WO2020043835A1

WO2020043835A1 - Novel recombinant newcastle disease virus

Info

Publication number: WO2020043835A1
Application number: PCT/EP2019/073113
Authority: WO
Inventors: Gerhard Noss
Original assignee: Thaller, Arno
Priority date: 2018-08-31
Filing date: 2019-08-29
Publication date: 2020-03-05
Also published as: EP3844268A1

Abstract

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 in a human.

Description

NOVEL RECOMBINANT NEWCASTLE DISEASE VIRUS

TECHNICAL FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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. (1994) In Webster R G, Granoff A (ed), Encyclopedia ofVirology. Academic Press, London; Lorence R M et al. (1994) Cancer Res 54: 6017-6021). In fact, observations in humans suggest that NDV possess significant oncolytic activity and, thus, can be a potent tool for treating various tumor diseases (see for example Zamarin & Palese 2012, Future Microbiol. 3: 347-367).

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. In addition, 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 glycoprotein, hemagglutinin-neuraminidase (HN), protrudes from the envelope allowing the virus to contain both hemagglutinin and neuraminidase activities. The fusion glycoprotein (F), which also interacts with the viral membrane, is first produced as an inactive precursor, then cleaved post-translationally to produce two disulfide linked polypeptides. 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. In association with the nucleocapsid are the P and L proteins. 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.

The replication of all negative- strand RNA viruses, including NDV, is complicated by the absence of cellular machinery required to replicate RNA. Additionally, the negative-strand genome cannot be translated directly into protein, but must first be transcribed into a positive- strand (mRNA) copy. Therefore, upon entry into a host cell, the genomic RNA alone cannot synthesize the required RNA-dependent RNA polymerase. The L, P and NP proteins must enter the cell along with the genome on infection.

Following transcription, virus genome replication is the second essential event in infection by negative-strand RNA viruses. As with other negative-strand RNA viruses, virus genome replication in Newcastle disease virus (NDV) 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). These plus-stranded copies (anti-genomes) differ from the plus- strand mRNA transcripts in the structure of their termini. Unlike the mRNA transcripts, 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). Both the NDV negative strand genomes (vRNAs) and antigenomes (cR As) are encapsidated by nucleocapsid proteins; the only unencapsidated RNA species are virus mRNAs. For NDV, 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.

Reverse genetics systems which allow the genetic manipulation of the NDV genome from recombinant or cloned DNA have been described (Peeters B P et al. (1999) J Virol 73: 5001- 5009; Romer-Oberdorfer A et al. (1999) J Gen Virol 80: 2987-2995; Krishnamurthy S et al. (2000) Virology 278: 168-182; Nakaya T et al. (2001) J Virol 75: 11868-11873). In all these systems, the necessary viral proteins of the non-segmented genome (NP, P/V, M, F, HN and L) are encoded from a single plasmid or recombinant nucleic acid on a single RNA molecule, thus mimicking the viral genome organization. Recently, however, reverse genetics systems have also been used to generate recombinant NDV with a segmented genome (Gao et al. 2008 (J. Virol. 82: 2692-2698)).

Genetically engineered NDV can potentially be used in vitro for expressing heterologous proteins encoded by the recombinant viral genome. In fact, 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. However, any of these uses are presently significantly limited by a titer of NDV particles which is well below of what is desirable.

Thus it was an object of the present invention to provide more infectious NDV particles which replicate to a higher virus titer when expressed in cell culture.

SUMMARY OF THE INVENTION

In one aspect, 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. In one aspect, 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.

In another aspect, 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.

In yet another aspect, 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, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine. In a preferred embodiment, said L protein may have a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.

In another aspect, 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.

In another aspect, 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 or 3 ofthe sequence listing. In another aspect, the nucleic acid comprises the nucleic acid sequence according to SEQ ID

NO: 4 or 5 of the sequence listing.

In another aspect, the HN protein of the present invention comprises the amino acid sequence according to SEQ ID NO: 6 of the sequence listing.

In another aspect, the M protein of the present invention comprises the amino acid sequence according to SEQ ID NO: 7 of the sequence listing.

In another aspect, the L protein of the present invention comprises the amino acid sequence according to SEQ ID NO: 8 or 14 of the sequence listing.

