenza virus or RSV, e.g. hPIV3 F and HN or hRSV F and G.
  • the virus according to the invention can contain one or more, e.g. two or three, sequences coding for a heterologous gene product.
  • Sequences coding for heterologous gene products can be inserted in the genome of the recombinant virus.
  • sequences coding for homologous gene products e.g. genes F and/or HN
  • sequences coding for heterologous gene products e.g. chimeric gene products. Combinations of inserted and substituted transgenes are also possible.
  • sequences of a Sendai virus can be replaced completely or partially with heterologous sequences of other negative-strand RNA viruses, e.g. with sequences of parainfluenza viruses, e.g. hPIV3, and/or with sequences of RSV.
  • Use of chimeric sequences is especially preferred, i.e. sequences comprising segments of the base virus genome and segments of a heterologous virus genome.
  • chimeric genes F and/or HN can be inserted in the virus genome, which comprise sequences of the base virus genome, e.g. Sendai virus and heterologous sequences, e.g. from human parainfluenza viruses such as hPIV3, and/or RSV.
  • the recombinant virus can contain one or more different transgenes. If several transgenes are present, these can be of the same or of different origin, which can be derived for example from a single or from several different pathogens, e.g. viruses. Thus, transgenes from several, e.g. 2 or 3 different pathogens, preferably viruses, and especially negative-strand RNA viruses, can be present.
  • transgenes are inserted in the 3′ region of the viral genome.
  • Insertion is effected for example in the form of transcription cassettes, with one or more transcription cassettes with singular restriction sites for integration of the respective reading frames inserted directly after the leader region at the vector level (i.e. at the level of the vector, e.g. of a plasmid vector, which codes for the negative-strand RNA). Integration of several transgenes is preferably effected in independent transcription cassettes in each case.
  • a transcription cassette preferably contains the sequence coding for the heterologous gene product in operational linkage with a transcription start sequence and a transcription termination sequence and preferably translation signals.
  • a further object of the present invention is a single-stranded or double-stranded DNA molecule, e.g. a cDNA molecule, which codes for a recombinant negative-strand RNA virus genome according to the invention or a precursor thereof or the virus-antigenome or a precursor thereof.
  • the term “precursor” means in this context that the DNA molecule does not yet contain a sequence coding for a heterologous gene product, but only a cloning site for insertion of such a sequence.
  • the cloning site can be a restriction site, for example a singular or non-singular restriction site in the DNA or a multiple cloning site, containing several consecutive restriction sites, preferably singular restriction sites.
  • the DNA molecule coding for the virus genome and/or the complementary sequence is preferably in operational linkage with suitable expression control sequences.
  • the DNA molecule is preferably a vector, for example a plasmid vector, which is suitable for propagation in a suitable host cell, i.e. in a vector or plasmid amplification cell, preferably in a prokaryotic cell, but also in a eukaryotic cell, especially in a mammalian cell, and has the necessary genetic elements for this, such as replication origin, integration sequences and/or selectable marker sequences.
  • a vector for example a plasmid vector, which is suitable for propagation in a suitable host cell, i.e. in a vector or plasmid amplification cell, preferably in a prokaryotic cell, but also in a eukaryotic cell, especially in a mammalian cell, and has the necessary genetic elements for this, such as replication origin, integration sequences and/or selectable marker sequences.
  • the DNA molecule contains the sequence coding for the recombinant virus or the complementary sequence, preferably under the control of a transcription signal, so that during transcription with a DNA-dependent RNA polymerase in a host cell suitable for the initial production of the virus, i.e. in a virus production cell, the viral negative-strand RNA can be formed.
  • the transcription signal is selected to permit efficient transcription of the DNA in the host cell used in each case. It is also possible to use a heterologous transcription signal for the particular cell, e.g. a bacteriophage promoter, such as the T7 or SP6 promoter, and then the virus production cell must also contain a corresponding heterologous DNA-dependent RNA polymerase, e.g.
  • the DNA molecule further contains, preferably at the 3′ end of the sequence coding for the recombinant virus, a transcription terminator and a ribozyme sequence, which permits cleavage of the transcript at the end of the viral sequence.
  • the virus production cell is preferably a eukaryotic cell and especially a mammalian cell.
  • the virus production cell according to the invention also contains helper sequences, whose gene products permit assembly of the recombinant virus RNA according to the invention in trans.
  • the cell can for example additionally contain one or more vectors which produce the N protein, the P protein and/or the L protein in trans. This makes assembly of nucleocapsids of the recombinant virus according to the invention possible in the production cell.
  • Multiplication of the recombinant virus initially assembled in the virus production cell takes place in a virus multiplication cell, which is infected with the virus according to the invention.
  • the virus multiplication cell contains helper sequences as mentioned above, for production of the N protein, the P protein and/or the L protein in trans.
  • a virus multiplication cell is used in which there is stable expression of the helper sequences, e.g. by genomic integration.
