EP3844268A1 - Neuartiges rekombinantes morbus-newcastle-virus - Google Patents

Neuartiges rekombinantes morbus-newcastle-virus

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Publication number
EP3844268A1
EP3844268A1 EP19762750.8A EP19762750A EP3844268A1 EP 3844268 A1 EP3844268 A1 EP 3844268A1 EP 19762750 A EP19762750 A EP 19762750A EP 3844268 A1 EP3844268 A1 EP 3844268A1
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EP
European Patent Office
Prior art keywords
amino acid
protein
nucleic acid
leucine
ndv
Prior art date
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Pending
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EP19762750.8A
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English (en)
French (fr)
Inventor
Gerhard Noss
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Noss Gerhard Willi Michael
Thaller Arno
Original Assignee
Noss Gerhard Willi Michael
Thaller Arno
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Application filed by Noss Gerhard Willi Michael, Thaller Arno filed Critical Noss Gerhard Willi Michael
Publication of EP3844268A1 publication Critical patent/EP3844268A1/de
Pending legal-status Critical Current

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a new recombinant paramyxovirus which can be used as a vector for gene expression in a eukaryotic cell or for treating a cancerous disease.
  • Virus families containing enveloped single-stranded RNA with negative-sense genome are classified into groups having non-segmented genomes (Paramyxoviridae, Rhabdoviridae, Bomaviridae and Filoviridae) or those having segmented genomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae).
  • the Paramyxoviridae family described in detail below includes Newcastle disease virus (NDV).
  • Newcastle disease virus is a negative strand RNA virus which belongs to the genus Rubulavirus of the family Paramyxoviridae, of the order of the Mononegavirales. This virus is an avian pathogen and several NDV strains have been isolated which are characterized by different levels of virulence in birds. Virulent (velogenic) strains of NDV cause a highly pathogenic disease in poultry. However, a virulent (mesogenic and lentogenic) strains of NDV cause mild or asymptomatic infections and they are currently used as live vaccines in domestic poultry against Newcastle disease. Humans are not the usual hosts for NDV, but the virus has been administered to humans and been found to be safe (Emmerson, P. T.
  • Newcastle disease virus is an enveloped virus containing a linear, single-strand, nonsegmented, negative sense RNA genome.
  • the molecular organization of the NDV genome is similar to that of other Paramyxoviridae and Rhabdoviridae viruses.
  • the genomic RNA contains genes in the order of 3'-NP-P-M-F-HN-L-5'.
  • the genomic RNA also contains a leader sequence at the 3' end. Sequences at the end of the genome are involved in transcription and replication of the RNA by the viral RNA-dependent RNA polymerase.
  • intergenic junctions contain gene-end, polyadenylation and gene-start signals.
  • the structural elements of the virion include the virus envelope which is a lipid bilayer derived from the cell plasma membrane.
  • the active F protein is involved in penetration of NDV into host cells by facilitating fusion of the viral envelope with the host cell plasma membrane.
  • the matrix protein (M) is involved with viral assembly, and interacts with both the viral membrane as well as the nucleocapsid proteins.
  • the main protein subunit of the nucleocapsid is the nucleocapsid protein (NP) which confers helical symmetry on the capsid.
  • NP nucleocapsid protein
  • P phosphoprotein
  • L L protein
  • the phosphoprotein (P) which is subject to phosphorylation, is thought to play a regulatory role in transcription.
  • the L gene which encodes an RNA-dependent RNA polymerase, is required for viral RNA synthesis together with the P protein.
  • the L protein which takes up nearly half of the coding capacity of the viral genome is the largest of the viral proteins, and plays an important role in both transcription and replication.
  • virus genome replication is the second essential event in infection by negative-strand RNA viruses.
  • virus genome replication in Newcastle disease virus is mediated by virus-specified proteins.
  • the first products of replicative RNA synthesis are complementary copies (i.e., plus-polarity) of NDV genome RNA (cRNA).
  • cRNA NDV genome RNA
  • anti-genomes differ from the plus- strand mRNA transcripts in the structure of their termini.
  • the anti- genomic cRNAs are not capped and methylated at the 5' termini, and are not truncated and polyadenylated at the 3 ' termini.
  • the cRNAs are coterminal with their negative strand templates and contain all the genetic information in each genomic RNA segment in the complementary form.
  • the cRNAs serve as templates for the synthesis of NDV negative-strand viral genomes (vRNAs).
  • vRNAs NDV negative-strand viral genomes
  • vRNAs NDV negative-strand viral genomes
  • vRNAs antigenomes
  • cR As antigenomes
  • RNA species are virus mRNAs.
  • the cytoplasm is the site of virus RNA replication, just as it is the site for transcription. Assembly of the viral components appears to take place at the host cell plasma membrane and mature virus is released by budding.
  • NP non-segmented genome
  • M plasmid or recombinant nucleic acid
  • F HN and L
  • reverse genetics systems have also been used to generate recombinant NDV with a segmented genome (Gao et al. 2008 (J. Virol. 82: 2692-2698)).
  • NDV Genetically engineered NDV can potentially be used in vitro for expressing heterologous proteins encoded by the recombinant viral genome.
  • NDV since a human infection with NDV is generally asymptomatic, NDV could also be a potential tool for human vaccination or for killing cancer tissue which is targetable by NDV.
  • any of these uses are presently significantly limited by a titer of NDV particles which is well below of what is desirable.
  • the present invention relates to an infectious Newcastle disease virus (NDV), comprising a viral genome comprising a nucleic acid comprising a nucleic acid sequence encoding a hemagglutinin-neuraminidase protein (HN) having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the viral genome comprises a nucleic acid comprising a nucleic acid sequence encoding a matrix protein (M) having a Tryptophan in amino acid position 165 of the M protein.
  • M matrix protein
  • the viral genome comprises a nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine, (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and (iii) a histidine in amino acid position 1717 of the L protein.
  • L large polymerase protein
  • the viral genome comprises a nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine, (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and (iii) an uncharged, polar amino acid in amino acid position 1551 of the L protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyros
  • the viral genome comprises a nucleic acid comprising a nucleic acid sequence encoding a fusion protein (F) having (i) an uncharged, polar amino acid in amino acid position 117 of the F protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and (ii) a hydrophobic apolar amino acid in amino acid position 190 of the F protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • F fusion protein
  • nucleic acid comprises a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence according to SEQ ID NO: 1, 2 or 3 ofthe sequence listing. In another aspect, the nucleic acid comprises the nucleic acid sequence according to SEQ ID NO: 1, 2 or 3 ofthe sequence listing.
  • the HN protein of the present invention comprises the amino acid sequence according to SEQ ID NO: 6 of the sequence listing.
  • the M protein of the present invention comprises the amino acid sequence according to SEQ ID NO: 7 of the sequence listing.
  • the L protein of the present invention comprises the amino acid sequence according to SEQ ID NO: 8 or 14 of the sequence listing.
  • the F protein of the present invention comprises the amino acid sequence according to SEQ ID NO: 9 of the sequence listing.
  • the present invention also relates to a nucl eic acid comprising a nucleic acid sequence encoding an infectious Newcastle disease virus, said nucleic acid comprising a nucleic acid sequence encoding a HN protein having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the nucleic acid of the present invention comprises a nucleic acid sequence encoding an M protein having a tryptophan in amino acid position 165 of the M protein.
