BRPI0709602A2 - recombinant mononegaviral virus vector, and, vaccine against a microbial pathogen - Google Patents

Abstract

RECOMBINANT MONONEGAVIRALES VIRUS VECTOR, AND, VACCINE AGAINST A MICROBIAL PATHOGEN. The invention relates to a recombinant Mononegaviral (MV) virus vector comprising a foreign gene that is flanked by non-coding regions of a MV virus gene.

Description

"RECOMBINANT MONONEGAVIRAL VIRUS VECTOR AND VACCINE AGAINST A MICROBIAN PATHOGEN"

This invention relates to a recombinant Mononegaviral Virus vector hosting an additional transcription unit comprising a foreign gene operably linked with an upstream Mononegaviral Virus gene (GS) start sequence and a downstream Mononegaviral Virus gene (GE) sequence as well as umavacin comprising such a recombinant Mononegaviral virus vector.

Live viruses that are able to replicate in an infected host induce a strong long-lasting immune response against their expressed antigens. They are effective in generating both cell and humoral immune responses as well as stimulating cytokine and chemokine routes. Therefore, live attenuated viruses offer distinct advantages over either inactive or subunit immunogen-based vaccine compositions that typically greatly only stimulate the humoral branch of the immune system.

Over the past decade recombinant DNA technology has evolved the field of genetic engineering of both DNA and RNA virus genomes. In particular, it is now possible to introduce foreign genes into the genome of a virus such that upon replication of the novel vector virus in a host animal a foreign protein is expressed which may exert biological effects on the host animal. Likewise, recombinant vector viruses have been exploited not only for the control and prevention of microbial infections, but also for the development of target therapies for non-microbial diseases such as malignancies and gene therapy.

The generation of non-segmented negative strand RNA virus (Mononegavirales virus) entirely from cDNA cloned by a technique called "reverse genetics" first reported in 1994 (Schnell et al., EMBO J 13, 4195-4203, 1994), has also made possible the use of Mononegavirales (MV) viruses as vectors. Since then, studies have been published describing the use of many MV disorder viruses as viral vectors to express foreign antigens of a pathogen aiming at the development of vaccines against that pathogen.

The order of Mononegavirales is classified into four main families: Paramyxoviridae, Rhabdoviridae, Filoviridae and Bornaviridae. Viruses belonging to these families have genomes that are represented by a single negative sense RNA molecule (-), ie the polarity of the RNA genome is opposite to messenger RNA polarity (mRNA) which is designated as positive sense (+). The classification of the major MVveterinary and human viruses is presented in the table below:

Table 1: Virus classification within the order of MononegaviraIes

<table> table see original document page 3 </column> </row> <table>

The genomic organization and life cycle details of the MV order viruses are well understood today and are reviewed by several authors (Neumann et al., J. Gen. Virology 83, 2635-2662, 2002; Whelan et al., Curr Top Microbiol Immunol 203, 63119, 2004; Conzelmann, K., Curr Top Microbiol Immunol 203, 1-41, 2004). Mononegaviralese emboraviruses have different hosts and different morphological and biological properties, have many common characteristics, such as genomic organization and essential elements for their typical mode of replication and gene expression, illustrating that they originate from a common ancestor. These are enveloped viruses that replicate in cell cytoplasms and produce unedited mRNAs.

A Mononegaviral virus consists of two larger functional units, a ribonucleoprotein complex (RNP) and an envelope. The complete genome sequences for viruses representative of the genera of all families mentioned above have been determined. Genomes range in size from about 9,000 nucleotides to about 19,000 and contain from 5 to 10 genes. The structure and organization of MV virus genomes are very similar and are governed by their particular mode of gene expression. All MV virus genomes comprise three coding nucleus genes: a nucleoprotein (N or NP), a phosphoprotein (P), and an RNA-dependent RNA polymerase (L). The viral envelope is composed of a matrix protein (M) and one or more transmembrane glycoproteins (eg G, HN and F proteins) that play a role in assembling / germinating viruses as well as in virus cell attachment and / or virus entry into the cell. Depending on the genre, the protein repertoire is extended by accessory proteins that exhibit certain specific regulatory functions in virus transcription and replication or that are involved in host-virus reactions (e.g., C, V and NS proteins). The order of the MV virus gene is highly conserved with the N and P nucleus genes at or near the 3 'terminus and the genegrande (L) at the 5' distal position. OM, the surface glycoprotein genes, as well as the other accessory genes, are located between the Ν, P and L genes.

In the RNP complex, genomic or antigenomic RNA is tightly encapsulated with protein N and is associated with RNA-dependent RNA polymerase consisting of protein L and P. After infection of a cell, the RNP complex, but not the RNA genome. nu, serves as a model for two distinct RNA synthesis functions, ie, subgenomic demRNAs transcription and full length genomic RNA replication.

All serially arranged genes are separated by so-called "gene junction" structures. A gen junction comprises a conserved "gene end" (GE) sequence, a non-transcribed "intergenic region" (IGR), and a conserved "gene start" (GS) sequence. These sequences are both sufficient and necessary for gene transcription. During transcription each gene is sequentially transcribed into mRNA by RNA-dependent viral RNA polymerase which initiates the transcription process at the 3 'end of genomic RNA in the first GS sequence. At each transcription gene junction is disrupted as a result of disengagement of RNA polymerase in the GE sequence. Resumption of transcription occurs in the subsequent GS sequence, although with reduced efficiency. As a result of this disrupted process, also called a "interrupt-initiation" process, transcription attenuation occurs at each gene junction as a result of which proximal 3 'genes in an MV virus genome are transcribed more abundantly than the genes a. successive downstream. The modular form of MV virus gene transcription in which each gene is part of a cistronseparate or separate transcriptional unit makes these viruses extremely suitable for insertion and expression of foreign genes. Each transcriptional unit in an MV virus genome comprises The following elements: 3'-GS-open reading array (ORF) -GE-5 \

At the -3'- and -5 'genomic terminations all MV devirus genomes have a short non-transcribed region called the "leader" (about 40-50 nt) and "tow" (about 20-600 nt), respectively. The leader and trailer sequences are essential sequences that control A-genomic RNA replication, viral encapsidation, and packaging.

Reverse genetics technology and MVinfective virus rescue have made it possible to manipulate your RNA genome through your cDNA copy. The minimal replication initiation complex required to synthesize viral RNA is the RNP complex. MVinfective virus can be rescued by intracellular coexpression of (anti) genomic RNAs and the appropriate carrier proteins of (T7) RNA polymerase-related plasmids. Since the initial report in 1994 by Schnell et al., 1994 (supra), reliable recovery of many species of MV virus has been achieved based on the original protocol (or its slight variations).

Newcastle disease and avian influenza are major poultry diseases that can cause severe economic losses in the poultry industry worldwide. The Newcastle disease virus is a non-segmented negative strand RNA virus within the order of MV. The genome, which is about 15 kb in length, contains six genes that encode nucleoprotein (NP), phosphoprotein and protein V (P / V), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein ( HN) and RNA or large protein (L) dependent RNA polymerase. NDV genes are sequentially arranged in the order of 3'-NP-PMF-HN-L-5 ', and are separated by intergenic regions of different length. All genes are preceded by a gene start sequence (GS) that is followed by a non-coding region, the NDV protein open-coding reading matrix, a second non-coding region, and gene end sequence (GE). The length of the NDV genome is a multiple of yours that has to be considered for the introduction of foreign genes.

Avian Influenza (AI) is a poultry disease characterized by mild respiratory signs to severe disease with high mortality. The causative agent is an avian influenza A virus (AIV) belonging to the family Orthomyxoviridae. AIV contains eight negative-polarity genomic RNA segments that encode 10 proteins. Based on the antigenicity of hemagglutinin (HA) and neuraminidase (N) surface glycoproteins, AI viruses are of subtype. To date, 16 hemagglutinin subtypes (H1 - H16) and nine neuraminidase subtypes (NI - N9) are known. HeN antibodies are important in the humoral immune response and inhibit or prevent disease.

Newcastle disease and avian influenza viruses can be grouped into two distinct pathotypes according to their virulence. Symptoms caused by pathogenic low AIV (LPAI) or lentogenic NDV are considered of minor relevance. In contrast, highly pathogenic avian influenza (HPAI) and Newcastle disease caused by high virulent virus (NDV: mesogenic and velogenic strains) are notifiable diseases.

Although routine NDV vaccination with slow NDV strains is performed to protect chicken from highly virulent NDV strains, HPAI vaccination is not performed in most countries because HPAI is controlled by an eradication strategy. However, vaccination can be used as a strategy to minimize losses and reduce the incidence of disease. Vaccine-induced immunity is subtype-specific, which means that an H5 subtype vaccine can protect against H5 AIV but not other H subtypes. Normally, replication of influenza virus is restricted to the lungs because hemagglutinin devoid of LPAI can be cleaved only by tryptase. Clara, the serine proteas restricted to the lungs. So far, all HPAI viruses have been subtypesH5 and H7. These HPAI viruses contain multiple basic amino acids at the H cleavage site so that it can be cleaved by ubiquitous subtilisin and furin-like enzymes into the HA1 and HA2 subunits. Such viruses can therefore grow in other organs.