  • the virus multiplication cell is preferably a mammalian cell.
  • An especially preferred multiplication cell is cell H29, derived from a 293 cell, of a human embryonic renal fibroblast cell line, or a cell derived from that.
  • Cell H29 was deposited on 11.05.2004 (DSM ACC 2702) in accordance with the provisions of the Budapest Treaty with the Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, Braunschweig, Mascheroder Weg. Vero cells, of a renal cell line from the African green monkey, or cells derived from LLCMK2 cells, of a renal cell line from the rhesus monkey, which have been stably transfected with corresponding helper sequences, e.g. SeV N and P genes, are also suitable.
  • helper sequences e.g. SeV N and P genes
  • the invention therefore further relates to a cell, preferably a eukaryotic cell and especially preferably a mammalian cell, which contains (i) a DNA molecule, which codes for the genome of the recombinant virus according to the invention and/or the complementary sequence thereof or a precursor thereof, and/or (ii) an RNA genome of the virus according to the invention.
  • the cell can be a vector multiplication cell, a virus production cell or a virus multiplication cell, as explained previously.
  • the cell is a vector multiplication cell, e.g. a plasmid multiplication cell
  • a vector multiplication cell e.g. a plasmid multiplication cell
  • any cell that is suitable for multiplication of the corresponding vector can be used, e.g. also a prokaryotic cell such as a transformed E. coli cell.
  • the cell is a virus production or multiplication cell, it contains helper sequences for production of the virus proteins N, P and/or L in trans.
  • the DNA inserted in a virus production cell is preferably under the control of a heterologous transcription signal, and advantageously the cell further contains a DNA that codes for a heterologous DNA-dependent RNA polymerase, which recognizes the heterologous transcription signal and effects transcription of the DNA coding for the recombinant negative-strand RNA virus.
  • the cell is a virus multiplication cell, it is infected with a genomic viral RNA molecule, e.g. in the form of a nucleocapsid, and contains the helper sequences in stably expressible form.
  • the present invention further relates to a method of production of a recombinant negative-strand RNA virus according to the invention comprising the steps: (a) preparation of a virus production cell, which is transfected with a DNA molecule that codes for the genome of a negative-strand RNA virus, containing a mutation in at least one of the genes N, L and P, which leads to loss of the capacity for genome replication without loss of the capacity for transcription, and optionally at least one sequence coding for a heterologous gene product, and (b) cultivation of the host cell in conditions such that transcription of the DNA molecule according to (a) takes place and the recombinant negative-strand RNA virus is formed initially.
  • the host cell is preferably capable of producing the N protein, the P protein and/or the L protein in trans, e.g. by transfection with the corresponding helper plasmids which contain sequences coding for the proteins N, P and/or L.
  • a cell which stably expresses, constitutively or inducibly, the proteins N, L and/or P, preferably at least protein P of a negative-strand RNA virus.
  • the invention thus also relates to a method of multiplication of a recombinant negative-strand RNA virus according to the invention, comprising the steps: (a) preparation of a virus multiplication cell, which is infected with the genome of a negative-strand RNA virus, containing a mutation in at least one of the genes N, L and P, which leads to loss of the capacity for genome replication without loss of the capacity for transcription, and optionally at least one sequence coding for a heterologous gene product, and (b) cultivation of the host cell in conditions such that multiplication of the virus takes place.
  • the present invention further relates to a pharmaceutical composition, which contains a recombinant replication-deficient and transcription-competent negative-strand RNA virus, as stated previously, or its nucleocapsid as active substance and optionally as pharmaceutically usual vehicles and/or excipients.
  • the pharmaceutical composition is suitable for applications in human and veterinary medicine. It can be used in particular as vaccine or for antitumor therapy, in particular for application in high-risk patients, such as children, the elderly and/or persons with a damaged or weak immune system.
  • the pharmaceutical composition can contain the negative-strand RNA virus in its native viral envelope.
  • vaccine is especially preferred, e.g. as vaccine against pathogens such as viruses, bacteria or protozoa.
  • pathogens such as viruses, bacteria or protozoa.
  • the recombinant virus contains a transgene or several transgenes of the same origin, e.g. from a single pathogen, it is a monovalent vaccine.
  • the recombinant virus contains transgenes of various origins, it can be used as a polyvalent vaccine, e.g. as bivalent or trivalent vaccine.
  • it is possible to produce a polyvalent vaccine against several pathogenic viruses e.g. against several pathogenic negative-strand RNA viruses, such as human parainfluenza virus and RSV.
  • a vaccine according to the invention is capable of triggering a humoral immune response, preferably the formation of neutralizing antibodies, and/or a T-cell immune response. Especially preferably, a humoral immune response and a T-cell immune response are triggered.
  • the pharmaceutical composition can be in the form of a solution, a suspension, a lyophilizate or in any other suitable form.
  • the composition can contain agents for adjusting the pH value, buffers, agents for adjusting tonicity, wetting agents and the like, and adjuvants. It can be administered by the usual routes, e.g. oral, topical, nasal, pulmonary etc., in the form of aerosols, liquids, powders etc.