In another aspect, 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.

In one aspect, 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.

In another aspect, 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.

In yet another aspect, 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, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine. In a preferred embodiment, said L protein may have a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.

In another aspect, 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.

In another aspect, 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.

In another aspect, the 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, 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; and (d) 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

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, 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, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine; and (d) 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 ln a preferred embodiment, said L protein may have a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910. In one aspect, 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. In one aspect, 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.

Furthermore, 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.

In one aspect, 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 melanoma, squamous cell carcinoma of the skin, oropharyngeal carcinoma, oesophageal carcinoma, glioblastoma multiforme, astrocytoma, ependymoma, osteosarcoma, Ewing sarcoma.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention Is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention wall be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unl ess the context indicates otherwise.

Preferably, 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).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word“comprise”, and variations such as“comprises” and“comprising”, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The terms“a” and“an” and“the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non- cl aimed element essential to the practice of the invention.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

In the following, definitions will be provided which apply to all aspects of the present invention.

In a first aspect, 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 term“infectious Newcastle disease virus” as used herein 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. Preferably the term’’Infectious Newcastle disease virus” encompasses any Newcastle disease virus that is capable of producing a titer of at least lxlO³ TCID50/ml, at least IxlO⁴ TCID50/ml, at least lxlO⁵ TCID50/ml, at least IxlO⁶ TCID50/ml, at least lxlO⁷ at least lxlO⁸ TCID50/ml, at least lxlO⁹ TCID50/ml, at least lxlO¹⁰ TCID50/ml, TCID50/ml or at least lxlO¹¹ TCID50/ml.

As used herein, 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%.

Preferably, 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 NaH₂P0₄ (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodium citrate, pH 7. After hybridization, 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.

In a preferred embodiment, 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. Preferably 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. Preferably 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. Preferably said 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. Preferably said 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.

The term“variant” refers to a sequence variation with respect to a reference sequence. Regarding the present invention, the reference sequence is that of SEQ ID NO: 1 or 2 of the sequence listing. In a preferred embodiment, 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. The term“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. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (in particular BLASTN in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). 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.

Preferably, 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”. Preferably sequence identity is calculated over the entire length of the genomic sequence. Preferably 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%.

Preferably, 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”. Preferably sequence identity is calculated over the entire length of the amino acid sequence of a protein. Preferably 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%.

As used herein, 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. In other words, according to the present teaching, 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.

Preferably, 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. In other words, the viral genome may comprise one, two, three, four, five or six nucleic acid molecules. Preferably, the viral genome consists of minus strand RNA. Each transcription unit may comprise a 3’ transcription start sequence and a 5’ transcription stop signal.

The term“recombinant genome” as used herein 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. Preferably 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).

Preferably 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). Preferably, the viral protein is one of the viral proteins described herein below. Preferably the expression of said recombinant genome results in a cell culture supernatant comprising recombinant Newcastle disease virus at a TCID50 of at least lxl O⁸ TCID50/ml, preferably at least lxl 0⁹ TCID50/ml, at least lxlO¹⁰ TCID50/ml, or at least lxlO¹¹ TCID50/ml. Preferably 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. 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 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. In a preferred embodiment, 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.

The term“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. In a preferred embodiment, 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. In a preferred embodiment, 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. In a preferred embodiment, 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, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, (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. In a preferred embodiment, the L protein is encoded by nucleotides 8381 to 14995 of SEQ ID NO: 4 or 5 of the sequence listing or is encoded by the nucleotide sequence according to SEQ ID NO: 12 of the sequence listing.

The term“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, Glycine, Alanine and

Proline, wherein 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. In a preferred embodiment, 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. In a preferred embodiment, 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.

The term “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 is preferably a Leucine. In a preferred embodiment, the F protein comprises the amino acid sequence of SEQ ID NO: 9 of the sequence listing. According to the present teaching, 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. The term 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.

As used herein, the term 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.

The term 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. Preferably a tumor-associated antigen is a protein that is not detectably expressed in a healthy somatic tissue. Preferably, a tumor associated antigen is only expressed in cancer cells or cancer tissue.

In one embodiment, 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.