H5 and H7 subtype vaccines may provide protection against misalignments and turkeys against clinical signs and death after HPAI infection. In addition to conventional oil-based whole AIV vaccines, it has been shown experimentally that the vector virus, protein-subunit and DNA vaccines are effective for immunization against AI. From the advent of reverse genetics to different viruses, generation of recombinant viruses for use as vaccine vectors is an important application. Different recombinant negative strand RNA viruses expressing foreign proteins have been constructed. Also, AIV hemagglutinin has been inserted into different vector viruses such as infectious laryngotracheitis virus (ILTV) (Luschow et al., Vaccine 19, 4249-59, 2001), Bovine Plague virus (Walsh etal., J. Virol. 74 , 10165-75, 2000) and vesicular stomatitis virus (VSV) (Roberts et al, J. Virol. 247, 4704-11, 1998).

Bovine Plague Virus has also been used as a vector virus for expression of the VP1 capsid protein of the mouth-and-foot disease virus (Baron et al, 1999, J. of Gen. Virol, vol. 80, p. 2031- 2039).

Tao et al. (1998, J. of Viral, vol. 72, p. 2955-2961) describes the construction of a chimeric human parainfluenza virus (hPIV) type 3, in which hPIV type 1 HN and F genes were used for a replacement of (not an addition in) endogenous hPIV type 3 HN and F genes.

Also NDV was used for hemagglutinin expression of AIV. The influenza A / WSN / 33 hemagglutinin gene was inserted between the NDV Hitchner BL strain P and M genes. This recombinant protected mice from lethal infection although there was detectable weight loss in mice that fully recovered within 10 days. Nakaya et al. ( J. Virol 75, 11868-73, 2001). Another recombinant NDV with the same insertion site for the foreign gene expressed H7 from an LPAI but only 40% of vaccinated chickens were protected from both HPAI and NDVvelogenic (Swayne et al., Avian Dis. 47, 104750, 2003).

These publications, however, do not describe any advantageous effects of so-called non-coding regions of MVendogen genes on the expression of additional foreign genes in the MV vector genome.

An object of this invention is to provide a recombinant MMV vector that exhibits a higher level of expression of a protein encoded by a foreign gene inserted into the genome of the vector virus and / or which shows stronger immunogenicity than that of existing MV virus vectors.

The present inventors have found that this objective can be achieved by a recombinant Mononegaviral virus vector according to the invention. Therefore, the present invention provides a recombinant Mononegaviral virus vector hosting an additional transcriptional unit comprising a foreign gene operably linked with an upstream Mononegaviral virus gene (GS) initiation sequence and a downstream Mononegaviral virus gene (GE) sequence characterized by the fact that between the GS sequence and a foreign gene initiation codon and between a foreign gene termination codon and the GE sequence, a 3 'non-coding region and a 5' (genome sense) coding region. a Mononegaviral virus gene are located, respectively.

It is noted that the indications here of the nucleic acid strand polarity and throughout the text are given in the genome (-) sense, except in the context of cDNA and mRNA sequences. It has been found that the presence of the 3 'non-coding regions and 5 'of an MV virus gene in a transcription unit comprising a foreign gene inserted into the genome of an MV virus has a positive effect on transcription and / or expression of the foreign gene.

It is shown in Figure 3 that engineering the non-coding regions of an MV virus umgene between a GS sequence and an avian influenza hemagglutinin (HA) gene and between the AIV HA gene and a GE sequence increases the amount of HA mRNA synthesized by an MV vector virus harboring that AIV HA gene. A positive effect is also conserved at protein expression level: Comparison of MVV virus vectors only showed intense immunological staining with AIV HA specific antiserum in case the MV virus vector hosting the foreign gene AIVHA was flanked by non-coding regions ( Figure 4). Therefore, it has been found that while the inventors do not wish to link to any theory or model that may explain these observations, the presence of non-coding MV virus regions flanking a foreign gene has a positive effect on the performance of the resulting MV virus vector.

A foreign gene is a polynucleotide molecule that encodes a polypeptide or protein and is not present in the genome of the recipient MV virus genome.

As already described in detail above, a common feature of the MV virus genomic organization is its modular form of transcription in which serially arranged transcription units are successively transcribed. In a wild-type MV virus the transcribed genes are flanked (i) at its 3 'end with a GS sequence and (ii) at its 5' end with a nucleotide sequence also designated in the art as a "non-coding region" and a GE sequence. . Therefore, the term "non-coding region" (3 'or 5') as used herein defines a nucleotide sequence that is located upstream (3 ') or downstream (5') of a natural MV virus gene and encompasses the region between the GS virus sequence and the initiation codon (ATG) of the MV virus and the region between the codon determining (TAA, TAG or TGA) of the MV virus gene and the GE sequence, respectively. The noncoding regions used herein are derived from a gene of the same virus as the vector virus (i.e. the noncoding regions are homologous to the MV virus vector).

Detailed information on the MV virus genomic organization is known in the art, including the nucleotide sequences of the various MV virus genes and their transcriptional control sequences (GS and GE), and noncoding sequences flanking the genes. Such training is, for example, available from the National Center for Biotechnology Information (NCBI) database, e.g. via its web page; see Tables 2 and

3) ·

Non-coding sequences which are preferably used in this invention are derived from natural MV virus genes, replacement of one or more nucleotides in a non-natural coding region is also considered to be within the invention. In particular, nucleotide substitutions that are located immediately are contemplated. upstream or downstream of the strange dogene initiation / termination codon, respectively, and result from the introduction of artificial restriction enzyme declining sites that allow genetic manipulation of these regions.

In a preferred recombinant MV virus vector according to the present invention non-coding regions of a gene encoding a MV virus envelope protein, in particular an M, G, F or HN protein, or an RNP protein, in particular, a protein Ν, P or L.

In a particularly preferred recombinant MV virus according to the invention the non-coding regions are from a F or HN protein genecoder. Nucleotide sequences specific for non-coding regions to be used in a recombinant MV virus vector according to the invention are presented in Table 3.

Table 2: Genome and vector information on Mononegaviral viruses

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The GS and GE sequences to be used in this invention as the transcriptional control sequences are preferably those derived from the natural MV virus genes. These sequences are believed to modulate RNA polymerase activity during the transcription process, particularly in the process of transcription initiation and 5 'end modification of mRNA and in the control of 3' end transcription termination and polyadenylation. For each of the MV viruses, the start of cadagene is marked by a sequence of about 10 nucleotides. While in some MV virus species the GS sequences are the same as for cadagene, the GS sequences in the genome of other MV virus species may exhibit subtle differences. In view of their common function in transcription termination and end-tail poly-A formation 3 'of mRNA, GE sequences and MV viruses share common sequence characteristics. A typical GE sequence comprises a U-region of between 4 and 8 nucleotides in length. Furthermore, there is a strong conservation of a C-residue directly upstream of the U-region and which is preceded by a denucleotide segment that is rich in A / U. In various GE sequences the 4 nucleotides directly upstream of the U-region are composed of 3'-AUUC-5 '.

Transcriptional control sequences that define the gene edges or gene junctions of the MV virus genes have been identified for many MV virus genes by comparing the nucleotide sequences in the genomic model and the nucleotide sequences present at the mRNA terminations. In addition many studies have identified the GS and GE consensus sequences that are required for efficient gene expression. General features and specific examples GS and GE sequences can be derived from NCBI sequence database information (see Table 2 for NCBI access numbers) and are also reviewed by Neumann et al. (J. Virol. 83, 2635-2662 , 2002), eWhelan et al. (Current Topics Microbiol. Immunol. 203, 63-119, 2004).

Specifically preferred GS and GE sequences to be used in a recombinant MV virus vector according to the invention are listed in Table 3, although it is recognized that a more precise border between the GS, NCR and GE sequences cannot always be determined. This sequence information is described in the NCBI database (see accession in Table 2). For NDV, reference is made to Yl8898 EMEM accession number, for RV to M31046 GenBank accession number. Table 3: Gene junction sequence information of various MV genes (sense +)

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In a preferred recombinant MV virus vector of the invention the GS5 GE sequences and non-coding regions derived from the same virus virus gene.

Methods for preparing a recombinant MVvirus vector hosting an additional transcription unit comprising a foreign gene are well known in the art. For example, Table 2 refers to the documents describing the preparation of such recombinant viruses for various MV virus species. In principle, the method used in the present invention is the same as that in the prior art, except that the foreign gene to be inserted into the MV virus genome is flanked by the appropriate 3 'and 5' non-coding regions as defined above.