  • the nucleic acid of the present invention comprises a nucleic acid sequence encoding an L protein having (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine, (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and (iii) a histidine in amino acid position 1717 of the L protein.
  • the nucleic acid of the present invention comprises a nucleic acid sequence encoding an L protein having (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine, (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and (iii) an uncharged, polar amino acid in amino acid position 1551 of the L protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cystein
  • the nucleic acid of the present invention comprises a nucleic acid sequence encoding a fusion protein (F) having (i) an uncharged, polar amino acid in amino acid position 1 17 of the F protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and (ii) a hydrophobic apolar amino acid in amino acid position 190 of the F protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • F fusion protein having (i) an uncharged, polar amino acid in amino acid position 1 17 of the F protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine
  • the nucleic acid of the present invention comprises a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence according to SEQ ID NO: 1, 2, 3, 4 or 5 of the sequence listing.
  • nucleic acid of the present invention comprises the nucleic acid sequence according to SEQ ID NO: 4 or 5 of the sequence listing.
  • the present invention also relates to a nucleic acid comprising a nucleic acid sequence, wherein said nucleic acid sequence is selected from the group consisting of: (a) a nucleic acid sequence encoding a hemagglutinin-neuraminidase protein (HN) having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine; (b) a nucleic acid sequence encoding a matrix protein (M) having a tryptophan in amino acid position 165 of the M protein; (c) a nucleic acid sequence encoding an L protein having (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine
  • the present invention also relates to a nucleic acid comprising a nucleic acid sequence, wherein said nucleic acid sequence is selected from the group consisting of: (a) a nucleic acid sequence encoding a hemagglutinin-neuraminidase protein (HN) having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine; (b) a nucleic acid sequence encoding a matrix protein (M) having a tryptophan in amino acid position 165 of the M protein; (c) a nucleic acid sequence encoding an L protein having (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine
  • the nucleic acid of the present invention comprises a nucleic acid sequence which is at least 70% identical to a nucleic acid sequence selected from the group consisting of the nucleic acid sequence according to any one of SEQ ID NOs: 1 to 5 and SEQ ID NOs: 10 to 13 of the sequence listing.
  • the present invention also relates to a vector comprising the nucleic acid of the present invention.
  • the vector encodes an infectious NDV.
  • the present invention also relates to a composition comprising the nucleic acid or the vector of the present invention.
  • the present invention also relates to a host cell comprising the nucleic acid or vector of the present invention.
  • the present invention also relates to a polypeptide or protein encoded by the nucleic acid of the present invention or having an amino acid sequence selected from the group consisting of the amino acid sequence according to any one of SEQ ID NOs: 6 to 9.
  • the present invention also relates to a method of producing a composition comprising NDV particles, comprising a step of (a) infecting a cell with the NDV of the present invention or transfecting a cell with the vector of the present invention; and (b) harvesting cell culture supernatant comprising said NDV particles.
  • the present invention also relates to a composition comprising NDV of the present invention or obtainable from the present invention’s method of producing a composition comprising NDV particles.
  • the present invention also relates to a method of treating or preventing a disease comprising administering to a patient the NDV of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host cell of the present invention or the composition of the present invention.
  • the present invention also relates to the use of the NDV of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host cell of the present invention or the composition of the present invention for the preparation of a medicament for the treatment of a disease.
  • the disease is a cancer disease which is preferably selected from the group consisting of ovarian carcinoma, colorectal carcinoma, neuroendocrine carcinoma, clear cell renal carcinoma, ovarian cancer, neuroendocrine carcinoma, follicular thyreoidal carcinoma, pancreatic cancer, stomach cancer, duodenal carcinoma, breast carcinoma, breast carcinoma, pancreatic cancer, urothelial bladder carcinoma, pancreatic cancer, stomach cancer, renal cancer, neuroendocrine carcinoma, breast cancer, colorectal cancer, terato carcinoma, thymus carcinoma, hepatocellular carcinoma, mesothelioma, papillar thyreoidal carcinoma, small cell lung carcinoma, squamous cell lung carcinoma, adeno carcinoma of the lung, carcinoid tumors, Hodgkin sarcoma, non Hodgkin sarcoma and lymphoma, chronic lymphatic leukemia, acute lymphatic leukemia, acute myeloid leukemia, chronical myeloid leukemia, malignant melanom
  • the terms used herein are defined as described in“A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).
  • the practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (c£, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).
  • the present invention relates to an infectious Newcastle disease virus (NDV), comprising a viral genome comprising a nucleic acid comprising a nucleic acid sequence encoding a hemagglutinin-neuraminidase protein (HN) having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and
  • Proline wherein the amino acid is preferably a Leucine.
  • infectious Newcastle disease virus refers to a Newcastle disease virus that is capable of replicating its genomic RNA and that is capable of entering from a first host cell into a second host cell.
  • An infectious NDV may be cell free or cell associated. Infection can be monitored by following expression in a tissue culture. To this end, the presence and/or amount of cell associated viral protein or RNA can be detected or determined or followed over time.
  • Viral protein can be detected by any suitable method such as SDS gel electrophoresis, Western Blotting, immunofluorescence, immunoprecipitation, FACS, and the like. Viral RNA can be detected or quantified for example in a Northern Blot analysis or by an RT PCR.
  • Infection can also be detected or quantified by determining the presence or amount of physical virus particles in the cell culture supernatant or by determining the presence or amount of infectious particles in the cell culture supernatant.
  • Infectious particles are preferably determined in a TCID50 assay on HeLa cells.
  • the term“Newcastle disease virus” or“NDV” includes any paramyxovirus which has a genomic nucleotide sequence which is at least 70% identical to the nucleotide sequence of Newcastle disease virus strain Mukteswar according to GenBank reference EF201805.1 or JF950509.1. SEQ ID NO: 1 of the sequence listing shows the genomic nucleotide sequence of JF950509.1.
  • the term“Newcastle disease virus” or“NDV” includes all known velogenic, mesogenic and lentogenic NDV strains, wherein mesogenic and lentogenic strains are preferred according to the teaching of the present invention.
  • the term“at least 70%” includes at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.
  • the nucleotide sequences are capable of hybridizing and forming a stable duplex with one another, with hybridization preferably being carried out under conditions which allow specific hybridization between polynucleotides (stringent conditions).
  • stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, F.M.
  • Ausubel et al. Editors, John Wiley & Sons, Inc., New York and refer, for example, to hybridization at 65 ° C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH 2 P0 4 (pH 7), 0.5% SDS, 2 mM EDTA).
  • SSC is 0.15 M sodium chloride/0.15 M sodium citrate, pH 7.
  • the membrane to which the DNA has been transferred is washed, for example, in 2 x SSC at room temperature and then in 0.1 -0.5 x SSC/0.1 x SDS at temperatures of up to 68 ° C.
  • a Newcastle disease virus which has a genomic nucleotide sequence of at least 70% includes a variant which encodes at least one of the sequence variants described herein below which affect the HN protein, the M protein, the L protein or the F protein.
  • said sequence variant of the HN protein is represented by the amino acid sequence according to SEQ ID NO: 6 or is encoded by the nucleotide sequence according to SEQ ID NO: 10 of the sequence listing.