In another embodiment, the heterologous protein or polypeptide is a protein or polypeptide or fragment thereof selected from the group consisting of apoptin, IL12, B18R. In another embodiment, 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

Nivolumab, Ipilimumab, NS1 (Influenza), Bevacizumab (anti-VEGF), Atezolizumab (anti- PD1-L), Irilumab (anti-KIR), Relatlimab (anti-LAG3), TRX518 (anti-GITR), BMS986178 (anti-OX40). Alternatively, the heterologous protein or polypeptide or fragment thereof is EGFP, CD80, IL12 or (ns)hIL12.

Alternatively, the heterologous polypeptide or protein is a viral or bacterial protein. Preferably 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.

In one aspect of the present invention, 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.

In one aspect, 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 In a preferred embodiment, the 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.

In another aspect, the viral genome may comprise 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/or

(iii) a histidine in amino acid position 1717 of the L protein. In another aspect, the viral genome may comprise a nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having

(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 serine in amino acid position 1551 of the L protein.

In a preferred embodiment, said L protein may have a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.

In a preferred embodiment, 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.

In another aspect, the viral genome may comprise

(a) 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; and

(b) a nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having

(iii) a histidine in amino acid position 1717 of the L protein.

In yet another aspect, the viral genome may comprise

(a) 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; and (b) a nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having

(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, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine.

In a preferred embodiment said L protein may have a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.

In another aspect, the viral genome may comprise 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/or

(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,

Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a

Leucine.

In a preferred embodiment, 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.

In another aspect, the viral genome may comprise

(b) 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, lsoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine, and/or

(iii) a histidine in amino acid position 1717 of the L protein; and

(c) a nucleic acid comprising 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/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, lsoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.

In another aspect, the viral genome may comprise

(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,

(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 ofTryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine,

(c) 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 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.

In a preferred embodiment, the L protein may have a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.

In another aspect, 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.

In yet another aspect, said nucleic acid comprises the nucleic acid sequence according to SEQ ID NO: 3, 4 or 5 of the sequence listing.

Preferably, 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.

In another aspect, 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.

In one aspect, the nucleic acid encodes the infectious Newcastle disease virus of the present invention. Preferably 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.

In another aspect, 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.

In one aspect, 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.

Preferably 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)). Preferably the nucleic acid contains all regulatory elements for RNA replication, transcription, polyadenylation, and packaging of RNA into NDV particles.

In another aspect, 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).

In another aspect, 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. In a preferred embodiment, 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.

In another aspect, 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.

In another aspect, 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. In a preferred embodiment, 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.

In another aspect, the present invention relates to a nucleic acid which 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/or

(iii) a histidine in amino acid position 1717 of the L protein.

In yet another aspect, the present invention relates to a nucleic acid which comprises a nucleic acid sequence encoding an L protein having

In a preferred embodiment, the L protein has a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.

In a preferred embodiment, 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.

In another aspect, the present invention relates to a nucleic acid comprising:

(a) a nucleic acid sequence encoding an M protein having a tryptophan in amino acid position 165 of the M protein; and

(b) 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

(c) a nucleic acid sequence encoding an L protein having

(iii) a histidine in amino acid position 1717 of the L protein.

In another aspect, the present invention relates to a nucleic acid comprising:

(c) a nucleic acid sequence encoding an L protein having

(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 ofTryptophan, Tyrosine, Glutamine, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine.

In a preferred embodiment, said L protein has a histidine in amino acid position 1717 and/or a Lysine in amino acid position 1910.

In another aspect, the present invention relates to a nucleic acid which 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 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.

In a preferred embodiment, 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.

In another aspect, the present invention relates to a nucleic acid comprising

(b) 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;

(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, Valine, Phenylalanine, Leucine, Isoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably an Isoleucine, and/or

(iii) a histidine in amino acid position 1717 of the L protein; and

(d) 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/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. In another aspect, the present invention relates to a nucleic acid comprising

(c) a nucleic acid sequence encoding an L protein having

(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, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine, and

(d) a nucleic acid sequence encoding a fusion protein (F) having

In another aspect, 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. In another aspect, 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. Preferably, 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:

(a) a nucleic acid sequence encoding a hem agglutinin-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

(iii) a histidine in amino acid position 1717 of the L protein; and/or

(d) a nucleic acid sequence encoding a fusion protein (F) having

(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;

(c) a nucleic acid sequence encoding an L protein having

(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, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine; and/or

(d) 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

In one aspect, 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.