In a general method according to the invention the recombinant MV vector virus is prepared by inserting an isolated nucleic acid molecule comprising (i) a foreign gene flanked by the 3 'and 5' non-coding regions as defined above and (ii) the sequences appropriate transcriptional control in the MV virus genome such that the resulting MV virus vector the foreign gene is both preceded and followed by an MV virus gene junction, in particular by a genomic nucleotide sequence fragment comprising the GE-GR elements. -GS. The presence of upstream and downstream elements ensures proper translation not only of the inserted foreign genes, but also of homologous MV virus genes that are located downstream of the inserted foreign gene.

More particularly, in this method the nucleic acid molecule and the MV virus genome are used in their cDNA (sense +) form. This allows for easy manipulation and insertion of desired nucleic acid molecules into the viral genome.

In general, various parts of the genome could be used for foreign gene insertion between two genes, ie in intergenic regions (IGRs), 3 'or 5' non-coding regions of a gene as well as 3 'proximal promoter ends (before the N / NP genes) or 5 'distal (after the L genes) of a genome.

The foreign gene could advantageously be inserted before dogene N / NP, between NP-P5 P-M, M-G / F, G / F-HN, HN-L and after the L gene.

The simplest way is to use a restriction enzyme recognition sequence (RE) existing at one of these sites by cutting with the enzyme and introducing an appropriate transcription cassette. Since naturally existing restriction enzyme recognition sequences are not always located at the desired location, RE recognition sites could be introduced into the genome conventionally by site-directed mutagenesis or PCR.

Examples of suitable IGRs for foreign gene insertion can be found in Table 3.

The composition of the transcription cassette to be inserted depends on the insertion site. For example, in the case of a transcription cassette inserted into an IGR, the cassette may comprise the following elements: 3'-RE-GS recognition site-non-coding region-ORF (non-coding region) -GS non-coding region Alternatively, in the case of a transcription cassette being introduced into a 5 'non-coding region of an MVnatural virus gene, the cassette may consist of: 3' - RE-recognition site. GE-IGR-GS non-coding region-ORF (foreign gene) non-coding region-RE-5 'recognition site.

Similarly, in the case of a transcription cassette being introduced into a 3 'non-coding region of a MVnatural virus gene, the expression cassette may consist of: 3' - non-coding RE-region-ORF recognition site (gene) non-coding region-GE-IGR-GS-RE-5 'recognition site.

The preparation of such transcription cassettes and their insertion into an MV virus genome involves only molecular molecular biology techniques, as exemplified in the literature references listed in Table 2, and in the present Examples. In particular, site-directed and PCR such mutagenesis techniques can be used for this purpose (Peeters et al., 1999, supra; Current Protocols in Molecular Biology, eds .: FM Ausubel et al., Wiley NY, 1995 edition, pages 8.5. (8.5.5; Ekunkel et al., Methods in Enzymology Vol. 154, 376-382, 1987).

More particularly, a recombinant MV virus vector according to the present invention may be prepared by the well-established "reverse genetics" method which allows genetic modification of the negative, non-segmented MV strand RNA virus (reviewed for example). by Conzelmann, KK, Current Topics Microbiol. Immunol 203, 1-41, 2004; and Walpita et al., FEMS Microbiol. Letters 244, 9-18, 2005).

In this method, an appropriate cell is co-transfected by a vector comprising a cDNA molecule comprising a nucleotide sequence encoding a full length genome, or preferably an antigenome (positive sense) of an MV virus, and one or more vectors comprising the cDNA molecules comprising nucleotide sequences encoding the required carrier proteins under conditions sufficient to permit transcription and co-expression of the MV (anti) genome and carrier proteins and the production of a recombinant MV vector. In this method, said full length MV (anti) virus genome nucleic acid molecule comprises an additional transcription unit as defined above.

By vector is meant a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be ligated to replicate the ligated DNA segment and its transcription and / or expression in a cell transfected with it. vector.

Preferably, the full-length genome transcription vector is a plasmid comprising a cDNA coding sequence for the MV (anti) genome, flanked by the T7 polymerase promoter at its 5 'end, and a ribozyme (hepatitedelta) sequence at its end. 3 ', although a T3 or SP6 RNApolymerase promoter may also be used.

For intracellular expression of the appropriate support proteins, use is preferably made of plasmids comprising the cDNA sequence encoding these proteins, under the control of appropriate expression control sequences, e.g. a T7 polymerase promoter.

In a particularly preferred method of preparing a recombinant MV virus vector according to the present invention, expression plasmids encoding the MV virus N (or NP), PeL proteins are used.

The amounts or ratios of transfected support plasmids to be used in this reverse genetic technology cover a wide range. Ratios for N: P: L support plasmids may range from about 20: 10: 1 to 1: 1: 2 and efficient transfection protocols for each virus known in the art.

An exact copy of the genomic RNA is prepared in a transfected cell by the combined action of the ribozyme T7 RNA polymerase promoter, and this RNA is subsequently packaged and replicated by the viral support proteins provided by the co-transfected expression plasmids.

It is preferred that a T7 polymerase enzyme be provided by a recombinant vaccinia virus that infects the transfected cell, in particular by the vTF73 vaccinia virus, as well as other recombinant virus vectors, such as the contagious epithelioma virus, egfpEFLT7pol, or other viral vectors. be used for the expression of T7 RNA polymerase.

Separation of rescued virus from vaccinia virus can easily be accomplished by simple physical techniques such as filtration. For rescue of the Sendai or NDV virus, rescue can be performed by inoculating the supernatant of transfected cells in embryonated eggs.

In an even more preferred embodiment stranded cell lines are used for transfection of the transcription and expression vectors that constitutively express the (T7) RNA polymerase and / or one or more of the required carrier proteins.

For example, measles virus rescue can be performed in a human embryo kidney cell line, 293-3-46, which expresses both T7 RNA polymerase and the measles virus N and P support proteins (Radecke et al., EMBO J. 14, 5773-5784, 1995). Very useful cell alignment that can be advantageously used in the present invention is based on BSR cells expressing T7 RNA polymerase, i.e. BSR-T7 / 5 cell line (Buchholz et al., J. Virol. 73, 251-259, 1999).

Furthermore, more detailed information regarding the reverse degenetic technology to be used for the preparation of an MV virus according to the present invention is described in the review by Conzelmann, K.K. (supra) and Example 1.

The ability of recombinant MV virus vectors to expressably express foreign genes has resulted in the development of vectors for both prophylactic and therapeutic applications.

In a recombinant MV virus vector according to the present invention the foreign gene may vary depending on the specific MV virus devector species and the application of the vector virus.

The foreign gene may encode an antigen of a (other) microbial pathogen (e.g., a virus, a parasite bacterium), especially the foreign gene encodes an antigen of a pathogen that is capable of producing a protective immune response.

For example, heterologous gene sequences that may be inserted into the virus vectors of the invention include, but are not limited to, influenza virus glycoprotein genes, in particular avian influenza virus hemagglutinin H5 and H7 genes, genes derived from Bursal Disease Virus Infectious (IBDV), specifically VP2 of (IBDV), genes derived from Infectious Bronchitis Virus (IBV), feline leukemia virus, canine distemper virus, equine infectious anemia virus, rabies virus, ehrlichia organism, in particular Ehrlichia canis, respiratory syncytial viruses, parainfluenza virus, measles virus and human metapneumovirus.

Alternatively, the foreign gene may encode a polypeptide immune modulator that is capable of enhancing or modulating the immune response to virus infection, for example by co-expressing a cytokine such as an interleukin (eg IL-2, IL-12, 1FN- γ, TNF-α or GM-CSF).

The order of MV viruses includes both viruses that are capable of replicating in humans and animals, or both (e.g., rabies virus and Newcastle disease virus). Therefore, the foreign gene may be selected from a wide variety of human veterinary microbial pathogens.

Although all viruses may be used as a virus of the present invention, in a preferred embodiment of the invention the recombinant MV vector virus is a virus of the Rhabdoviridae family, preferably of the genus Lyssavirus or Novirhabdovirus, more preferably of the rabies virus or IHNV, respectively.

In a further preferred embodiment the MVrecombinant virus is a virus of the Paramyxoviridae family, preferably Respovirus dog, in particular the hPIV3 or bPIV3 species; Morbillivirus, in particular the CDV species; Pneumovirus, in particular the RV species; eAvulavirus, in particular the NDV species.

In a particularly preferred manner of the present invention there is provided a recombinant MV virus vector in which the virus is Newcastle disease virus (NDV). As NDV is capable of replication in both humans and animals, in particular poultry, more particularly chickens, a recombinant NDV vector according to the invention may comprise a foreign gene encoding a pathogen antigen, in particular a respiratory pathogen, or an immune modulator that is capable of producing an immune response in humans or any of these animals.