  • the therapeutically effective dose of the virus is administered to the patient, and this dose depends on the particular application (e.g. virotherapy or vaccine), on the type of disease, the patient's weight and state of health, the manner of administration and the formulation etc.
  • virus particles especially preferably about 10 4 to 10 6 virus particles are administered per application.
  • virus particles can be administered together, e.g. in the case of combination vaccinations.
  • Administration can be single or multiple, as required.
  • Preferred fields of application are for example the prevention or treatment of respiratory viral diseases.
  • FIG. 1 shows the morphology of a Sendai virus (SeV) according to Fields (Virology. Lippincott, Williams and Wilkins (2001), 4th edition; modified).
  • the genome comprises a single-stranded RNA, which has the proteins N, P and L in the form of a nucleocapsid.
  • the nucleocapsid is surrounded by a membrane envelope, in which the proteins HN and F (each consisting of one F 1 and F 2 subunit) are incorporated.
  • Protein M is associated with the inside of the membrane, and is also bound to the nucleocapsid components at the same time.
  • the single-stranded negatively oriented RNA genome of the wild-type Sendai virus comprises 15384 nucleotides.
  • the genes of the 6 structural proteins are located thereon in the order 3′-N-P/C-M-F-HN-L-5′ ( FIG. 2 ). Between the genes there are transitions of 50-80 nucleotides, each containing a highly conserved region of 22 nucleotides: the termination signal of the preceding gene, an intergenic sequence and the start signal for the next gene.
  • a unit comprising start signal, open reading frame (ORF), optionally untranslated regions and termination signal is called a transcription cassette.
  • a leader sequence (ld) 55 nucleotides long, which is transcribed, but not translated.
  • the L gene is followed by a trailer sequence (tr) 54 nucleotides long.
  • the ld and tr regions function as genomic and antigenomic promoters for the replication of genome or antigenome.
  • the mRNA molecules formed by transcription are monocistronic.
  • the multiplication cycle of the Sendai virus comprises entry into the host cell, transcription and translation plus replication and virus maturation followed by release of newly produced viruses.
  • the proteins N, P and L are involved in the transcription process, with L representing the viral RNA-dependent RNA polymerase with all catalytic activities.
  • L representing the viral RNA-dependent RNA polymerase with all catalytic activities.
  • the genome of the Sendai virus is in negative-strand orientation, the viral RNA cannot be converted to proteins directly.
  • FIG. 3 is a schematic representation of the transcription mode of the Sendai virus.
  • the polymerase complex comprising an L protein and a homotetramer of P proteins, migrates along the RNA packed with N proteins toward the 5′ end.
  • the genetic information on the genomic negative-strand RNA is read off and transcribed into positive-strand mRNA.
  • Replication of the genome comprises the production of new virus genomes with negative polarity. For this, first antigenomes are formed, which then serve as matrixes for the formation of the genomes. As transcription begins at the 3′ end of the genome (ld), switching from transcription to replication mode is required. This switching is determined by the amount of free N protein in the cell. Replication cannot take place until sufficient N protein has been formed after translation of mRNA molecules. Once an antigenome, which is complexed with N proteins over its entire length, is present, this can serve as a matrix for the production of further genomes. These are also packed directly with N proteins. Once again the proteins N, P and L are responsible for the process of replication ( FIG. 4 ).
  • RNA and viral proteins are transported in the form of secretory vesicles to the cytoplasmic membrane, where enveloping with viral surface proteins and budding of virus particles occur.
  • recombinant virus mutants are prepared, in which the functions of transcription and replication are decoupled, i.e. the viruses are transcription-competent, but replication-deficient.
  • the missing genome replication function must, for the production of virus particles and/or their nucleocapsids, be compensated by helper cells which complement the missing or functionally deficient viral protein in trans.
  • helper cell of this kind is the cell line H29 (Willenbrink and Neubert, J. Virol. 68 (1994), 8413-8417).
  • this cell line was deposited under reference DSM ACC2702 on 11.05.2004 in accordance with the provisions of the Budapest Treaty.
  • FIG. 5 is a schematic representation of the P protein with its N-terminal and C-terminal domains (PNT, PCT), the tetramerization domain (amino acids 320-446), the P:L domain (amino acids 411-445) and the P:RNP binding domain (amino acids 479-568).
  • PNT N-terminal and C-terminal domains
  • amino acids 320-446 the tetramerization domain
  • P:L domain amino acids 411-445
  • P:RNP binding domain amino acids 479-568.
  • helper cell line H29 (DSM ACC2702), which among other things provides the required wild-type P protein, multiplication of the virus mutant can be achieved.
  • the efficiency of virus multiplication is approx. 45% compared with wild-type Sendai viruses.
  • the virus mutant according to the invention is able to express virus-encoded transgenes in infected cells.