In another aspect, 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.

In one aspect, the vector of the present application comprises the nucleic acid of the present invention and encodes an infectious NDV. In a preferred embodiment of the present invention, 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. Preferably, the buffer may be free of RNAase or comprise an inhibitor of RNAse.

In one aspect, the composition is a kit which comprises at least one nucleic acid or vector of the present invention. In a preferred embodiment, 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 5⁵ transcription stop signal.

Optionally, 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.

In one embodiment, the host cell of the present invention comprises an unsegmented recombinant viral genome as described herein above. In another embodiment, 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.

In a preferred embodiment, 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.

The term“transfection” includes for example electroporation, lipofection or calcium phosphate transfection. As used herein, 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. These 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.

In a preferred embodiment, 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.

In a preferred embodiment, the cell is a cancer cell. Preferably, 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 myeloid leukemia, malignant melanoma, squamous cell carcinoma of the skin, oropharyngeal carcinoma, oesophageal carcinoma, glioblastoma multiforme, astrocytoma, ependymoma, osteosarcoma, Ewing sarcoma.

In another preferred embodiment, 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.

In a preferred embodiment, the method comprises culturing the cell in a cell culture medium comprising human serum, preferably of human blood group AB Rhesus positive.

In another preferred embodiment, the method comprises a step of filtering the cell culture supernatant. Preferably, the filter has a filter diameter of 0.45 p .

In a preferred embodiment, 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.

Furthermore, 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. The term“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.

Moreover, 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.

Furthermore, 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.

The term“prevention” includes the vaccination of a human or an animal.

In a preferred embodiment, 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 leukemia, malignant melanoma, squamous cell carcinoma of the skin, oropharyngeal carcinoma, oesophageal carcinoma, glioblastoma multiforme, astrocytoma, ependymoma, osteosarcoma, Ewing sarcoma. The following Examples illustrate the invention:

Example 1 : Preparation of a stock solution of MTH68

Lyophilized MTH68 grown in embryonated eggs containing about 10⁹ EID50/ml (medium egg infecting dose) a vaccine strain prepared by Csatary, L. et al, Budapest were used for preparing a stock solution of MTH68/H (further called MTH68), a genotype III strain that is closely related to the mesogenic vaccine strain Mukteswar. When prepared under standard conditions, virus stock typically reached a TCID50 of lxlOVml.

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.

Example 2: Infection of Hela cells with NDV MTH68

IOOmI of the MTH68 stock solution was used to infect a confluent tissue culture of 10⁷ 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. The TCID50 of the cell culture supernatant was determined in a TCID50 assay and showed a TCID50 of lxl0⁵/ml (h=10 for each dilution).

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.

When using a 100 mΐ 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.

The TCID50 of the cell culture supernatant initially obtained from Hela cells was determined in a TCID50 assay on HeLa cells and surprisingly showed a TCID50 of lxl 0⁸/ml (n=10 for each dilution). When the infection with the HeLa cell culture supernatant was repeated on HeLa cells and on cell cultures of the cells shown in Tables 3 and 4 (below), a TCID50 of between lxl0^{7 5}/ml and lxl 0^{8 7}/ml was consistently determined (n=T0 for each dilution). Parallel infections with MTH68 resulted in a TCID50 of <l0⁵/ml.

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.

Taken together, these results demonstrate that that an 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 and titer is typically only achieved in eggs.

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 mΐ of the cell culture supernatant of Example 3 (”NDV-MutHu”, TCID50 of l lOVml) was used for infecting the tumor cells.

After three days of infection, a 100 mΐ aliquot of the cell culture supernatant was used to infect a fresh confluent tissue culture obtained from the same tumor biopsy. This procedure was repeated for five times. Subsequently, the tumor 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 tumor cells of the tissue culture. The TCID50 of the cell culture supernatant was determined in a TCID50 assay on HeLa cells and surprisingly showed a TCID50 of 0.5-2xl0^u/ml (four independent experiments, h=10 for each dilution).

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. However, 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.