Reverse genetics methods for the genetic manipulation of NDV have been described for NDV by Peeters et al. (J. Virology 73, 50 O-5009, 1999), Romer-Oberdorfer et al. (J. Gen. Virol. 80, 2987-2995, 1999), and review by Conzelmann, K.K. (supra). In addition, it is also known that NDV can be used as a vector for foreign gene expression, for example, to produce an immune response in animals infected with NDV vector (Nakaya et al., 2001, supra) and Swayne et al., Avian Dis. 47, 1047-50, 2003). A foreign gene may be advantageously introduced into an NDV genome at various positions as generally described for the MMV viruses above. In particular, in a recombinant NDV vector according to the invention, a foreign gene (as part of an appropriate transcriptional unit) may be inserted between the following NDV genes: NP-P, PM, MF, F-HN, HN-L and proximal 3 'and 5' distal locus (Zhao et al., 2003, supra; Nakaya et al., 2001, supra), preferably in the proximal 3 ', PM, MF, and F-HN regions, the F-HN region being most preferred.

Furthermore, in a recombinant NDV vector according to the present invention non-coding regions flanking the foreign gene may be derived from all naturally occurring NDV genes, in particular from the Ν, Ρ, M, F or genes genes, the preferred HN gene. .

In a particular embodiment of the invention a recombinant NDV vector is provided herein in which the additional transcriptional unit is located between the F-HN genes and the inserted foreign gene is flanked by non-coding regions of the NDV HN gene.

More specifically, an NDV vector is provided in which 3'- and 5'-NCR (and optionally GS and GE sequences) have a nucleotide sequence as shown in SEQ ID NO: 1 and 2 or 3 and 4.

A recombinant NDV vector according to the present invention may be advantageously used to induce a domestic emave immune response, in particular chickens, against other pathogens. Therefore, the recombinant NDV ovator preferably comprises a foreign gene that encodes a protective antigen of an avian pathogen, in particular influenza virus, Marek's disease virus (MDV), infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), infectious bush disease virus (IBDV), chicken anemia virus (CAV), reovirus, avian retrovirus, avian adenovirus, turkey rhinotracheitis virus (TRTV), Ε. coli, Eimeria species, Cryptosporidia, Mycoplasms such as M.gallinarum, M. synoviae and M. meleagridis, Salmonella, Campylobacter, Ornithobacterium (ORT) or Pasteurella sp.

More preferably, the recombinant NDV vector comprises a foreign gene encoding an AIV, MDV, ILTV, IBV, TRTV, E. coli, ORT or Mycoplasma antigen.

In particular, the mutant recombinant NDV vector comprises a hemagglutinin (HA) gene from an influenza virus, preferably from an avian influenza virus (AIV), more preferably from a highly pathogenic AVI, in particular from A5 H5 or H7.

In principle, the HA gene of all strains of influenza (avian) can be used in this invention. Nucleotide sequences from many HA genes have been described in the art and relevant HA genes can be retrieved from nucleic acid sequence databases, such as GenBank or EMBL databases.

The highly pathogenic AIVA / chicken / Italy / 8/98 hemagglutinin (HA) subtype H5N2 gene can be advantageously used as a foreign gene in the present invention as described above. The gene is reverse transcribed, cloned into the eukaryotic expression vector pcDNA3 (Invitrogen), and sequenced (Lüschow et al., Vaccine, vol. J_9, p. 4249-4259,2001, and AJ305306 GenBank Accession No.). From the obtained expression plasmid pCD-HA5 the HA gene can be obtained by amplification by specific primers that generate the SRsartificial recognition site that allows the insertion of the HA gene into the NDV genomic sequences.

In another embodiment, the highly pathogenic A7 A / chicken / Italy / 445/99 HA subtype HA gene may be used as a foreign gene in the present invention as described above. The HA gene is reverse transcribed and PCR amplified. The 1711 bp product is cloned into the SmaI digested (Amersham) pUC 18 vector and sequenced (Veitset al., J. Gen. Virol. 84, 3343-3352, 2003; and GenBank Accession No. of AJ580353).

In a particularly advantageous recombinant MV virus vector according to the present invention the MV vector virus is attenuated, that is, the vector virus is not pathogenic to the target animal or exhibits a substantial reduction in virulence compared to the wild type virus. MV used herein as virus vectors have a long safety record as live attenuated vaccines such as the measles and NDV virus, whereas other viruses such as SeV and VSV are considered non-pathogenic to humans. In addition, there are commercial techniques for obtaining and attenuated virus screening that show limited potential for infectivity or replication. Such techniques include serial (cold) passaging of a virus on a heterologous substrate and chemical mutagenesis.

A recombinant NDV vector according to the invention may be derived from any conventional ND vaccine strain. Examples of suitable NDV samples present in commercially available ND vaccines are: Clone-30®, La Sota, Hitchner BI, NDW, C2 and AV4; Clone-30® is the preferred strain.

It has also been found by the present inventors that a recombinant MV virus vector according to the present invention is capable of inducing a protective immune response in animals.

Therefore, in another embodiment of this invention there is provided a vaccine against a microbial pathogen comprising a recombinant MV vector virus as defined above in a living or inactivated form, and a pharmaceutically acceptable diluent or carrier.

A vaccine according to the invention may be prepared by conventional methods such as those commonly used for commercially available inactivated or live MV virus vaccines. Briefly, a susceptible substrate is inoculated with the recombinant MV virus receptor and propagated until the replicated virus has the desired titer. after which the virus-containing material is harvested. Subsequently, the harvested material is formulated into a pharmaceutical preparation with immunizing properties.

Each substrate that is capable of supporting recombinant MV virus vector replication can be used in the present invention. As a substrate host cells can be used from both prokaryotic and eukaryotic origins, depending on the MV virus. Suitable host cells may be vertebrate, e.g., primate cells. Examples are human HEK, WI-38, MRC-5 or H-239 human cell lines, Vero simian cell line, CHO rodent cell line, BHK, MDCK canine cell line or ECF avian cell lines. or CEK.

A particularly suitable substrate on which a recombinant NDV vector according to the present invention may be propagated refers to SPF embryonated eggs. Embryonated eggs may be inoculated with, for example, 0.2 ml NDV-containing allantoic fluid comprising at least 10 'EID50 per egg. Preferably, 9- to 11-day-old embryonated eggs are inoculated with about 10 50 EID 50 and subsequently incubated at 37 ° C for 2-4 days. After 2-4 days the ND virus product may preferably be collected by collecting allantoic fluid. The fluid may then be centrifuged at 2,500 g followed by filtration of the supernatant through a filter (100 μm).

The vaccine according to the invention comprises the recombinant MV virus vector together with a pharmaceutically acceptable diluent or carrier commonly used for such compositions.

The vaccine containing the live virus may be prepared and marketed as a suspension or in a lyophilized form. Vehicles include stabilizers, preservatives, and buffers. Diluents include water, aqueous buffer and polyols.

In another aspect of the present invention there is provided a vaccine comprising the recombinant MV virus vector in an inactivated form. The biggest advantages of an inactivated vaccine are its safety and the high levels of long lasting protective antibodies that can be induced.

The purpose of inactivating viruses harvested after the payment stage is to eliminate virus reproduction. In general this can be achieved by well known physical or chemical means.

If desired, the vaccine according to the invention may contain an adjuvant. Examples of suitable compounds and compositions with adjuvant activity for this purpose are aluminum hydroxide, aluminum phosphate or aluminum oxide, oil-in-water or water-in-oil emulsion in, for example, a mineral oil, such as Bayol F®. or Marcol52® or vegetable oil such as vitamin E acetate and saponins. Administration of a vaccine according to the invention may be in any of the well known forms and may depend on the type of virus vector. Suitable modes of administration include parenteral, intranasal, oral and spray vaccination.

An NDV vector vaccine according to the invention is preferably administered by inexpensive mass application techniques commonly used for vaccination against NDV. For ND vaccination Technical techniques include spray vaccination and drinking water.

The vaccine according to the invention comprises an effective dosage of the recombinant MV virus vector with the active component, i.e. a quantity of immunizing MV virus material that will induce immunity to challenge vaccinated vessels by a virulent microbial organism.

Immunity is defined herein as the induction of a significantly higher level of protection in a human or animal population against mortality and clinical symptoms after vaccination compared with an unvaccinated group. In particular, the vaccine according to the invention protects a large proportion of animals or humans vaccinated against clinical symptoms of disease and mortality.

Typically, the live vaccine may be administered at a dose of 10 ^ 2.0 -10 ^ 8.0 tissue culture / embryo infectious dose (TC / EID50), preferably at a dose ranging from 10 ^ 4.0-10 ^ 7.0 TC / EID ^ 50. Inactivated vaccines may contain the antigen equivalent of 10 ^ 4.0-10 ^ 9.0 TC / EID50.

The invention also includes combination vaccines comprising, in addition to the recombinant MV virus vector according to the invention, a vaccine strain capable of inducing protection against another pathogen.

Example 1: Generation of a Recombinant MV Virus Vector Expressing an Avian Influenza Virus HA (NDV / AIVH5) gene

□ Viruses and cells

Rescued recombinant NDV and influenza A / chicken / Italy / 8/98 virus isolate were propagated in pathogen-free age-embryonic (SPF) embryonated chicken eggs. The Herts33 / 56 Velogenic NDV strain and the Clone 30 NDV vaccine (Nobilis®) were used.