  • said sequence variant of the M protein is represented by the amino acid sequence according to SEQ ID NO: 7 or is encoded by the nucleotide sequence according to SEQ ID NO: 11 of the sequence listing.
  • sequence variant of the L protein is represented by the amino acid sequence according to SEQ ID NO: 8 or 15 or is encoded by the nucleotide sequence according to SEQ ID NO: 12 of the sequence listing.
  • sequence variant of the F protein is represented by the amino acid sequence according to SEQ ID NO: 9 or is encoded by the nucleotide sequence according to SEQ ID NO: 13 of the sequence listing.
  • variant refers to a sequence variation with respect to a reference sequence.
  • the reference sequence is that of SEQ ID NO: 1 or 2 of the sequence listing.
  • the reference sequence is the sequence according to SEQ ID NO: 3 of the sequence listing.
  • a sequence variation is for example the substitution, addition or deletion of at least one nucleotide.
  • “at least one nucleotide” means up to 1 nucleotide, up to 2 nucleotides, up to 3 nucleotides, up to 4 nucleotides, up to 5 nucleotides, up to 6 nucleotides, up to 7 nucleotides, up to 8 nucleotides, up to 9 nucleotides, up to 10 nucleotides or up to 20 nucleotides.
  • the variation may result in an amino acid substitution, wherein the amino acid substitution can be a conservative or a non-conservative amino acid substitution.
  • The“percentage identity” is obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length of either the genomic nucleotide sequence or the nucleotide sequence encoding a particular viral gene product such as the HN protein, the M protein, the L protein, the F protein.
  • Sequence comparisons between two sequences are conventionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out by segment or by“window of comparison” in order to identify and compare local regions of sequence similarity.
  • the optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981 , Ads App. Math.
  • the percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared and multiplying the result obtained by 100 so as to obtain the percentage identity between these two sequences.
  • sequence identity of a nucleotide sequence is determined using BLASTN, preferably BLASTN in standard settings as provided by the website of the U.S. National Library of Medicine“https://blast.ncbi.nlm.nih.gov”.
  • sequence identity is calculated over the entire length of the genomic sequence.
  • the degree of sequence identity of a nucleotide sequence referred to herein is at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%.
  • sequence identity of an amino acid sequence is determined using BLASTP, preferably BLASTP in standard settings as provided by the website of the U.S. National Library of Medicine“https://blast.ncbi.nlm.nih.gov”.
  • sequence identity is calculated over the entire length of the amino acid sequence of a protein.
  • the degree of sequence identity of an amino acid sequence referred to herein is at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%.
  • the term“viral genome” refers to an unsegmented or a segmented genome and includes a recombinant genome. Recombinant unsegmented genomes of NDV have been described for example in Nakaya et al. 2001 (J. Virol. 75: 11868-11873). Recombinant segmented genomes ofNDV have been described for example in Gao et al. 2008 (J. Virol. 82; 2692-2698).
  • a segmented genome may comprise at least two nucleic acids which are not covalently linked.
  • the term“at least two” means two, three, four, five or six, or up to two, up to three up to four, up to five or up to six.
  • the six transcriptional units, i.e. NP, P, M, F, HN and L of an unsegmented NDV may be divided into two or more segments, so that recombinant, naturally non-segmented NDV is generated containing two or more RNA segments or recombinant RNA molecules.
  • the transcriptional units preferably comprise (a) a binding site for the NDV L protein described herein and (b) signals required for viral-mediated replication and transcription.
  • Each transcription unit preferably comprises a 3’ transcription start sequence and a 5’ transcription stop signal.
  • the viral genome is a recombinant genome comprising one or more nucleic molecules.
  • the recombinant genome preferably comprises at least six transcriptional units that encode the nucleocapsid protein (NP), phosphoprotein and V protein (P/V), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN), and large polymerase protein (L).
  • the transcriptional units may be arranged on the same nucleic acid or on separate nucleic acids.
  • the viral genome may comprise one, two, three, four, five or six nucleic acid molecules.
  • the viral genome consists of minus strand RNA.
  • Each transcription unit may comprise a 3’ transcription start sequence and a 5’ transcription stop signal.
  • recombinant genome encompasses any kind of derivative of a naturally occurring NDV genome.
  • Such derivative may be a genomic derivative comprising a single or multiple nucleotide substitutions, deletions and/or additions.
  • the recombinant genome encodes at least one viral protein with a single or with multiple amino acid substitutions, deletions and/or additions with respect to the reference genome according to SEQ ID NO: 1 or 2, in a preferred aspect with respect to the reference genome according to SEQ ID NO: 3.
  • the viral protein may be selected from the group consisting of nucleocapsid protein (NP), phosphoprotein and V protein (P/V), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN), and large polymerase protein (L).
  • NP nucleocapsid protein
  • P/V phosphoprotein and V protein
  • M matrix protein
  • F fusion protein
  • HN hemagglutinin-neuraminidase
  • L large polymerase protein
  • the recombinant genome encodes at least one viral protein with at least one amino acid substitution, wherein the viral protein with the at least one amino acid substitution is selected from the group consisting of hemagglutinin-neuraminidase (HN), matrix protein (M), large polymerase protein (L) and fusion protein (F).
  • HN hemagglutinin-neuraminidase
  • M matrix protein
  • L large polymerase protein
  • F fusion protein
  • the viral protein is one of the viral proteins described herein below.
  • the expression of said recombinant genome results in a cell culture supernatant comprising recombinant Newcastle disease virus at a TCID50 of at least lxl O 8 TCID50/ml, preferably at least lxl 0 9 TCID50/ml, at least lxlO 10 TCID50/ml, or at least lxlO 11 TCID50/ml.
  • TCID50 of at least lxl O 8 TCID50/ml, preferably at least lxl 0 9 TCID50/ml, at least lxlO 10 TCID50/ml, or at least lxlO 11 TCID50/ml.
  • the term“cell culture” does not include allantoic fluid.
  • The“hemagglutinin-neuraminidase protein” or“HN protein” of the present invention is a polypeptide which is encoded by the open reading frame (ORF) of nucleotides 6412 to 8127 of SEQ ID NO: 1, 2, 3, 4 or 5.
  • any other functionally equivalent polypeptide encoded by a nucleic acid of the present invention which is at least 70% identical in sequence to the nucleotide sequence SEQ ID NO: 1, 2, 3, 4 or 5, said polypeptide having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the apolar amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the HN protein is encoded by nucleotides 6412 to 8127 of SEQ ID NO: 3, 4 or 5 of the sequence listing or is encoded by the nucleotide sequence according to SEQ ID NO: 10 of the sequence listing.
  • hemagglutinin-neuraminidase protein or“HN protein” of the present invention also encompasses a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence according to SEQ ID NO: 6 of the sequence listing, preferably at least 75%, more preferably at least 80%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%, said polypeptide preferably having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the apolar amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the HN protein comprises the amino acid sequence of SEQ ID NO: 6 of the sequence listing.
  • The“matrix protein” or“M protein” of the present invention is a polypeptide which is encoded by the ORF of nucleotides 3290 to 4384 of SEQ ID NO: 1, 2, 3, 4 or 5. Also encompassed by the present teaching is any other functionally equivalent polypeptide encoded by a nucleic acid of the present invention, which is at least 70% identical in sequence to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4 or 5, said polypeptide having a tryptophan in amino acid position 165 of the M protein.