Taken together, these results demonstrate that that an 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 5-2xl0ⁿ/ml) exceeds the level of infectivity typically achieved in eggs by several orders of magnitude.

Example 5: 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.

Table 2 provides summary of exemplary results

Subsequently, the cell culture supernatant of Example 4 was used for infecting cell cultures of the cells shown in Tables 3 and 4 (below). Surprisingly, a TCID50 of between 6xl0⁹/ml and 2xlO^u/ml was consistently determined (h=10 for each dilution) for each of these infections. However, when tested for hemagglutination (HA) with erythrocytes, the preparations of NDV variants propagated in the human cell cultures, despite their high titer, did not result in a HA titer.

Table 3: permanent growing tumor cell lines susceptible to Newcastle disease virus (n=30)

Table 4: primary tumor cultures susceptible to Newcastle disease virus (NDV): n=28

Example 6: Recombinant expression of NDV-MutHu, NDV-mutHu-1 and NDV-mutHu-

2

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.

For the generation of infectious virus particles, 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. The TCID50 of the cell culture supernatant of these cultures was determined in a TCID50 assay on HeLa cells and showed a TCID50 of lxl0ⁿ/ml (h=10 for each dilution) for NDV -mutHu- 1 and NDV-mutHu-2. In a parallel experiment, the TCID50 of the virus stock of NDV-MutHu was determined as lxl0⁸/ml (n=10 for each dilution).

These results confirmed that the recombinant NDV constructs, when transfected under appropriate conditions into cells expressing NDV NP, P and P protein, resulted in the production of infectious virus particles. These infectious NDV particles showed the same level of infectivity as observed from the NDV variants isolated from the tissue culture supernatant after propagation in HeLa cells (NDV-MutHu) or from the tissue culture supernatant after extensive propagation in tissue cultures derived from human cancer tissue (NDV-MutHu- 1 and NDV-MutHu-2). These results demonstrate that the recombinant NDV-MutHu- 1 and NDV- MutHu-2 described herein is stable in tissue culture and that the nucleic acid of the present invention can be used as a vector for recombinant gene expression or as an efficient oncolytic tool for treating cancer patients. Example 7: Generation of recombinant NDV and establishing their Intracerebral pathogenicity

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.

Based on the NGS results the 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

rgNDV-MutHu-2F2

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. To this end, 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). In the subsequent 8-day period, the chickens were observed for clinical signs of pathogenicity. Each day, the animals were inspected and scored, wherein a score of 0 was assigned to normal chicken, a score of 1 was assigned to sick chickens and a score of 2 was assigned to dead chickens. At the end of the 8 -day period, all scores were summed up and divided by 80 (the number of observations). The theoretical minimal score is 0 (all animals normal) and the theoretical maximum score is 2.0, i.e. all animals dead on the first day: 8 (days) x 2 (score) x 10 (animals) = 160/80 = 2.0. score ICPI

0

12

96

108 1,35 score ICPI

0

23

72

95 1,19 score ICPI

0

27

36

63 0,79 score ICPI

0

31

30

61 0,76

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. Surprisingly, the 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. Since a low ICPI is crucial for the use of an NDV strain for vaccination and clinical application, the above data establishes that both rgNDV-MutHu-2F2L3 and rgNDV -MutHu-2F2 are useful for vaccination and that both strains are significantly superior in this aspect over either NDV-MTH68 or NDV-MutHu. The experimental observations furthermore allow the conclusion that an NDV strain can be improved by introducing a mutation into the F gene resulting in an amino acid exchange at position 117 or 190 of the F protein, or at both positions. This modification clearly has the potential to significantly reduce the intracerebral pathogenicity of an NDV infection of poultry.

Claims

1. 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.

2. The NDV of claim 1, said the viral genome comprising 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.

3. The NDV of claim 1 or 2, said the viral genome comprising a nucleic acid comprising a nucleic acid sequence encoding a large polymerase protein (L) having

(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, Cysteine, Serine and Threonine, wherein the amino acid is preferably a Serine; and optionally

(iv) a histidine in amino acid position 1717 of the L protein and/or a Lysine in amino acid position 1910 of the L protein.