  • the shortened protein P produced by the virus mutant gives sufficient support for secondary viral mRNA synthesis. Synthesis of virus-encoded proteins continues over several days in the infected cells and is only reduced by a factor of approx. 10 relative to the wild-type virus, so that a sufficient immune response can reliably be expected on using the mutant as vaccine.
  • PCR X I and PCR X II were prepared for the production of pSeV-X ( FIG. 6 ).
  • PCR X I (370 bp) comprises the sequence of the T7 promoter (T7-prom.), the leader (ld) sequence, the N-gene start with 5′-NTR up to before the start codon of the N-ORF (open reading frame).
  • T7-prom. T7 promoter
  • leader (ld) sequence the N-gene start with 5′-NTR up to before the start codon of the N-ORF (open reading frame).
  • reverse primer X I (+) Table 3
  • 24 nucleotides of the N-gene stop sequence of PCR X I inserted by the mutagenic primer X I (+), serve in the subsequent fusion step as the region overlapping PCR X II.
  • PCR X II (970 bp) comprises the sequence of the N-gene start and the first third of the N-ORF. Via the forward primer X II, the sequence of the 3′-NTR N and the gene-stop sequence of N, as well as the intergenic region (IR) were attached.
  • the reverse primer XII (+) binds in the first third of the N-ORF just behind the singular SphI site in the SeV genome.
  • the amplicon PCR X I was complementary, in the 3′ region, to the 5′ region of PCR X II. Through this overlapping region, the two PCR fragments X I and X II could be fused.
  • the fusion product (1310 bp) could be inserted, by restriction cleavage with the enzymes MluI and SphI, in the vector pSeV, also treated with MluI and SphI.
  • plasmid-DNA was isolated by plasmid preparation, and verified by restriction analysis and sequencing for correct insertion of the transcription cassette. The cDNA construct pSeV-X was thus made available.
  • the gene for the enhanced green fluorescent protein (eGFP) was now inserted in the empty cassette of pSeV-X.
  • the eGFP-ORF was amplified by PCR from the expression plasmid pEGFP-N1 (from Clontech), maintaining the “rule of six” and achieving attachment of two flanking NotI sites by means of mutagenic primers.
  • the resultant 771-bp PCR fragment was cleaved with the restriction enzyme NotI and a 738-bp fragment was isolated by gel elution, and was inserted via the NotI site of pSeV-X in its “empty” transcription cassette pSeV-X.
  • the cDNA construct pSeV-eGFP was made available.
  • pSeV-X-X With the construct pSeV-X-X, two additional transcription cassettes were to be made available, in which two transgenes can be incorporated.
  • the use of pSeV-X-X as base vector for the production of the replication-deficient vectors should make it possible to equip the vector with multivalent, e.g. trivalent, properties.
  • pSeV-X-X was produced via a PCR reaction, in which pSeV-X served as template ( FIG. 7 ).
  • the primer XX-forward hybridizes with pSeV-X in the region of the NotI site and the 3′-NTR of the second transcription cassette that is to be integrated.
  • a singular SgrAI restriction site was introduced by means of the XX-forward primer between the NotI site and the 3′-NTR. It serves as singular restriction site for the later insertion of the ORF of a transgene. Gene stop, intergenic region (IR), gene start and 5′-NTR follow in the PCR product XX.
  • the singular restriction sites FseI and Nru I were inserted by the primer XX (+), which hybridizes with the 5′-NTR.
  • the FseI site serves for incorporation of the ORF of a second transgene.
  • the singular Nru I site was cloned-in prospectively, so as to be able to integrate a third transcription cassette if necessary.
  • Primer XX (+) hybridizes in the 3′ region with the sequence of the NotI site of pSeV-X.
  • the PCR product XX (220 bp) was treated with the restriction enzyme NotI and a fragment of 144 bp was isolated by gel extraction.
  • the two transcription cassettes (X) of pSeV-X-X were provided with reading frames of two different fluorescent proteins.
  • the reading frame for the fluorescent protein eGFP from the expression plasmid pEGFP-N1 was amplified by PCR while observing the “rule of six”, attaching two flanking SgrAI sites by means of mutagenic primers. After restriction cleavage with SgrAI and gel elution, the approx. 738-bp fragment could be incorporated in the first transcription cassette of pSeV-X-X (pSeV-eGFP-X).
  • cDNA constructs pSeVV-eGFP- ⁇ N, - ⁇ P and - ⁇ L were produced, which code for replication-deficient Sendai viruses, in each of which the gene for the protein N, P and L has been deleted.
  • N, P or L had to be deleted while observing the rule of six, and a non-coding transcription cassette was to be retained at the corresponding position ( FIG. 8A ).
  • an additional functional transcription cassette into which a further transgene can be inserted if required, was to be made available, for later applications, in each cDNA construct pSeVV-eGFP- ⁇ N, - ⁇ P and - ⁇ L.