  • the M protein is encoded by nucleotides 3290 to 4384 of SEQ ID NO: 3, 4, or 5 of the sequence listing or is encoded by the nucleotide sequence according to SEQ ID NO: 11 of the sequence listing.
  • the term“matrix protein” or“M protein” of the present invention also encompasses a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence according to SEQ ID NO: 7 of the sequence listing, preferably at least 75%, more preferably at least 80%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%, said polypeptide having a tryptophan in amino acid position 165 of the M protein.
  • the M protein comprises the amino acid sequence of SEQ ID NO: 7 of the sequence listing.
  • The“large polymerase protein” or“L protein” of the present invention is a polypeptide which is encoded by the ORF of nucleotides 8381 to 14995 of SEQ ID NO: 1, 2, 3, 4 or 5.
  • the term also includes any other functionally equivalent polypeptide encoded by a nucleic acid of the present invention, which is at least 70% identical in sequence to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4 or 5, said polypeptide having (a) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an isoleucine, (b) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine
  • “large polymerase protein” or“L protein” of the present invention also encompasses a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence according to SEQ ID NO: 8 or 14 of the sequence listing, preferably at least 75%, more preferably at least 80%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%, said polypeptide having (a) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valin, Phenylalanin, Leucine, Isoleucine, Glycin, Alanin and Prolin, wherein the amino acid is preferably an isoleucine, (b) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine
  • the amino acid is preferably a Leucine, and/or (c) a histidine in amino acid position 1717 of the L protein, and/or (d) an uncharged, polar amino acid in amino acid position 1551 of the L protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine.
  • the L protein comprises the amino acid sequence of SEQ ID NO: 8 or 14 of the sequence listing.
  • The“fusion protein” or“F protein” of the present invention is a polypeptide which is encoded by the ORF of nucleotides 4544 to 6205 of SEQ ID NO: 1, 2, 3, 4 or 5 or by the ORF of nucleotides 4442 to 6205 of SEQ ID NO: 1 , 2, 3, 4 or 5.
  • the term also includes any other functionally equivalent polypeptide encoded by a nucleic acid of the present invention, which is at least 70% identical in sequence to the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4 or 5, said polypeptide having (a) an uncharged, polar amino acid in amino acid position 117 of the F protein, wherein the amino acid is selected from the group consisting ofTryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and/or (b) a hydrophobic apolar amino acid in amino acid position 190 of the F protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the F protein is encoded by nucleotides 4544 to 6205 of SEQ ID NO: 4 or 5 or by nucleotides 4442 to 6205 of SEQ ID NO: 4 or 5 of the sequence listing or is encoded by the nucleotide sequence according to SEQ ID NO: 13 of the sequence listing.
  • fusion protein or“F protein” of the present invention also encompasses a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence according to SEQ ID NO: 9 of the sequence listing, preferably at least 75%, more preferably at least 80%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%, said polypeptide having (a) an uncharged, polar amino acid in amino acid position 117 of the F protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and/or (b) a hydrophobic apolar amino acid in amino acid position 190 of the F protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid
  • the F protein comprises the amino acid sequence of SEQ ID NO: 9 of the sequence listing.
  • the recombinant genome may comprise a heterologous nucleotide sequence.
  • the heterologous nucleotide sequence preferably comprises a 3’ transcriptional start sequence and a 5’ transcriptional stop signal.
  • the heterologous nucleotide sequence may encode a regulatory RNA or a heterologous amino acid sequence.
  • a regulatory RNA may be for example an siRNA or an antisense RNA, preferably an RNA capable of reducing or inhibiting the expression of a cellular protein.
  • heterologous amino acid sequence refers to a sequence of at least 6 consecutive amino acids which are not found in any known NDV protein or polypeptide or fragment thereof.
  • fragment refers to a peptide or polypeptide comprising at least 6 amino acids, at least 8 amino acids, at least 10 amino acids, at least 12 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids or at least 50 amino acids.
  • a fragment may be a peptide or polypeptide comprising an amino acid sequence of up to 50 amino acids, of up to 100, 200, 300, 500, or of up to 1000 amino acids.
  • At least 6 consecutive amino acids preferably comprises a sequence of at least 6 amino acids, at least 8 amino acids, at least 10 amino acids, at least 12 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids or at least 50 amino acids.
  • the heterologous amino acid sequence preferably defines a heterologous polypeptide or a heterologous protein which is either fused to a viral protein or which is expressed separately.
  • the heterologous polypeptide or protein may comprise an amino acid sequence of up to 50 amino acids, of up to 100, 200, 300, 500, or of up to 1000 amino acids.
  • the heterologous polypeptide or protein may be an antigen used for vaccination, in particular a tumor-associated antigen.
  • a tumor-associated antigen is a protein that is not detectably expressed in a healthy somatic tissue.
  • a tumor associated antigen is only expressed in cancer cells or cancer tissue.
  • the heterologous protein or polypeptide is a protein or polypeptide or fragment thereof selected from the group consisting of Her2/neu, NKG2D, CS1 , GD2, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-l, GAGE, CD52, CD30, MUC5AC, c-Met, EGFR, FAB, WT-1, PSMA, NY- ESOl, AFP, CEA, CTAG1B, CD19 and CD33.
  • the heterologous protein or polypeptide is a protein or polypeptide or fragment thereof selected from the group consisting of apoptin, IL12, B18R.
  • the heterologous protein or polypeptide is an inhibitor of CTLA-4, PD-1 or PD- L1 , Preferably the inhibitor is an antibody or antigen-binding fragment of an antibody such as
  • the heterologous protein or polypeptide or fragment thereof is EGFP, CD80, IL12 or (ns)hIL12.
  • the heterologous polypeptide or protein is a viral or bacterial protein.
  • the protein is a protein expressed by chickenpox, hepatitis A, hepatitis B, hepatitis C , haemophilus influenza, in particular haemophilus influenza type B, human immunodeficiency virus, in particular HIV-l or HIV-2, human papillomavirus, in particular of serotypes 6, 1 1, 16, or 18, influenza virus, preferably influenza strains H1N1, H3N2, and Type-B strains, meningococcus, in particular neisseria meningitidis, measles virus, mumps virus, rubella virus, streptococcus pneumoniae, poliovirus, rotavirus, or Tetanus.
  • the heterologous amino acid sequence is immunogenic in human, i.e. it induces the production of antibodies or the production of T cells, preferably cytotoxic T cells.
  • the viral genome may comprise a nucleic acid comprising a nucleic acid sequence encoding a matrix protein (M) having a tryptophan in amino acid position 165 of the M protein
  • M protein comprises an amino acid sequence according to SEQ ID NO: 7 of the sequence listing or is encoded by a nucleic acid sequence according to SEQ ID NO: 11 of the sequence listing.
  • the viral genome may comprise a nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine,
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or
  • the viral genome may comprise a nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine,
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and
  • said L protein may have a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.
  • the L protein comprises an amino acid sequence according to SEQ ID NO: 8 or 14 of the sequence listing or is encoded by a nucleic acid sequence according to SEQ ID NO: 12 of the sequence listing.