BSR-T7 / 5 cells stably expressing the phage T7 RNA polymerase were used to recover infectious cDNA NDV.

□ NDV antigen RNA encoding cDNA construction containing AIV H5 gene

Numbering in parentheses as used herein to identify nucleotide positions in the NDV genome and amino acid residues in NDV proteins is as described by Römer Oberdörfer etal. (J. Gen. Virol. 80, 2987-2995, 1999, accession number in EMBL from Y888). Plasmid pfINDV, expressing Clone 30 total-length antigenomic RNA (Römer Oberdörfer et al., Supra) was used to introduce the AIV H5 gene that had been amplified from the CD-HA5 plasmid (Luschow et al., Supra) by site-specific primers. Artificial MluI Restrictions (PH5F1: 5'-cta agggg taa aat gga gaa aat agt gc -3 '(SEQ ID NO: 5) and PH5R1: 5'-tcg gac gcg SEQ ID NO: 6), MluI sites are underlined) for rNDV / AIVH5-A and NcoI or AflII commissions (PH5F2: 5'-cct tcc atg gag aaa ata gtg ctt c -3 '(SEQ IDNO: 7) and PH5R2: 5'- cct cct taa gta taa ttg act caa tta aat gca aat tct gca ctgcaa tga tcc -3 '(SEQ ID NO: 8), restriction sites are underlined) to NDV / AIVH5-B. Introduction of H5 into the Clone 30 antigenome (Fig. 1A) was done using the MluI sites as previously described for GFP insertion (Engel-Herbert et al., J. Virol. Methods 108, 19-28, 2003). Briefly, for construction of the full length plasmid pfINDV / AIVH5-A to H5 ORF was amplified with primers containing artificial MluI sites (see above) that were used to insert the minimal gene H5 ORF between the NDV F and HN genes (Fig. 1 A). Construction of the full length plasmid containing the AIV H5 gene for rNDV / AIVH5-B gene is given in Figure 1B. Mutagenesis reactions were performed using the Quik Change® IIXL site-directed mutagenesis kit (Stratagene). To this end, a pUC 18 plasmid (pUCNDVI) containing the NotI / BsiWI fragment (nt 4953 - 8852) from the Clone 30 genome and the following primers were used: MPl (5'-gac aac agt cct caa cg tgg accgcg CCg -3 ') (SEQ ID NO: 9) and MP2 (5'-ctg gct agt tga gtc aat tct taa gga gttgga aag atg gc -3') (SEQ ID NO: 10) for mutagenesis A (Fig. 1B) resulting in plasmid pUCNDVla with newly created NcoI and AflII sites (primer restriction sites are underlined). After digestion with NcoI and AflII aHN ORF of Clone 30 was replaced by amplified AIV H5 ORF. Paramutagenesis B to create SgfI and SnaBI sites in the intergenic region of the pUCNDVH5 Lde gene resulting in pUCNDV / AIVH5_lb (Fig. 1 B) primersMP3 (5'-caa aac age tca tgg tac gta ata cgg gta gga cat gg -3 ') (SEQ ID NO: 11) and MP4 (5'-gta agt ggc aat gcg up to gea ggc aaa aca gct cat gg -3 ') (SEQ IDNO: 12) were used. Mutagenesis C was done with MP3 and MP5 (5'-gaaaaact acc ggc gat cgc tga cag ag gac gat ata cgg g -3 ') (SEQ ID NO: 13) resulting in plasmid pUCNDV_lc to obtain SgfI and SnaBI sites that were used for the introduction of the Clone 30 HN gene in the intergenic region of the L gene in plasmid pUCNDVH5_lb. Finally, the NotI / BsiWI fragment from pflNDV-1 was replaced by the NotI / BsiWI depUCNDVH5_lc fragment (Fig. 1B). The lengths of the newly generated total length genomes represent a multiple of six (16938 nt for nNDV / AIVH5-A and 17196 nt for rNDV / AIVH5-B).

□ Virus transfection and spread

Transfection experiments, virus propagation, and confirmation of infectious virus recovery were performed as previously described (Römer-Oberdörfer et al., Supra; Engel-Herbert et al., Supra). The only difference was that a total amount of 20μg DNA (10μg of full length genome containing plasmid, 6μg of pCiteNP, 2μg of pCiteP, and 2μg of pCiteL) was used for transfection.

□ Results

The AIV H5 open reading template was inserted between the F and HN genes of the previously described plasmid pflNDV (Römer-Oberdörferet al., Supra). To this end, AIV H5 ORF from AIVA / chicken / Italy / 8/988 (H5N2) isol was amplified from plasmid pCD-HA5 (Luschow et al., Supra) by specific primers with MluI restriction sites that were used for insertion of AIV H5 ORF at pflNDVoligol singular MluI restriction site; (Engel-Herbert et al., Supra) resulting in plasmid pflNDV / AIVH5-A (Fig. 1A). In this construct the AIV H5 ORF is outlined by the artificial gene end (GE) and gene start (GS) sequences in the intergenic region between the NDV F and HN genes. Full length plasmid pfNDV / AIVH5-B to HN ORP from plasmid pUCNDVIa was replaced by amplified H5 ORF as a NcoI / AflII- fragment (Fig. 1B). In the resulting plasmid pUCNDVH5 restriction sites SgfI and SnaBI were created in the intergenic region of the H5 gene resulting in plasmid pUCNDVH5_lb (Fig. 1B). The constrained sites SgfI and SnaBI were used to introduce the HN gene from plasmid pUCNDVlc, in which the HN gene was also flanked by the SgfI and SnaBI restriction sites (Fig. 1 B). Finally, the resulting plasmid pUCNDVH5_1c was used to replace the pflNDV NotI / BsWI fragment (Fig. 1). The constructed pflNDV / AIVH5 -B differs from plasmidopflNDV / AIVH5-A in that the non-coding regions of NDV HN were additionally inserted between the transcriptional control elements (GS, GE) and the H5 ORF.

Recombinant NDV rNDV / AIVH5-A and rNDV / AIVH5-B were recovered from BSR-T7 / 5 cells transfected with the respective full-length cDNA and support plasmids as previously described (Römer-Oberdörfer et al., Supra; Engel-Herbert et al. ., supra). For virus recovery, transcription supernatants were injected with allantoic hatches of 10-day-old embryonated chicken eggs and incubated for 5 days. Allantoic fluid was collected and analyzed for virus by hemagglutination or indirect immunofluorescence (IF) testing. Virus-containing allantoic fluids were used for a second egg passage for further virus propagation. The presence of H5 dogene inserted into the viral genomes of rNDV / AIVH5-A and rNDV / AIVH5-B was confirmed by reverse transcription PCR and sequencing (data not shown). Figures 2A and 2B show the nucleotide sequence of regions flanking HN ORF in NDV and H5 ORF in the NDV vector.

Example 2: In vitro Characterization of NDV / AIVH5 Vector □ RNA Analysis

CEF cells were infected with NDV Clone 30, rNDWAIVH5-A, rNDV / AIVH5-B and AIV A / chicken / Italy / 8/98 (H5N2) at a multiplicity of infection (MOI) of 10 per cell and incubated for 8 h at 37 ° C. Ç. Total RNA from infected and uninfected cells was prepared, separated on denaturing agarose gels and hybridized with radiolabeled cRNAs. Plasmids pCD-HA5 and pCD-NDVHN which contain AIV open reading matrix / chicken / Italy / 8/98 (H5N2) H5 and NDVClone 30 HN, respectively, were used for in vitro transcription of P-labeled detRNA (SP6 / T7 Transcription kit, Roche).

To verify the transcription of the AIV H5 gene inserted into rrNDV / AIVH5-A and -B Northern blot analyzes were performed with total RNA from recombinant NDV / AIVH5 infected primary chicken embryo fibroblasts. RNA preparation from cells infected with NDV Clone 30 and AIV A / chicken / Italy / 8/98 (H5N2) was used as a control. Transcription of the inserted AIV H5 gene was detected for rNDV / AIVH5-Abem as for NDV / AIVH5-B with Gene-specific antisense tRNA (Fig. 3). It can be seen that the AIV-H5B transcript is extended by about 81 nt and is more abundantly present than the AIV H5A transcript.

□ Western blot analysis

CEK cells were infected with NDV Clone 30, rND V / AI VH5-A, rNDV / AIVH5-B and AIV A / chicken / Italy / 8/98 (H5N2) and incubated for 20 h at 37 ° C. Infected and uninfected control cell lysates were separated by SDS-PAGE (ca. 104 cells per lane), and transferred to nitrocellulose filters (Trans-Blot® SD cell, Bio-Rad). Blots were incubated with a polyclonal rabbit anti-NVV antiserum, or a polyclonal chicken antiserum against H5 subtype IVA in dilutions of 1: 200.00 and 1: 2,500, respectively. Peroxidase-conjugated species-specific antibody binding was detected by chemiluminescence using SuperSignal® West Pico ChemiluminescentSubstrate (Pierce) on X-ray films (Hyperfilm® MP, Amersham).