  • the deletion mutant pSeVV-eGFP-P ⁇ 2-77 was produced, which codes for an N-terminal-shortened P protein lacking amino acids 2 to 77 ( FIG. 8B ).
  • the clonings pSeVV-eGFP- ⁇ N, - ⁇ P and - ⁇ L were all carried out according to the same principle. As an example, the cloning of pSeVV-eGFP- ⁇ P will be described in detail in the next section. Then just the differences in the clonings of pSeVV-eGFP- ⁇ N, and - ⁇ L will be presented in a table.
  • the ORF of the P protein was removed from the cDNA construct of the replication-competent virus pSeV-eGFP, to produce the new cDNA pSeVV-eGFP- ⁇ P, coding for the replication-deficient vector.
  • An XhoI restriction site was used instead of the P-ORF.
  • PCR ⁇ P I For the cloning of pSeVV-eGFP- ⁇ P, two PCR fragments named PCR ⁇ P I and PCR ⁇ P II were produced and then fused. pSeV-eGFP served as template for both PCR reactions.
  • fragment PCR ⁇ P I (1272 bp)
  • hybridization with the template in the region of the N ORF was achieved before a singular SphI site.
  • the reverse primer ⁇ P I (+) hybridizes with the template in the 5′-NTR region of the P-gene up to before the ATG codon of P and inserts the restriction site XhoI there.
  • the fragment PCR ⁇ P II comprises 1938 bp, and pSeV also serves as template here.
  • the forward primer ⁇ P II hybridizes with a portion of the 5′-NTR P sequence and attaches an XhoI site.
  • the reverse primer of PCR ⁇ P II (+) binds in the ORF of the F gene after a singular Eco47III site and additionally has an artificial MluI site.
  • the two PCR fragments ⁇ P I and ⁇ P II were combined via the XhoI site.
  • the fusion product comprising a partial sequence of the N ORF, the non-coding P-transcription cassette with inserted XhoI restriction site, the M plus a quarter of the F ORF—was cleaved with the restriction enzymes SphI and MiuI, intercloned and sequence-verified.
  • An SphI-Eco47III fragment with a size of 3006 bp was cut out of a subclone with correct sequence and was ligated in the identically treated vector pSeV-eGFP.
  • a corresponding pSeVV-eGFP- ⁇ P clone (genomic viral cDNA) was now ready, after sequence verification, for the production of the replication-deficient SeVV-eGFP- ⁇ P ( FIG. 9 ).
  • a PCR with two mutagenic primers was employed for constructing the deletion mutant pSeVV-eGFP-P ⁇ 2-77.
  • the forward primer “XhoI P ⁇ 2-77” contains an XhoI site, followed by an ATG start codon plus codons for the amino acids 78 to 86 of the P protein.
  • the reverse primer “P ⁇ 2-77 (+) XhoI” contains the last 10 codons of the P protein and an XhoI site.
  • the reading frame of the P protein shortened by 76 amino acids at the N-terminal was produced by PCR, starting from the template pSeV, observing the rule of six.
  • the XhoI-cleaved, 1488-bp fragment was inserted via two cloning steps into the non-coding transcription cassette of pSeVV-eGFP- ⁇ P at the position of the original P-ORF. After sequence verification, a genomic cDNA clone was now also ready for production of the replication-deficient SeVV-eGFP-P ⁇ 2-77.
  • fusion PCR products were cleaved with restriction enzymes which occur singly in pSeV-eGFP and allow the insertion of the corresponding fusion product in pSeV-eGFP (e.g.: NarI when cloning pSeVV-eGFP- ⁇ N, see Table 1).
  • restriction enzymes which occur singly in pSeV-eGFP and allow the insertion of the corresponding fusion product in pSeV-eGFP (e.g.: NarI when cloning pSeVV-eGFP- ⁇ N, see Table 1).
  • the purified cleavage product was inserted by ligation in the vector pSeV-eGFP, which was also digested with the corresponding enzymes.
  • E. coli cells were transformed with a portion of the ligation preparations and plasmid DNA of the clones obtained was isolated by plasmid mini-preparation. After verification of the correct sequence by restriction analysis and sequencing, a plasmid preparation was prepared from one positive clone in each case (DNA Maxi Prep-Kit, Qiagen), and the various pSeVV-eGFP- ⁇ X were thus ready for the production of recombinant deletion mutants.
  • SeVV-eGFP- ⁇ X Replication-deficient SeV vectors
  • the plasmid-encoded cDNA of pSeVV-eGFP- ⁇ N, - ⁇ P and - ⁇ L lacks the genomic information of one of the genes N, P or L in each case. Accordingly the nucleocapsids initially produced are only able to express two of the genes N, P or L in each case. The amount of the missing protein required for multiplication of SeVV-eGFP- ⁇ N, - ⁇ P and - ⁇ L nucleocapsids must therefore be provided exclusively via the T7-promoter-controlled expression of the plasmid-encoded genes N, P and L.