  • the viral genome may comprise
  • nucleic acid comprising a nucleic acid sequence encoding a matrix protein (M) having a tryptophan in amino acid position 165 of the M protein;
  • nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine,
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or
  • the viral genome may comprise
  • nucleic acid comprising a nucleic acid sequence encoding a matrix protein (M) having a tryptophan in amino acid position 165 of the M protein; and (b) a nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine,
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or
  • amino acid position 1551 of the L protein an uncharged, polar amino acid in amino acid position 1551 of the L protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine.
  • said L protein may have a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.
  • the viral genome may comprise a nucleic acid comprising a nucleic acid sequence encoding a fusion protein (F) having
  • amino acid position 117 of the F protein (i) an uncharged, polar amino acid in amino acid position 117 of the F protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine,
  • amino acid is preferably a Serine, and/or
  • amino acid position 190 of the F protein (i) a hydrophobic apolar amino acid in amino acid position 190 of the F protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine,
  • amino acid is preferably a
  • the F protein comprises an amino acid sequence according to SEQ ID NO: 9 of the sequence listing or is encoded by a nucleic acid sequence according to SEQ ID NO: 13 of the sequence listing.
  • the viral genome may comprise
  • nucleic acid comprising a nucleic acid sequence encoding a matrix protein (M) having a tryptophan in amino acid position 165 of the M protein;
  • nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine,
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, lsoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or
  • nucleic acid comprising a nucleic acid sequence encoding a fusion protein (F) having
  • amino acid position 1 17 of the F protein (i) an uncharged, polar amino acid in amino acid position 1 17 of the F protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and/or
  • amino acid position 190 of the F protein (ii) a hydrophobic apolar amino acid in amino acid position 190 of the F protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, lsoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the viral genome may comprise
  • nucleic acid comprising a nucleic acid sequence encoding a matrix protein (M) having a tryptophan in amino acid position 165 of the M protein;
  • nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, lsoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an lsoleucine,
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, lsoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or
  • amino acid position 1551 of the L protein an uncharged, polar amino acid in amino acid position 1551 of the L protein, wherein the amino acid is selected from the group consisting ofTryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine,
  • nucleic acid comprising a nucleic acid sequence encoding a fusion protein (F) having (i) an uncharged, polar amino acid in amino acid position 117 of the F protein, wherein the amino acid is selected from the group consisting ofTryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and/or
  • amino acid position 190 of the F protein a hydrophobic apolar amino acid in amino acid position 190 of the F protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the L protein may have a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.
  • the nucleic acid comprises a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence according to SEQ ID NO: 1, 2, 3, 4 or 5 of the sequence listing.
  • said nucleic acid comprises the nucleic acid sequence according to SEQ ID NO: 3, 4 or 5 of the sequence listing.
  • the nucleic acid sequence of the nucleic acid of the present invention is at least 70% identical to the nucleic acid sequence according to SEQ ID NO: 1, 2, 3, 4 or 5 of the sequence listing.
  • the HN protein of the present invention comprises the amino acid sequence according to SEQ ID NO: 6 of the sequence listing.
  • the M protein of the present invention comprises the amino acid sequence according to SEQ ID NO: 7 of the sequence listing.
  • the L protein of the present invention comprises the amino acid sequence according to SEQ ID NO: 8 or 14 of the sequence listing.
  • the F protein comprises the amino acid sequence according to SEQ ID NO: 9 of the sequence listing.
  • the present invention also relates to a nucleic acid comprising a nucleic acid sequence encoding an infectious Newcastle disease virus, said nucleic acid comprising a nucleic acid sequence encoding a HN protein having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the nucleic acid encodes the infectious Newcastle disease virus of the present invention.
  • the nucleic acid comprises a nucleic acid sequence according to SEQ ID NO: 3, 4 or 5 of the sequence listing or a nucleotide sequence which is at least 70% identical to the nucleic acid sequence according to SEQ ID NO: 3, 4 or 5 of the sequence listing.
  • the nucleic acid of the present invention comprises a nucleic acid sequence encoding a HN protein of the present invention as defined herein, a nucleic acid sequence encoding an M protein of the present invention as defined herein, a nucleic acid sequence encoding a L protein of the present invention as defined herein, and a nucleic acid sequence encoding an F protein of the present invention as defined herein.
  • the nucleic acid of the present invention can be DNA or RNA.
  • the nucleic acid is RNA.
  • the RNA may be capped or uncapped.
  • the RNA may be mRNA.
  • the RNA may be genomic RNA (vRNA), i.e. negative strand RNA.
  • the RNA may be a plus stranded copy of the genomic RNA, i.e. RNA which is complementary to the negative strand RNA (cRNA) and which represents an anti-genome.
  • the cRNA is preferably not capped and not methylated at the 5’ terminus, the cRNA is preferably not truncated and/or polyadenylated at the 3’ terminus.
  • the nucleic acid of the present invention is organized in six transcriptional units, preferably in the order 3'-NP-P-M-F-HN-L-5', wherein“3”’ refers to the 3’ end of the nucleic acid,“5”’ refers to the 5’ end of the nucleic acid,“NP” refers to the nucleoprotein,“P” refers to the phosphoprotein,“M” refers to the matrix protein,“F” refers to the fusion protein,“HN” refers to the hemagglutinin-neuraminidase, and”L” refers to the large polymerase protein.
  • Each transcriptional unit may contain a major open reading frame flanked by short 5' and/or 3 untranslated regions (UTRs), which may be followed by a transcriptional initiation sequence (so called“gene start” (GS)) and/or a termination control sequence (“gene end”, (GE)).
  • UTRs transcriptional initiation sequence
  • GE termination control sequence
  • the nucleic acid contains all regulatory elements for RNA replication, transcription, polyadenylation, and packaging of RNA into NDV particles.
  • the nucleic acid is DNA.
  • the DNA may be single stranded or double stranded.
  • the single stranded DNA may be negative strand DNA or positive strand DNA.
  • the transcriptional units may be flanked by a promoter and/or a terminator for a DNA directed RNA polymerase such as bacteriophage T7, T3 or SP6 polymerase or by an appropriate eukaryotic polymerase such as polymerase I.
  • the nucleic acid may also comprise a ribozyme, for example a hepatitis delta virus ribozyme (Pattnaik et al. 1992, Cell 69: 101 1-1020) to ensure that the 3’ end of the transcript corresponds to the exact terminal nucleotide of the cloned DNA fragment (Peeters et al. 1999).
  • the nucleic acid of the present invention comprises a nucleic acid sequence encoding a HN protein having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the nucleic acid sequence encoding the HN protein comprises the nucleotide sequence according to SEQ ID NO: 10 of the sequence listing or encodes a HN protein comprising the amino acid sequence according to SEQ ID NO: 6 of the sequence listing.
  • the nucleic acid of the present invention comprises a nucleic acid sequence encoding an M protein having a tryptophan in amino acid position 165 of the M protein.
  • the nucleic acid of the present invention comprises a nucleic acid sequence encoding an M protein having a tryptophan in amino acid position 165 of the M protein and a nucleic acid sequence encoding a HN protein having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the amino acid in amino acid position 277 of the HN protein is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the nucleic acid sequence encoding the M protein comprises the nucleotide sequence according to SEQ ID NO: 1 1 of the sequence listing or encodes an M protein comprising the amino acid sequence according to SEQ ID NO: 7 of the sequence listing.