In Western blot analyzes H5 protein was detectable only in cells infected with rNDV / AIVH5-B. The AIY subtype H5 specific antiserum detected two prominent proteins of approximately 70 and 50 kDa and one hardly visible protein of ca. 25kDa, which were not found in cells infected with NDV Clone 30 (Fig. 4).

□ Indirect immunofluorescence (IF) tests

For indirect IF tests CEF cells were infected at low MOI with NDV Clone 30, rNDV / AIVH5-A, rNDV / AlVH5-B and AIVA / chicken / Italy / 8/98 (H5N2) for 20 h. After fixation with methanol and acetone (1: 1) the cells were subsequently incubated with either a rabbit NDV anti-polyclonal antiserum or an H5 subtype AIV polyclonal chicken antiserum at 1: 3,000 and 1: 100 dilutions respectively. .After incubation with F (ab) 2 fragment of anti-rabbit IgG and fluorescein-conjugated IgGanti-chicken antibodies samples were analyzed by conventional fluorescence microscopy.

H5 expression was examined by indirect IF testing of infected CEF cells. Following incubation with a pronounced fluorescence-specific antiserum was detectable in cells infected with NDV Clone 30, rNDV / AIVH5-A and rNDV / AIVH5-B, but not in cells infected with AIV A / chicken / Italy / 8/98 (H5N2) or in uninfected cells (Fig. 5, right panel). Incubation with an A5 subtype H5 specific antiserum showed a marked fluorescent in AIV infected cells. Comparing with two recombinants, rNDV / AIVH5-B showed a more intense H5-specific fluorescence than rNDV / AIVH5-A indicating a higher level of H5 protein expression (Fig. 5, left-hand panel).

Viral particles were adsorbed to form coated copper grids for 7 min. The crates were washed four times with PBS containing 0.5% bovine serum albumin and subsequently incubated with either an IVA subtype H5-specific or NDV-specific antiserum for 45 min. After several PB washes, the crates were incubated for a further 45 min with protein A - gold (10 nm, PAG 10, Biocell International) or anti-chicken gold rabbit (10 nm, RCHL 10, Biocell International). After final washings with CP the virus particles were counterstained with phosphotungstic acid (PTA, pH 7.2) and examined with an electron microscope.

When NDV Clone 30 or rNDV / AIVH5-A virions were examined, staining was observed only with NDV specific antiserum. In contrast, for rNDV / AIVH5-B staining was observed using NDV antisera and also by the use of AIV antisera, demonstrating that rNDV / AIVH5-B virions contain H5 hemagglutinin. Gold particles were found predominantly along the surface. rNDV / AIVHS-B virions, indicating that hemagglutinin was stained in the viral membrane.

Example 3: In vivo Characterization of NDV / AIVH5 Vector

□ Protection evaluation by rNDV / AIVH5-A and rNDV / AIVH5-Brecombinantes:

One-day-old chickens were randomly assigned to two groups and vaccinated oculonasally with 10 6 EID50 derNDV / AIVH5-A or with commercial NDV Clone 30 vaccine (Nobilis®, Intervet, NL) via spray. At 28 days of age a second immunization was given in the same manner. On day 12 after the second sanue immunization was collected to assess the presence of antibodies to NDV and AIVH5 HI test. Two weeks after the second vaccination the immunized groups were divided and part of each group was oculally challenged with 10 EID50 of highly pathogenic A / chicken / Italy / 8/98 (H5N2) isolate. The remaining chickens were used to evaluate the protective efficacy of velogenic NDV vaccines. Therefore the additional control birds and animals received 10 'EID5O of NDV strain Herts33 / 56 intramuscularly.

After immunization and challenge infection all birds were observed daily for a period of 10 days for clinical signs and classified as healthy (0), sick (1; one of the following signs: respiratory signs, depression, diarrhea, cyanosis, edema, nervous signs), severely ill (2; more than one of the following signs: respiratory signs, depression, diarrhea, cyanosis, edema, nervous signs), or dead (3). A clinical score representing the average value of all poultry per group for this period was calculated. Finally, three weeks after challenge blood samples were taken from all surviving animals to evaluate antibody titers against AIV and NDV.

The recombinant NDV rNDV / AIVH5-B was tested in a separate animal of almost identical experimental design. The only differences were that immunizations with 10 6 EID50 of either NDV / AIVH5-B vaccination or NDV Clone 30 vaccine were administered to both groups oculonasally and no challenge infection of NDV was performed.

All data from these experiments are summarized in Table 4 and Figure 8. Sera from immunized chickens were analyzed on this HI three weeks after vaccination, but HA-specific serum antibodies could not be detected in either case. All animals in both groups developed high-level NDV-specific antibodies already after the first immunization (mean HI titers of 2 -2) and the animals were fully protected against a velogenic NDV infection, while all control animals died within 4 weeks. diasexhibiting typical signs of ND. As expected, AIV challenge infection caused severe disease in NDV Clone 30 immunized chickens with a 90% mortality rate. Animals in the group immunized with rNDV / AIVH5-A survived with a high dose of highly pathogenic AIV, but all chickens exhibited varying signs of avian influenza with a clinical score of 0.64 indicating significant yet partial protection. However, rNDV / AIVH5- immunized chickens. B have been completely protected against any signs of disease following infection with a highly pathogenic avian influenza A virus.

Table. 4: Summary of animal experiments with rNDV / AIVH5-A and

<table> table see original document page 36 </column> </row> <table> <table> table see original document page 37 </column> </row> <table>

Example 4: Construction of an NDV / AIV-H5 vector with an NDV F-gene gene flanking vector.

Using essentially the same methods and materials as described above (Engel-Herbert et al, supra), an NDV vector construct was prepared by bringing the H5 AIV gene between the F and HN genes of the plasmidopflNDV previously described (Rõmer-Oberdörfer et al., Supra). ), but now insert H5 has been flanked with non-coding regions of the NDV F gene.

Briefly the various steps and materials used were:

A pUC plasmid with the 1.6 kb NotI-PstI fragment of NDVH5 (pUCIRA) was mutated to create a restriction enzyme site MluI using mutagenesis primers: pMPMLUIGRFHNF (5'-ggt tgt agatga cca aag gac gcg tta cgg gta gaa cgg taa gag agg -3 '; SEQ ID NO: 14) aned pMPMLUIGRFHNR (5'-cct ctc tta ccg ttc tac ccg taa cgc gtc ctt tgg tcatct aca acc -3'; underlined MluI site, with GSem sequence in bold), resulting in plasmid pUCIRAMLU.

Two oligo's were ringed: OFVOF: 5'- agg acg cgt tac gggtag aag att ctg gat ccc ggt tgg cgc cct cca ggt gca cca tgg ag -3 '(SEQ IDNO: 16, underlined MluI site) and OFVOR: 5'- ctc cat ggt get gca cct gga gggcgc caa ccg gga tcc aga to ttc tac ccg taa cac gtc ct -3 '(SEQ ID NO: 17, underlined siteMluI). These were then digested with MluI and NcoI.

Digestion of pUCIRAMLU Plasmid with MluI and NcoI.Linking of approximately 4.3 kb of pUCIRAMLU fragment with MluI-NcoL-Digested OFVOF / OFVOR The resulting plasmid was called pUCIRA2.

A pUC plasmid (pUCAROK) with the NDV NotI-BsiWI fragment with H5 instead of HN orf and pUCIRA2 was digested with NcoI and SgfI and the pUCAROK NotI-NcoI fragment was replaced by that of pUCIRA2, resulting in the pUCAROK2 plasmid.

HF-PCR (Roche) was performed to amplify the ncr of the NDV F gene behind the inserted F gene using the primers: PNCRFHIF: 5'-atactt aag ttc cct aat agt aat ttg tgt -3 '(SEQ ID NO: 18, site Underlined), ePNCRFHIR: 5, - cac gcg through gea ttg cca ctg tac att ttt tet taa ctc tet gaa ctg acagac tac c -3 '(SEQ ID NO: 19, SgfI underlined site), and pUCAROA plasmid (pUC with fragment) NDV NotI-SpeI). The ~ 100 resultant fragment was ligated into pGEMTeasy vector to give plasmidopGEMFncrhi.

Plasmids pUCAROK2 and pGEMFncrhi were digested with AfII and SgfI to replace the HN ncr behind plasmid pUCAROK2 H5 with that of pGEMFncrhi. The resulting plasmid was called depUCAROK4.

In the last step the NotI-SgfI fragment of the NDVH5 plasmid was replaced by that of pUCAROK4 resulting in the new full-length E18C plasmid, comprising the AIV H5 gene inserted into the NDV vector between F and HN, and flanked by the F gene ncr's.

With the experiments of transfection of new constructs, rec virus propagation and confirmation of infectious virus recovery were performed as described above. Then the virus was characterized biochemically and biologically.