  • Production of the replication-deficient SeVV-eGFP- ⁇ N, - ⁇ P and - ⁇ L was similar to the production of replication-competent SeV variants in HeLa cells (ATCC CCL2) or BSR-T7 cells. After incubation of the HeLa cells for 48 hours, we investigated whether viral particles of SeVV-eGFP- ⁇ N, - ⁇ P or - ⁇ L had been released into the culture supernatants, and how many initial deletion mutants had been produced.
  • SeVV-eGFP- ⁇ N, - ⁇ P or - ⁇ L have, in contrast to the recombinant SeV wt, the reporter gene for eGFP integrated in the 3′ region. This detection marker was now used for analyzing how many SeVV-eGFP- ⁇ X had been formed.
  • SeVV-eGFP- ⁇ N, - ⁇ P or - ⁇ L and SeV wt show that all three virus mutants SeVV-eGFP- ⁇ X can be produced initially and after initial production are also capable of infecting cells, which can be detected by a detectable eGFP transgene expression.
  • Co-infection of Vero cells with about 100 particles of the replication-competent virus SeV-eGFP and SeV wt was used as positive control.
  • multiplication of all three deletion mutants was observed: in each case, at first there was only fluorescence of individual Vero cells, which had been co-infected with SeVV-eGFP- ⁇ X and SeV wt. After about 24 h, newly synthesized virus particles were released from these cells, and were capable of penetrating nearby cells.
  • SeVV-eGFP- ⁇ X and SeV wt were also infected simultaneously with SeVV-eGFP- ⁇ X and SeV wt, there was also detectable fluorescence in those cells.
  • the multiplication of SeVV-eGFP- ⁇ N, - ⁇ P and - ⁇ L in cells co-infected with wt could be detected from this “tailing” of fluorescing cells 48 h p.i. and beyond.
  • the medium was replaced with DMEM+10% fetal calf serum (FCS)+cytosine-arabinoside (AraC) (100 ⁇ g/ml) and the cells were incubated for 72 h at 33° C., changing the medium daily, to add fresh AraC.
  • FCS fetal calf serum
  • AraC cytosine-arabinoside
  • SeVV-eGFP- ⁇ X The propagation of SeVV-eGFP- ⁇ X was analyzed via eGFP expression after 72 h. In this time, in the positive assays there was multiplication of green-fluorescing cells from one initial cell to 10-30 adjacent fluorescing cells.
  • a helper cell For the multiplication of SeVV-eGFP- ⁇ N, a helper cell must express the SeV proteins N and P simultaneously, expression of SeV P protein in the helper cell is sufficient for multiplication of SeVV-eGFP- ⁇ P, and the amplification of SeVV-eGFP- ⁇ L should be possible by cellular expression of the SeV proteins P and L.
  • H29 a derivative of human 293 renal cells
  • Vero cells renal cells of the African green monkey
  • LLCMK2 cells renal cells of the rhesus monkey
  • SeVV-eGFP- ⁇ P In the assay with SeVV-eGFP- ⁇ P, about a hundred initially infected individual cells could be detected 1 d p.i. About 70% of the fluorescing individual cells had developed to spots, with up to 30 fluorescing cells, 3 d p.i. Therefore propagation of SeVV-eGFP- ⁇ P to surrounding H29 cells could definitely be observed. Thus, it is possible for the first time to multiply a viral SeV vector whose P-ORF has been deleted. Characterization of the multiplication of SeVV-eGFP- ⁇ P will be discussed in the next subsection.
  • SeVV-eGFP- ⁇ P can be amplified by the SeV P proteins produced by H29 helper cells. The P-deletion mutants released are able to infect surrounding H29 cells. It was now necessary to analyze the propagation of SeVV-eGFP- ⁇ P in comparison with the propagation of the replication-competent SeV-eGFP.
  • H29 cells 1 ⁇ 10 6 H29 cells were infected with on average 100 SeVV-eGFP- ⁇ P or SeV-eGFP. 3, 5 and 10 days p.i., green-fluorescing cells were detected using the fluorescence microscope.
  • SeVV-eGFP- ⁇ P could be multiplied successfully by cellular supply of SeV P proteins. It was found, at all times of investigation, that SeVV-eGFP- ⁇ P and SeV-eGFP multiply efficiently on H29 cells.
  • SeVV-eGFP- ⁇ P propagation of SeVV-eGFP- ⁇ P to cells that do not supply the missing P protein (“target cells”, e.g. Vero cells) was not observed, which confirms that SeVV-eGFP- ⁇ P is replication-deficient (see Section 8).
  • this replication-deficient SeVV For a therapeutic application of this replication-deficient SeVV, the capacity for expression seems too weak, or disproportionately many particles of SeVV ⁇ P would have to be applied per patient. Therefore it is desirable to use a replication-deficient SeV variant that also performs a secondary transcription. This leads to the development of additional modified polymerase complexes, which cannot replicate the viral genome, but are capable of increased expression of the therapeutic gene or antigen.