4. The NDV of any one of claims 1 to 3, the viral genome comprising 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

5. The NDV of any one of claims 1 to 4, wherein said 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 of the sequence listing.

6. The NDV of any one of claims 1 to 5, wherein said nucleic acid comprises the nucleic acid sequence according to SEQ ID NO: 4 or 5 of the sequence listing.

7. The NDV of any one of claims 1 to 6, wherein said HN protein comprises the amino acid sequence according to SEQ ID NO: 6 of the sequence listing.

8. The NDV of any one of claims 2 to 6, wherein said M protein comprises the amino acid sequence according to SEQ ID NO: 7 of the sequence listing.

9. The NDV of any one of claims 3 to 6, wherein said L protein comprises the amino acid sequence according to SEQ ID NO: 8 or 14 of the sequence listing.

10. The NDV of any one of claims 4 to 6, wherein said F protein comprises the amino acid sequence according to SEQ ID NO: 9 of the sequence listing.

11. 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, lsoleucine, Glycine, Alanine and Proline, wherein the amino acid is preferably a Leucine.

12. The nucleic acid of claim 11 , wherein the nucleic acid comprises a nucleic acid sequence encoding an M protein having a tryptophan in amino acid position 165 of the M protein.

13. The nucleic acid of claim 11 or 12, wherein the nucleic acid comprises a nucleic acid sequence encoding an L protein having

14. The nucleic acid of any one of claims 11 to 13, wherein said nucleic acid comprises 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

15. The nucleic acid of any one of claims 11 to 14, wherein said 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.

16. The nucleic acid of any one of claims 11 to 15, wherein said nucleic acid comprises the nucleic acid sequence according to SEQ ID NO: 4 or 5 of the sequence listing.

17. Nucleic acid comprising a nucleic acid sequence, wherein said nucleic acid sequence is selected from the group consisting of:

(c) a nucleic acid sequence encoding an L protein having

(iv) a histidine in amino acid position 1717 of the L protein and/or a Lysine in amino acid position 1910 of the L protein; and

(d) a nucleic acid sequence encoding a fusion protein (F) having

(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 Pro line, wherein the amino acid is preferably a Leucine.

18. The nucleic acid of claim 17, wherein said nucleic acid 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.

19. Vector comprising the nucleic acid of any one of claims 11 to 18.

20. The vector of claim 19, which encodes an infectious NDV.

21. Composition comprising the nucleic acid of any one of claims 11 to 18 or the vector of claim 19 or 20.

22. Host cell comprising the nucleic acid of any one of claims 11 to 18 or the vector of claim 19 or 20.

23. Polypeptide encoded by the nucleic acid of any one of claims 11 to 18 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.

24. Method of producing a composition comprising NDV particles, comprising a step of

(a) infecting a cell with the NDV of any one of claims 1 to 10 or transfecting the cell with the vector of claim 19 or 20;

(b) harvesting cell culture supernatant comprising said NDV particles.

25. Composition comprising NDV according to any one of claims 1 to 10 or obtainable from the method of claim 24.

26. Method of treating or preventing a disease comprising administering to a patient the NDV according to any one of claims 1 to 10, the nucleic acid according to any one of claims 11 to 18, the vector according to claim 19 or 20, the host cell according to claim 22 or the composition according to claim 21 or 23.

27. Use of the NDV according to any one of claims 1 to 10, the nucleic acid according to any one of claims 11 to 18, the vector according to claim 19 or 20, the host cell according to claim 22, or the composition according to claim 21 or 23 for the preparation of a medicament for the treatment of a disease.

28. NDV according to any one of claims 1 to 10, the nucleic acid according to any one of claims 11 to 18, the vector according to claim 19 or 20, the host cell according to claim 22, or the composition according to claim 21 or 23 for use in therapy, preferably for treating a disease.

29. The method of claim 26, the use of claim 27, the NDV for use of claim 28, the nucleic acid for use of claim 28, the vector for use of claim 28, the host cell for use of claim 28, or the composition for use of claim 28, wherein 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 leukemia, malignant melanoma, squamous cell carcinoma of the skin, oropharyngeal carcinoma, oesophageal carcinoma, glioblastoma multi forme, astrocytoma, ependymoma, osteosarcoma, Ewing sarcoma.