Example 5: Generation of other NDV vector constructs and rec virus:

Using similar techniques several other inserts were prepared in the NDV vector using different inserted genes and different insertion sites.

With the details already provided here all are within easy search of the skills of the experienced person, so it is sufficient to present these in table form:

Table 5: Other NDV vector constructs according to the invention:

<table> table see original document page 39 </column> </row> <table>

Example 6: Generation of a recombinant rabies virus vector by expressing an EIAV envelope gene

To demonstrate that the advantageous effects of the invention (the use of non-coding regions of the MV gene to increase expression and / or presentation of foreign genes in rec MV virions) extend beyond the Paramyxoviridae family, rabies virus - a member of the Rhabdoviridae family - was Used as a vector to express an envelope protein derived from an unrelated virus, equine infectious anemia virus (EIAV).

In this example the construction is described of a rabies virus comprising the EIAV envelope protein inserted between the rabies G and L genes, where the env gene is flanked by the ncr's of the parental G-protein gene.

Rabies virus subclones were prepared on phagemidop Bluescript® SK + using the full length clone SAD-D29 (Mebatsion, 2001, J. Virol., Vol. 75, p. 11496-11502), which is referred to herein as ORA-D. To prepare a cloning vector, a pSK vector was first digested with Saci, blinded with Klenow enzyme, followed by HindIII digestion and gel purification of the ~ 3 kb fragment. The insert was prepared by digesting ORA-D with StuI and HindIII, purifying the resulting 1.3 kb fragment, and ligating into the prepared pSK vector to generate pNCR-b oplasmide.

Plasmid pNCR-b was digested with BstXI and HindIII and -4.0 kb deletion was purified and used for ligation with oligo'sBSSNH + and BSSNH- (see Table 6) to create plasmid pSSNsc containing minimal transcription and binding cassette. with RABGNCRI-4 oligos (Table 6) to generate the GNCR-b construct, containing non-coding regions in addition to the minimal transcription unit (Figure 7).

The EIAV env strain Wyoming strain was obtained by amplifying a 2052 nt synthetic gene that had been codon-optimized, RNA editing sites removed (Cook, et al. 2005, Vet.Micro., Vol. 108, p. 23 -37), and cracked by 134 amino acids in the 3 'coding region of the original gene.

Table 6: Oligo sequences used during the construction of recurrent hail viruses

<table> table see original document page 40 </column> </row> <table>

(*) 3 nt changed from original ORAD: GGAAAG GGACTGG, to: GGATACGTACTGG

The amplified env gene was kinased and subcloneed as follows: a ~ 2.0 kb amplicon, engineered to represent the entire truncated EIAV envelope protein, was generated using the EIAsynCDF + EIAsynCDstRR primer set (see Table 6). Amplicon was inserted into the GNCR-b subclone which had been digested with dephosphorylated SnaBI to generate the recombinant plasmid pGNCR-b: envG. The -2.0 kb fragment was also inserted into the SSNsc subclone which had been pre-digested with NheI, blunt end and dephosphorylated to generate recombinant oplasmid pSSNsc: env.

Each recombinant construct was digested with linked SphI and HindIIIe in the SSNsc subclone, which was pre-digested by CTAP-dephosphorylated Sphl / HindIII. Upon return to the SSNsc subcclone, the modified inserts were returned to the main support ORAD by digestion with SphI and MluI to generate recombinant RV-env and RV-envG rabies viruses (Figure 8). The 5 'and 3' ends of the constructs were verified by sequence analysis using the Big-Dye® Terminator Cycle Sequencingchemistry (Applied Biosystems) and analyzed using an Applied Biosystems3100-Avant Capillary Electrophoresis Sequencer.

Construction of a full-length cDNA clone based on the modified SAD rabies ORAD strain and generation of a recombinant rabies virus have been described (Schnell et al., 1994, EMBO J., vol. 13, p. 4195-4203; Mebatsion). , 2001, supra). Each recombinant rabies virus was transferred to BSR cells as previously described (Schnell, et al, 1994, supra) using Mirus Trans-IT-LT 1. Three days after transfection, cells and supernatant were harvested and passed. Subsequent passages were by supernatant only, using an estimated moi of 0.5. Each recombinant virus was passed a minimum of five times to check for stability.

Recombinant rabies infected BSR cells were fixed 40 hours after infection. These were analyzed by indirect immunofluorescence using FITC Monoclonal Globulin Anti-Rabies (FDI DiagnosticsInc.) Or employing anti-EIAV polyclonal horse sera. At 10-fold serial dilutions, the ratio of infected rabies virus producing cells was also compared to infected cells expressing EIAV to monitor virus stability and recombinant antigen expression.

Recombinant five-pass rabies viruses were inoculated into T-75 flasks with fresh BSR cells at a moi of 0.01. 24 hours after infection, the medium was changed to serum free. Supernatant was pelleted and clarified by centrifugation (10,000 χ g) at 72 hours after infection. Viral supernatants were purified by sucrose gradients for purified anlisarvirions.

Purified virions and total infected cellular protein were combined with 2x Laemmli reducing sample buffer and placed in boiling water for 5-10 minutes. Samples were loaded onto a 10% Tris-HCl deacrylamide gel (Bio-Rad) in SDS-PAGE Ix Running Buffer. SOS-PAGE gels were run at 20 mA until the dye front was close to or at the bottom of the gel. The separated proteins were transferred to the Immobilon-P (PVDF) membrane (IPVH10100, Immobilon) by blotting at 225 mA for 45 minutes. Blots were incubated in blocking buffer (PBS-Tween 20 + 1% skim powder bed) for one hour at room temperature and then washed 3 times for 5 minutes in PBS-Tween20. Rabies expression was detected using rabbit anti-rabies polyclonal sera directed against diluted 1: 20,000 and 1: 2,000 rabies nucleoprotein glycoprotein, respectively, in blocking buffer.

Simultaneously, EIAV env expression was detected using polyclonal anti-EIAV horse sera diluted 1: 500 in blocking buffer. Blots were incubated for one hour at room temperature and then washed 3 times for 5 minutes in PBS-Tween 20. Blots were placed in HRP-conjugated goat anti-rabbit IgG (H + L) and IgG (H + L) of HRP-conjugated goat anti-horse (Bethyl Labs), each diluted 1: 2,000 in blocking buffer and incubated for one hour at room temperature. Blots were then washed 3x5 minutes in PBS-Tween 20. Blots were incubated on TMB membrane peroxidase (KPL) substrate until development was complete, approximately 1-3 minutes. Blots were placed in distilled water to stop the reaction.

A typical result of such a Western blot experiment is shown in Figure 9, and demonstrates the composition of recombinant hail virus protein expressing EIAV envelope protein.

Track 1: Broadband PM Ladder (Bio-Rad);

Lane 2: ORAD main support virus;

Lane 3: RV-env, comprising the EIAV-env gene, inserted between rabies G and L genes, without flanking ncr's;

Lane 4: RV-envG, comprising the EIAV-env gene, flanked by the ncr regions of the rabies virus G protein.

Both recombinant rabies viruses gave comparable titer of infection and stably expressed EIAV-gene insert after multiple passages in vitro.

However, despite subjection of comparable amounts to Western blot, the recombinant RV-env, built in the modocustomer (therefore without flanking ncr's) exhibited only a very weak band corresponding to the EIAV-env protein; whereas the recombinant RV-envG virus, which had the non-G-protein coding regions flanking the inserted EIAV-protein, was expressed at a much higher rate: Figure 9, comparing the band for env protein in lanes 3-4.

Because the RV-envG and RV-env constructs and inserts are otherwise identical, this is strong evidence that non-coding regions have a positive effect on promoting high level of immune presentation and foreign protein production. SEQUENCE LISTING

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<220>

<221> NDV_final_geneHN <222> (167) .. (177) <400> 2

ttgagtcaat tataaaggag ttggaaagat ggcattgtat cacctatctt ctgcgacatc 60aagaatcaaa ccgaatgceg gcgcgtgctc gaattccatg ttgccagttg accacaatca 120

gccagtgctc atgcgatcag attaagcctt gtcaatagtc tcttgattaa gaaaaaa 177

<210> 3 <211> 133 <212> DNA <213> Artificial <220>

<223> NDV rec insert construct <220>

<221> rNDV_final_geneF <222> (1). . (11) <220>

<221> rNDV_F-HN_intergenic region

<222> (12) .. (42)

<220>

<221> rNDV_start_geneHN

<222> (43) .. (52)

<220> <221> rNDV_geneHN_coding_region_5_ <222> (53) .. (133) <400> 3

ttaagaaaaa actaccggtt gtagatgacc aaaggacgat atacgggtag aacggtaaga 60gaggccgccc ctcaattgcg agccaggctt cacaacctec gttctaccgc ttcaccgaca 120

acagtcctca acc 133

<210> 4 <211> 177 <212> DNA <213> Artificial <220>

<223> NDV rec insert construct <220>

<221> rNDV_geneHN_region_not_coder_3 '