  • the present invention relates to a nucleic acid which comprises a nucleic acid sequence encoding an L protein having
  • amino acid position 757 of the L protein wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine
  • amino acid position 1700 of the L protein wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or
  • the present invention relates to a nucleic acid which comprises a nucleic acid sequence encoding an L protein having
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine,
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or
  • amino acid position 1551 of the L protein an uncharged, polar amino acid in amino acid position 1551 of the L protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine.
  • the L protein has a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.
  • the nucleic acid sequence encoding the L protein comprises the nucleotide sequence according to SEQ ID NO: 12 of the sequence listing or encodes a L protein comprising the amino acid sequence according to SEQ ID NO: 8 or 14 of the sequence listing.
  • the present invention relates to a nucleic acid comprising:
  • a nucleic acid sequence encoding a HN protein having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine; and
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine,
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or
  • the present invention relates to a nucleic acid comprising:
  • a nucleic acid sequence encoding a HN protein having a hydrophobic apolar amino acid in amino acid position 277 of the HN protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine; and
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine,
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or
  • amino acid position 1551 of the L protein an uncharged, polar amino acid in amino acid position 1551 of the L protein, wherein the amino acid is selected from the group consisting ofTryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine.
  • said L protein has a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.
  • the present invention relates to a nucleic acid which comprises a nucleic acid sequence encoding a fusion protein (F) having
  • amino acid position 1 17 of the F protein (i) an uncharged, polar amino acid in amino acid position 1 17 of the F protein, wherein the amino acid is selected from the group consisting ofTryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and/or (ii) a hydrophobic apolar amino acid in amino acid position 190 of the F protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the nucleic acid sequence encoding the F protein comprises the nucleotide sequence according to SEQ ID NO: 13 of the sequence listing or encodes an F protein comprising the amino acid sequence according to SEQ ID NO: 9 of the sequence listing.
  • the present invention relates to a nucleic acid comprising
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine, and/or
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and
  • amino acid position 117 of the F protein (i) an uncharged, polar amino acid in amino acid position 117 of the F protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and/or
  • the present invention relates to a nucleic acid comprising
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine, and/or
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and
  • amino acid position 1551 of the L protein an uncharged, polar amino acid in amino acid position 1551 of the L protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and
  • amino acid position 117 of the F protein (i) an uncharged, polar amino acid in amino acid position 117 of the F protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and/or
  • amino acid position 190 of the F protein a hydrophobic apolar amino acid in amino acid position 190 of the F protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • said L protein has a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.
  • the nucleic acid of the present invention comprises a nucleic acid sequence which is at least 70% identical to the nucleic acid sequence according to SEQ ID NO: 1 or 2 of the sequence listing.
  • the nucleic acid of the present invention comprises the nucleic acid sequence according to SEQ ID NO: 3, 4 or 5 of the sequence listing.
  • the nucleic acid sequence of the nucleic acid of the present invention is at least 70% identical to the nucleic acid sequence according to SEQ ID NO: 1, 2, 3, 4 or 5 of the sequence listing.
  • the present invention also relates to a nucleic acid comprising a nucleic acid sequence, wherein said nucleic acid sequence is selected from the group consisting of:
  • HN hem agglutinin-neuraminidase protein
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine,
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or
  • amino acid position 1 17 of the F protein (i) an uncharged, polar amino acid in amino acid position 1 17 of the F protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and/or
  • amino acid position 190 of the F protein a hydrophobic apolar amino acid in amino acid position 190 of the F protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the present invention also relates to a nucleic acid comprising a nucleic acid sequence, wherein said nucleic acid sequence is selected from the group consisting of:
  • HN hemagglutinin-neuraminidase protein
  • amino acid position 757 of the L protein (i) a hydrophobic apolar amino acid in amino acid position 757 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine,
  • amino acid position 1700 of the L protein (ii) a hydrophobic apolar amino acid in amino acid position 1700 of the L protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or
  • amino acid position 1551 of the L protein an uncharged, polar amino acid in amino acid position 1551 of the L protein, wherein the amino acid is selected from the group consisting of Tryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine; and/or
  • amino acid position 117 of the F protein (i) an uncharged, polar amino acid in amino acid position 117 of the F protein, wherein the amino acid is selected from the group consisting ofTryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and/or
  • amino acid position 190 of the F protein a hydrophobic apolar amino acid in amino acid position 190 of the F protein, wherein the amino acid is selected from the group consisting of Methionine, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.
  • the L protein has a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.
  • the nucleic acid of the present invention comprises a nucleic acid sequence which is at least 70% identical to a nucleic acid sequence selected from the group consisting of the nucleic acid sequence according to any one of SEQ ID NOs: 1 to 5 and SEQ ID NOs: 10 to 13 of the sequence listing.
  • the present invention relates to a vector comprising the nucleic acid of the present invention.
  • the vector can be a DNA vector or an RNA vector.
  • the vector can be linear or circular.
  • the nucleic acids of the present invention may be functionally linked to promoter or terminator sequences, preferably of T7, T3 and/or SP6.
  • Vectors of the present invention may comprise the entire genome of the NDV described herein or may be a subgenomic vector comprising a fragment the genomic nucleic acid.
  • Subgenomic vectors may comprise a eukaryotic promoter such as the early immediate CMV promoter or the early SV40.
  • the vector of the present application comprises the nucleic acid of the present invention and encodes an infectious NDV.
  • the vector is a plasmid comprising the genomic cDNA described herein.
  • the present invention also relates to a composition comprising the nucleic acid of the present invention and/or the vector of the present invention.
  • the composition can be a liquid composition, a dried composition or a kit.
  • the composition may comprise a buffer such as Tris, TE or PBS or a salt such as NaCl.
  • a buffer such as Tris, TE or PBS or a salt such as NaCl.
  • the buffer may be free of RNAase or comprise an inhibitor of RNAse.
  • the composition is a kit which comprises at least one nucleic acid or vector of the present invention.
  • the kit comprises at least two nucleic acids or at least two vectors of the present invention, preferably at least three, at least four, at least five or at least six nucleic acids or vectors of the present invention.
  • the kit preferably comprises a segmented recombinant viral genome as described herein above, wherein the six transcriptional units of NDV, i.e. NP, P, M, F, HN and L, are divided into two or more segments, so that recombinant, naturally non-segmented NDV is generated containing two or more RNA segments or recombinant RNA molecules.
  • the transcriptional units preferably comprise (a) a binding site for the NDV L protein described herein and (b) signals required for viral-mediated replication and transcription. Each transcription unit preferably comprises a 3 transcription start sequence and a 5 5 transcription stop signal.
  • the composition may comprise a set of expression vectors for expressing the NDV NP protein, the NDV P protein and the NDV L protein or a set of expression vectors for expressing the NDV P protein and the NDV L protein.
  • the present invention also relates to a host cell comprising at least one of the nucleic acids of the present invention and/or at least one of the vectors of the present invention.
  • the host cell of the present invention comprises an unsegmented recombinant viral genome as described herein above.