  • SeVV-eGFP-P ⁇ 2-77 particles were generated and multiplied as in Section 4.1 and 5.4.
  • the viral-encoded P ⁇ 2-77 protein is synthesized together with the cellular-encoded P protein.
  • the supernatants of the individual assays were determined over a period of 120 h by a cell infection dose test of the titers of progeny viruses from the number of eGFP-expressing cells.
  • SeV-eGFP-P ⁇ 2-77 could be multiplied with about equal efficiency as SeVV-eGFP- ⁇ P: From infected H29 cells, after 120 h about 20 ⁇ 10 6 virus particles of SeVV-eGFP- ⁇ P or SeVV-eGFP-P ⁇ 2-77 are released, which corresponds to a number of about 40 released virus particles of the P mutants per H29 cell.
  • the transcription cassette into which the reporter gene eGFP in SeVV-eGFP-P ⁇ 2-77 was inserted, is to encode the antigen of a pathogen, e.g. of a desired virus. This antigen expression must be sufficient to elicit a protective immune response in the patient.
  • each SeVV-eGFP-P ⁇ 2-77 nucleocapsid that infects a target cell is able to perform a detectable transgene expression
  • the same number of H29 and Vero cells were infected with the same quantity of virus particles and the number of eGFP-expressing H29 or Vero cells were compared by FACS (fluorescence-activated cell sorting) analysis using a FACS-Calibur flow cytometer. The data were evaluated from a computer-generated histogram, plotting the fluorescence signals of infected cells against the total cell count.
  • the medium was replaced with DMEM+10% FCS. Then the cells were incubated at 33° C. for several days. At first, the transgene expression of both vector variants was monitored from the eGFP fluorescence. On day 5 and 9 p.i., HAD test series were performed, for analyzing the exposure of the viral HN proteins on the basis of binding of human erythrocytes.
  • the infected cells were further incubated at 33° C., during which the neuraminidase activity of the HN protein causes the erythrocytes bound to infected cells to be released.
  • the cells were washed to remove the released erythrocytes, and incubated for a further 4 d at 33° C.
  • a HAD test was performed again on day 9 p.i. About 30% HAD-positive Vero cells were now detected. The number of bound red blood cells dropped to 5-20 erythrocytes per cell.
  • a semi-quantitative assessment of eGFP expression of the replication-deficient SeV vector in comparison with replication-competent SeV-eGFP was carried out by Western blot analysis using serial dilution of total cellular protein.
  • the fluorescence protein eGFP was detected using Western blot analysis both in SeV-eGFP and in SeVV-eGFP-P ⁇ 2-77 infected Vero cells. It can be concluded, from comparison of the intensities of the eGFP signals, that expression—mediated by the replication-deficient SeVV-eGFP-P ⁇ 2-77—is reduced by about a factor of 16 compared with the replication-competent SeV-eGFP.
  • SeVV-eGFP-P ⁇ 2-77 can produce very efficient secondary transcription.
  • the SeV HN protein is a membrane-bound surface protein and is an important antigenic determinant.
  • a semi-quantitative assessment of HN expression of the replication-deficient SeV vector in comparison with the replication-competent SeV-eGFP was carried out by Western blot analysis using serial dilutions of total cellular protein.
  • the proteins were transferred to a PVDF membrane and the viral-encoded HN protein (60 kDa) was detected by means of a monoclonal HN-antibody ( FIG. 14 ).
  • the HN protein was detected efficiently in the case of SeV-eGFP infected Vero cells after both incubation times in all traces (16 to 2 ⁇ g total protein; traces 2-5 left and right).
  • the band of the HN protein is still visible in the traces with 16 and 8 ⁇ g total protein (trace 7, 8), though at lower intensity.
  • Relative quantification of HN expression in SeVV-eGFP-P ⁇ 2-77 infected Vero cells relative to SeV-eGFP was carried out by comparing the traces with 16 and 8 ⁇ g vs. 2 ⁇ g total protein (traces 7 and 8 vs.
  • Vero cells are infected with the replication-competent SeV-eGFP, in the next two days a spot comprising up to a thousand additional fluorescing cells forms around the initially infected, strongly fluorescing cell.
  • Vero cells were placed in a T75 flask. The cells had been seeded at high density at the beginning of the incubation phase and accordingly were no longer dividing actively. These Vero cells were infected with SeVV-eGFP-P ⁇ 2-77 at an MOI of 0.001. The medium was changed to DMEM with 5% FCS (for reduced activity of division) after incubation for 1 hour, and the Vero cells were incubated at 33° C. (P1). Two days p.i., according to the selected MOI, initially several thousand separate infected, fluorescing Vero cells were observed. Owing to the high cell density, over the next 4 days of incubation there was hardly any cell division, i.e.
  • SeVV-eGFP-P ⁇ 2-77 Propagation of SeVV-eGFP-P ⁇ 2-77 from the originally infected cells to surrounding Vero cells by production of new virus genomes and particles could thus be ruled out. Therefore SeVV-eGFP-P ⁇ 2-77 can be described as a replication-deficient viral vector.