<222> (1) .. (166)

<220>

<221> rNDV_final_geneHN <222> (167) .. (177) <400> 4

ttgagtcaat tctaagggag ttggaaagat ggcattgtat cacctatctt ctgcgacatc 60aagaatcaaa ccgaatgccg gcgcgtgctc gaattccatg ttgccagttg accacaatca 120

gccagtgctc atgcgatcag attaagcctt gtcaatagtc tcttgattaa gaaaaaa 177

<210> 5 <211> 29 <212> DNA <213> Artificial <220>

<223> Cloning initiator PH5F1 <400> 5

ctaaacgcgt aaaatggaga aaatagtgc 29

<210> 6 <211> 31 <212> DNA <213> Artificial <220>

<223> PH5R1 cloning initiator <400> 6

tcggacgcgt ttaaatgcaa attctgcact g

<210> 7 <211> 25 <212> DNA <213> Artificial <220>

<223> PH5F2 cloning initiator <400> 7

ccttccatgg agaaaatagt gcttc

<210> 8

<211> 54

<212> DNA

<213> Artificial

<220>

<223> PH5R2 cloning initiator <400> 8

ccttccatgg ãôâaastáât gcttc

<210> 9 <211> 30 <212> DNA <213> Artificial <220>

31

25

25 <223> MPl Cloning Initiator <400> 9

gacaacagtc ctcaaccatg gaccgcgccg

<210> 10 <211> 41 <212> DNA <213> Artificial <220>

<223> MP2 cloning initiator <400> 10

ctggctagtt gagtcaattc ttaaggagtt ggaaagatgg c

<210> 11 <211> 38 <212> DNA <213> Artificial <220>

<223> MP3 cloning initiator <400> 11

caaaacagct eatggtacgt aatacgggta ggacatgg

<210> 12 <211> 38 <212> DNA <213> Artificial <220>

<223> MP4 Cloning Initiator <400> 12

gtaagtggca atgcgategc aggcaaaaca gctcatgg

<210> 13 <211> 43 <212> DNA <213> Artificial <220>

<223> MP5 cloning initiator <400> 13

gaaaaaacta ccggcgatcg ctgaccaaag gacgatatac ggg

<210> 14

<211> 48

<212> DNA

<213> Artificial

<220>

<223> Mutagenesis Primer pMPMLUIGRFHNF <400> 14

ggttgtagat gaccaaagga cgcgttacgg gtagaacggt aagagagg

<210> 15 <211> 48 <212> DNA <213> Artificial <220>

<223> Mutagenesis Primer pMPMLUIGRFHNR <400> 15

cctctcttac cgttctaccc gtaacgcgtc ctttggtcat ctacaacc

<210> 16 <211> 65 <212> DNA <213> Artificial <220>

<223> OFVOF Cloning Oligo <400> 16

aggacgcgtt acgggtagaa gattctggat cccggttggc gccctccagg tgcagcaccatggag <210> 17 <211> 65 <212> DNA <213> Artificial <220>

<223> OFVOR Cloning Oligo <400> 17

ctccatggtg ctgcacctgg agggcgccaa ccgggatcca gaatcttcta cccgtaacgc 60gtcct 65

<210> 18 <211> 31 <212> DNA <213> Artificial <220>

<223> PNCRFHIF Cloning Initiator <400> 18

atacttaagt tccctaatag taatttgtgt g ??? 31

<210> 19 <211> 58 <212> DNA <213> Artificial <220>

<223> PNCRFHIR Cloning Initiator <4 00> 19

cácgcgatcg cattgccact gtacattttt tettaaetct ctgaactgac agactacc 58

<210> 20 <211> 31 <212> DNA <213> Artificial <220>

<223> BSSNH cloning initiator + <400> 20

Ctggtgaaaa aaactaacac ccctgctagc at 31

<210> 21 <211> 39 <212> DNA <213> Artificial <220>

<223> BSSNH- <400> Cloning Initiator 21

cgttgaccac tttttttgat tgtggggacg atcgttcga 39

<210> 22 <211> 50 <212> DNA <213> Artificial <220>

<223> RABGNCRoligol cloning initiator <400> 22

ctggtgaaaa aaactattaa catccctcaa aagactcaag gatacgtact SO

<210> 23

<211> 49

<212> DNA

<213> Artificial

<220>

<223> Cloning Initiator RABGNCRoligo2 <400> 23

gtatccttga gtcttttgag ggatgttaat agtttttttc accagttgc 49

<210> 24 <211> 50 <212> DNA-

<213> Artificial <220>

<223> RABGNCRoligo3 cloning initiator <400> 23

ggccgtcctt tcaacgatcc aagtcctgaa gatcacctcc ccttggggga

<210> 25 <211> 59 <212> DNA <213> Artificial <220>

<223> RABGNCRoligo4 cloning initiator <400> 23

agcttccccc aaggggaggt gatcttcagg acttggatcg ttgaaaggac ggccagtac

<210> 26

<211> 20

<212> DNA

<213> Artificial

<220>

<223> EIAsynCDF Cloning Initiator <400> 26

atggtgtcca tcgccttcta

<210> 27 <211> 21 <212> DNA <213> Artificial <220>

<223> EIAsynCDstopR cloning initiator <400> 27

tcagtgtatg ttgtgttggg c

Claims (23)

1. Recombinant Mononegaviral virus vector hosting an additional transcription unit comprising a foreign gene operably linked with an upstream Mononegaviral virus gene start (GS) sequence and a downstream Mononegaviral virus gene (GE) sequence, characterized in that that between the GS sequence and a foreign gene initiation codon and between a foreign gene termination codon and the GE sequence, a 3 'non-coding region and a 5' non-coding region (genome sense) of a Mononegaviral virus gene are located respectively. Recombinant Mononegaviral Virus Vector according to Claim 1, characterized in that the non-coding regions 3 'and 5' are from a gene encoding a Mononegaviral virus envelope protein, in particular from an M, G gene. , F or HN. Recombinant Mononegaviral Virus Vector according to Claim 1, characterized in that the non-coding regions 3 'and 5' are from a gene encoding a Mononegaviral virus RNP protein. Recombinant Mononegaviral virus vector according to claims 1-3, characterized in that the foreign gene encodes an antigen of a pathogen. Recombinant Mononegaviral virus vector according to claims 1-3, characterized in that the foreign gene encodes an immune modulator. Recombinant Mononegavirales virus vector according to claims 1-5, characterized in that the Mononegaviales virus vector is a virus of the Rhabdoviridae family. Recombinant Mononegaviral virus vector according to claim 6, characterized in that the Mononegavial virus virus vector is a rabies virus. Recombinant Mononegaviral virus vector according to claim 6, characterized in that the Mononegavial virus virus vector is an infectious hematopoietic necrosis virus. Recombinant Mononegaviral virus vector according to claims 6-8, characterized in that the 3 'and 5' non-coding regions are from a Ν, Ρ, M or G gene. Recombinant Mononegaviral virus vector according to claims 6-9, characterized in that the additional transcriptional unit is located proximally 3 'or between the genes P and Μ, M and G or G and L. Recombinant Mononegaviral virus vector according to claims 1-5, characterized in that the Mononegavial virus virus vector is a virus of the Paramyxoviridae family. Recombinant Mononegaviral virus vector according to claim 11, characterized in that the Mononegavial virus virus vector is a Newcastle disease virus, cinomosecanin virus or parainfluenza virus (bovine). Recombinant Mononegaviral virus vector according to claims 11-12, characterized in that the non-coding regions 3 'and 5' are from an NP, Ρ, M, F or HN gene. Recombinant Mononegaviral virus vector according to claims 11-13, characterized in that the additional transcriptional unit is located proximally 3 'or between the genes P and Μ, M and F, F and NH or NH and L. Recombinant Mononegaviral virus vector according to claims 11-14, characterized in that the Mononegavial virus virus vector is Newcastle disease virus. Recombinant Mononegaviral virus vector according to claim 15, characterized in that the additional transcriptional unit is located between the F and HN genes. Recombinant Mononegaviral virus vector according to claims 15-16, characterized in that the non-coding regions are from an HN gene. Recombinant Mononegaviral virus vector according to claims 15-17, characterized in that the foreign gene encodes an antigen of an avian pathogen. Recombinant Mononegaviral virus vector according to claims 15-18, characterized in that the foreign gene encodes a hemagglutinin (HA) of an influenza virus, preferably a H5 or H7 hemagglutinin. Recombinant Mononegaviral virus vector according to claims 1-19, characterized in that the Mononegavial virus vector is an attenuated virus. Vaccine against a microbial pathogen, characterized in that it comprises a recombinant Mononegaviral virus vector as defined in claims 1-20 in a living or inactivated form, and a pharmaceutically acceptable diluent or carrier. Vaccine according to claim 21, characterized in that it further comprises an adjuvant. Vaccine according to claims 21-22, characterized in that it comprises an additional vaccine strain.