  • the host cell comprises a segmented recombinant viral genome as described herein above, wherein the six transcriptional units ofNDV, i.e. the NP, P, M, F, HN and L of the present invention, are divided into two or more segments, so that recombinant, naturally non-segmented NDV is generated containing two or more two or more RNA segments or recombinant RNA molecules.
  • the transcriptional units preferably comprise (a) a binding site for the NDV L protein described herein and (b) signals required for viral-mediated replication and transcription. Each transcription unit preferably comprises a 3 transcription start sequence and a 5’ transcription stop signal.
  • the host cell is a cell expressing (a) the L protein and the P protein or (b) the L protein, the P protein and the NP protein, wherein the transcription of these proteins may be under control of a eukaryotic promoter or of a bacterial promoter such as T7, T3 or SP6.
  • the present invention also relates to a polypeptide encoded by the nucleic acid of the present invention or having an amino acid sequence according to any one of SEQ ID NOs: 6 to 9 or an amino acid sequence which is at least 70% identical to said amino acid sequence.
  • the present invention also relates to a method of producing a composition comprising NDV particles, comprising a step of (a) infecting a eukaryotic cell with the NDV of the present invention or transfecting a eukaryotic cell with the vector of the present invention; (b) harvesting cell culture supernatant comprising said NDV particles.
  • transfection includes for example electroporation, lipofection or calcium phosphate transfection.
  • the term also includes co -transfection of a first vector or plasmid comprising a full-length cDNA of the NDV of the present invention with one or more additional vectors.
  • additional vectors may be a subgenomic vector expressing NDV NP, P and/or L protein or a vector expressing the T7, T3 or SP6 polymerase.
  • the eukaryotic cell is a cell stably expressing NDV NP, P, L and/or the T7, T3 or SP6 polymerase. In another embodiment, the eukaryotic cell is a cell transiently expressing NDV NP, P, L and/or the T7, T3 or SP6 polymerase.
  • the cell is a cancer cell.
  • the cancer is selected from the group consisting of ovarian carcinoma, colorectal carcinoma, neuroendocrine carcinoma, clear cell renal carcinoma, ovarian cancer, neuroendocrine carcinoma, follicular thyreoidal carcinoma, pancreatic cancer, stomach cancer, duodenal carcinoma, breast carcinoma, breast carcinoma, pancreatic cancer, urothelial bladder carcinoma, pancreatic cancer, stomach cancer, renal cancer, neuroendocrine carcinoma, breast cancer, colorectal cancer, teratocarcinoma, thymus carcinoma, hepatocellular carcinoma, mesothelioma, papillar thyreoidal carcinoma, small cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma of the lung, carcinoid tumors, Hodgkin sarcoma, non Hodgkin sarcoma and lymphoma, chronic lymphatic leukemia, acute lymphatic leukemia, acute myeloid leukemia, chronical my
  • the cell is selected from the group consisting of HeLa, LN- 405, U-138-MG, HAT- 1080, PC-3, HCC-1143, BEN, HUP-T3, COLO-320, COLO-704, EFO- 27, HD-My-2, DU-145, CAL-72, Hep-G2, GAMG, MEL-Ho, EGI-l, LNCAP, CAKI-l , IMR- 32, HCC-33, RD-ES, MSTO-211H, RPMI8226, HCC1937, CAL-27, HCT-15, A549.
  • the method comprises culturing the cell in a cell culture medium comprising human serum, preferably of human blood group AB Rhesus positive.
  • the method comprises a step of filtering the cell culture supernatant.
  • the filter has a filter diameter of 0.45 p .
  • the cell transfected with the vector of the present invention expresses (a) the NDV L protein and the NDV P protein or (b) the NDY L protein, the NDV P protein and the NDV NP protein, wherein their transcription may be under control of a eukaryotic promoter or a heterologous promoter.
  • Said heterologous promoter may be a eukaryotic promoter or a T7, T3 or SP6 promoter.
  • the present invention also relates to a composition comprising the infectious NDV of the present invention or which is obtainable from the method of the present invention.
  • the present invention relates to a method of treating or preventing a disease comprising administering to a patient the NDV of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host cell of the present invention or the composition of the present invention.
  • patient includes any human or non-human subject.
  • Non-human includes mammalian and non-mammalian animals.
  • a non-mammalian animal is for example a bird, in particular a bird which is susceptible for an NDV infection such as chicken, duck, turkey, emu, Egyptian goose, goose, Indian peafowl, mute swan, ostrich, partridge, small-billed tinamou, pigeon, or quail.
  • a mammalian animal is for example cattle of the genus bos, in particular bos taurus or a domestic pig of the genus sus, in particular sus scrofa.
  • the present invention also relates to the use of the NDV of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host of the present invention, or the composition of the present invention for the preparation of a medicament for the treatment or prevention of a disease.
  • the present invention relates to the NDV of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host of the present invention, or the composition of the present invention for use in therapy, preferably for treating or preventing a disease.
  • prevention includes the vaccination of a human or an animal.
  • the disease is a cancer disease which is preferably selected from the group consisting of ovarian carcinoma, colorectal carcinoma, neuroendocrine carcinoma, clear cell renal carcinoma, ovarian cancer, neuroendocrine carcinoma, follicular thyreoidal carcinoma, pancreatic cancer, stomach cancer, duodenal carcinoma, breast carcinoma, breast carcinoma, pancreatic cancer, urothelial bladder carcinoma, pancreatic cancer, stomach cancer, renal cancer, neuroendocrine carcinoma, breast cancer, colorectal cancer, teratocarcinoma, thymus carcinoma, hepatocellular carcinoma, mesothelioma, papillar thyreoidal carcinoma, small cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma of the lung, carcinoid tumors, Hodgkin sarcoma, non Hodgkin sarcoma and lymphoma, chronic lymphatic leukemia, acute lymphatic leukemia, acute myeloid leukemia, chronical myeloid leuk
  • the genomic sequence of NDV strain Mukteswar is shown in SEQ ID NO: 1 of the sequence listing.
  • the genomic sequence of MTH68 is shown in SEQ ID NO: 2 of the sequence listing.
  • IOOmI of the MTH68 stock solution was used to infect a confluent tissue culture of 10 7 HeLa cells at a multiplicity of infection (MOI) of 0.01. After three days of infection, the cells were analysed by immunofluorescence with a polyclonal antibody raised in rabbit against the viral HN protein. The results demonstrated that only a few cells ( ⁇ 5%) in the tissue cell culture were infected with MTH68.
  • Example 3 Serial passages of NDV MTH68 in HeLa cells generates NDV-MutHu
  • HeLa cells were infected with MTH68 as describe in Example 2 and passaged serially 5 times. Subsequently, the HeLa cells in the tissue culture showed strong signs of pathogenicity including the formation of large syncytia that included 5 to 20 nuclei. When analysed by immunofluorescence with a polyclonal antibody against the viral HN protein, the HN protein could be detected in all HeLa cells of the tissue culture.
  • this cell culture When using a 100 m ⁇ aliquot of this cell culture supernatant to infect a fresh confluent tissue culture of HeLa cells for three days, this cell culture showed strong signs of pathogenicity including the formation of large syncytia that included 5 to 20 nuclei.