  • SeV vectors are replication-deficient in cells which do not supply the P protein in trans (so-called target cells), but they differ considerably in their capacity for gene expression.
  • This P gene-deficient SeVV displays similar weak expression as the analogous rabies ⁇ P variant (Shoji et al., supra). Both vectors are only capable of primary transcription in the infected target cell, via the polymerase complex that is supplied from the virus particle. However, stronger expression of the encoded transgene or antigen is desired for therapeutic application of the vector.
  • This condition can be fulfilled with the aid of the replication-deficient variant SeVV-eGFP-P ⁇ 2-77, which only gives a capacity for expression in the target cells that is reduced on average by a factor of 10 in comparison with replication-competent SeV. Owing to the presence of the gene for a P protein shortened at the N-terminal end in the vector genome, not only primary, but also secondary transcription is possible. This is realized with newly formed, modified polymerase complexes, which contain the vector-encoded P ⁇ 2-77 protein; this does not, however, support the replication mode of polymerase.
  • a replication-deficient SeV P ⁇ 2-77 was constructed, in which the genes of the original surface proteins F and HN were replaced with genes coding for chimeric F and HN proteins SeV/hPIV3.
  • the chimeric F protein contains 558 amino acids and comprises the extracellular domain of hPIV3 (493 amino acids), the transmembrane domain of SeV (23 amino acids) and the cytoplasmic domain of SeV (42 amino acids).
  • the chimeric HN protein has 579 amino acids and comprises the cytoplasmic domain of SeV (35 amino acids), the transmembrane domain of SeV (25 amino acids) and the extracellular domain of hPIV3 (519 amino acids).
  • the amino acid sequences of the chimeric F protein and of the chimeric HN protein are shown in the sequence listing as SEQ ID No. 27 and 28.
  • Inserting chimeric genes in the virus genome produces a novel antigenicity and in addition ensures efficient assembly of vaccine particles during their production.
  • the surface protein F of RSV was encoded in an additional expression cassette interposed between two viral genes, so that the construct was extended to a bivalent vaccine.
  • the replication-deficient vaccine prototype produced a definite induction of IgA antibodies specifically against hPIV3 ( FIG. 15A ), but there was less induction of anti-RSV IgA antibodies (not shown).
  • the induction of a humoral immune response to the surface antigens of both viruses produced comparable titers, and the amount of specific IgG differs by a factor of 2 ( FIG. 15B ).
  • Further analysis of the anti-hPIV3-IgG showed that the induced antibodies have neutralizing properties (titer 1/64). In contrast, as expected, no specific IgA or IgG induction was found in the control group.
  • the vaccine according to the invention was able to induce a specific mucosal and humoral immune response to heterologous viral antigens. Additional experiments showed that lymphocytes of immunized mice produced interferon- ⁇ , whereas IL-5 could not be detected. This finding indicates that the bivalent, replication-deficient RNA vaccine is able to trigger a T-cell immune response, which is a prerequisite for long-lasting immunity.

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US8911975B2 (en) 2011-02-08 2014-12-16 Mie University Method for producing virus vector for gene transfer
US8980634B2 (en) 2008-01-31 2015-03-17 Institut Pasteur Reverse genetics of negative-strand RNA viruses in yeast
US9309289B2 (en) 2007-12-27 2016-04-12 Universitat Zurich Replication-defective arenavirus vectors
US20160120974A1 (en) * 2013-06-10 2016-05-05 Amvac Ag Semi-live respiratory syncytial virus vaccine
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US9944952B2 (en) 2007-12-27 2018-04-17 Universität Zürich Replication-defective arenavirus vectors
US10655145B2 (en) 2007-12-27 2020-05-19 Universität Zürich Replication-defective arenavirus vectors
US11401528B2 (en) 2007-12-27 2022-08-02 Universität Zürich Replication-defective arenavirus vectors
US8980634B2 (en) 2008-01-31 2015-03-17 Institut Pasteur Reverse genetics of negative-strand RNA viruses in yeast
US9682136B2 (en) 2008-01-31 2017-06-20 Institut Pasteur Reverse genetics of negative-strand RNA viruses in yeast
US8911975B2 (en) 2011-02-08 2014-12-16 Mie University Method for producing virus vector for gene transfer
AU2013270018B2 (en) * 2012-05-30 2019-02-14 Amvac Ag Method for expression of heterologous proteins using a recombinant negative-strand RNA virus vector
US9809801B2 (en) 2013-03-15 2017-11-07 Université De Genève Anti-mycobacterial vaccines
US20160120974A1 (en) * 2013-06-10 2016-05-05 Amvac Ag Semi-live respiratory syncytial virus vaccine
US20160346378A1 (en) * 2013-06-10 2016-12-01 Amvac Ag Semi-live respiratory syncytial virus vaccine

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