  • Parallel infections with MTH68 resulted in a TCID50 of ⁇ l0 5 /ml.
  • NGS The genomic sequence of the virus isolate in the cell culture supernatant was determined by NGS and resulted in the nucleotide sequence according to SEQ ID NO: 3 of the sequence listing.
  • This virus isolate (hereinafter”NDV-MutHu” or“MutHu-origmal”) represents a variant of MTH68 comprising the nucleotide substitution G3782T and the nucleotide substitution T7240C, affecting the HN protein by the amino acid substitution F277L and the M protein by the amino acid substitution G165W.
  • Example 4 Serial passages of NDV-MutHu in cell lines derived from tumor tissue generates NDVmutHu-1 and NDVmutHu-2
  • a primary tumor culture was established by mincing a biopsy of a primary tumor of a human lung and subsequently plating the minced tissue on standard cell culture dishes (lOcm dish with 6 ml of medium. When the cells reached confluency, 100 m ⁇ of the cell culture supernatant of Example 3 (”NDV-MutHu”, TCID50 of l lOVml) was used for infecting the tumor cells.
  • the genomic sequence of the virus in the cell culture supernatant was determined by NGS. As evident from the results shown in Table 1 , the cell culture supernatant comprised a 1 :1 mixture of two variants.
  • the genomic nucleotide sequence of a first variant (hereinafter“NDV-MutHu- 1”) is shown in SEQ ID NO: 4 of the sequence listing and the genomic nucleotide sequence of a second variant (hereinafter“NDV-MutHu-2”) is shown in SEQ ID NO. 5 of the sequence listing.
  • Table 1 shows NDV variants with increased infectivity and titer production
  • Both virus isolates represent a variant of NDV-MutHu according to SEQ ID NO: 3 of the sequence listing and retain the nucleotide substitutions G3782T and T7240C which encode a variant of the HN protein with a Leucine at position 277 and a variant of the M protein with a Tryptophan in amino acid position 165.
  • the genomic nucleotide sequence of both variants i.e.
  • NDV-MutHu- 1 and NDV-MutHu-2 is characterized by nucleotide substitutions G 10649 A, G13479T and T13529C which encode a variant of the L protein with an Isoleucine at position 757, a Leucine at position 1700 and a histidine at position 1717.
  • NDV-MutHu-l comprises the additional nucleotide substitutions T4893C and T511 1C which encode a variant of the F protein with a Serine at position 117 and a Leucine at position 190.
  • NDV variant comprising a HN protein with a Leucine in position 277 and an M protein with a Tryptophan in position 165 shows a significantly increased infectivity and results in the production of exceptionally high virus titers.
  • This level of NDV infectivity can be significantly increased by variants in the L protein which have an Isoleucine at position 757, a Leucine at position 1700 and a histidine at position 1717.
  • the resulting level of NDV infectivity (TCID50 of 0 5-2xl0 n /ml) exceeds the level of infectivity typically achieved in eggs by several orders of magnitude.
  • NDV-mutHu-1 and NDV-mutHu-2 are capable of infecting a large number of human tissues
  • NDV-MutHu-l and NDV-MutHu-2 of Example 4 was further tested for virus replication in various tumor cell lines and primary tumor cultures.
  • Table 2 provides summary of exemplary results obtained from infections with the cell culture supernatant.
  • HA hemagglutination
  • NDV Newcastle disease virus
  • HeLa cells infected with NDV-MutHu, NDV-mutHu-l and NDV-mutHu-2 were used to assemble subgenomic overlapping cDNA fragments. These were cloned in 3’ -NP-P-M-F -HN- L-5’ orientation between a T7 RNA polymerase promoter and a hepatitis delta virus ribozyme and a T7 polymerase terminator. Essentially, the cloning strategy described in Peeters et al. 1999 (Journal of Virology 73: 5001-5009) was followed so that transcription of the resulting plasmid by using T7 polymerase generates antigenomic RNA. The integrity of the resulting constructs was determined by sequencing.
  • the plasmids were transfected into cells expressing T7 polymerase and the NDV NP, P and L proteins. Expression of was provided by co-transfecting plasmids encoding NDV NP, P and L proteins. Reliable virus production was observed when the expression of NDV NP, P and L proteins was under control of the T7 promoter and the T7 polymerase was either provided by infection with the recombinant FLV described by Britton et al. 1996 (Journal of General Virology: 77: 963-967) or by using cells stably expressing the T7 polymerase.
  • the initial lot of recombinant virus was produced by transfection into chicken embryo fibroblasts. In order to boost virus production, the supernatant was injected into the allantoic cavities of 9-11 days old embryonated eggs and the allantoic fluid was harvested after four days. Recombinant virus was subsequently propagated in HeLa cells to prepare a virus stock.
  • NDV-MutHu tissue culture supernatant after propagation in HeLa cells
  • NDV-MutHu- 1 and NDV-MutHu-2 tissue culture supernatant after extensive propagation in tissue cultures derived from human cancer tissue
  • Recombinant MutHu virus (rgND V -MutHu) was prepared as described above.
  • Recombinant MTH68 (rgND V -MTH68) was derived from the NDV-MutHu cDNA by reversing the mutation affecting the M protein (i.e. by introducing the mutation W165G) and by reversing the mutation affecting the HN protein (i.e. by introducing the mutation L277F) followed by virus rescue.
  • rgNDV-MutHu cDNA was modified to obtain the following recombinant rescued viruses: rgNDV-MutHu-2F2L3
  • F-protein FI 17S and F190L
  • L-protein V757I, F1551S and R1700L
  • F-gene F117S and F190L;
  • rgND V -MutHu-2F2 L3 and rgND V -MutHu-2F2 differ from rgNDV-MutHu in the encoded F protein by a Serine at position 117 and a Leucine at position 190.
  • rgNDV-MutHu-2F2L3 encodes an L protein with three additional amino acid modifications, i.e. an Isoleucine at position 757, a Serine at position 1551 and a Leucine at position 1700.
  • rgND V -MutHu-2F2L3 , rgNDV-MutHu-2F2, rgNDV-MutHu and rgNDV-MTH68 were used to determine their respective intracerebral pathogenicity index (ICPI) in one-day-old chickens.
  • ICPI intracerebral pathogenicity index
  • a total of 10 1-day old chickens were inoculated intracerebrally with recombinant virus (rgND V -MutHu-2F2L3 , rgND V -MutHu-2F2, rgNDV-MutHu or rgNDV-MTH68).
  • the chickens were observed for clinical signs of pathogenicity.
  • MTH68 is a Mukteswar strain and the prior art mentions ICPIs for Mukteswar vaccine strains in the range of 1.32-1.45. Neither of these strains, nor any other NDV strain adapted for replication on human cells, have been suitable for clinical application, as the observed high ICPI represents a significant danger for the poultry industry.
  • ICPI values observed for rgNDV -MutHu-2F2L3 (0.76) and rgND V -MutHu-2F2 (0.79) were significantly lower than the ICPI values for rgNDV-MutHu (1.19) and rgNDV -MTH68 (1.35) and further preliminary experimental observations indicate that the ICPI of rgNDV -MutHu-2F 2L3 and rgNDV-MutHu-2F2 can be further reduced to a level of well below 0.70 by adapting the cell culture conditions.

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