RU2441070C2 - Recombinant newcastle disease virus expressing h5 hemagglutinin of avian influenza virus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: recombinant vector of Newcastle disease virus contains an additional transcription unit including a gene coding HA protein of pathogenic avian influenza virus H5 operatively related with a gene start sequence of an initial portion of Newcastle disease virus and a gene end sequence of an end portion of Newcastle disease virus, characterised by the fact that said protein contains an elongation portion consisting of at least three essential amino acids where said elongation portion consists of arginine (Arg) and/or lysine (Lys) residues and contains at least one lysine with said gene not containing a sequence which can be recognised by a viral polymerase of Newcastle disease virus as an end gene sequence.

EFFECT: presented vector enables high expression level of HA protein of pathogenic avian influenza virus H5, as well as vaccine containing such vector provides an immune protection against Newcastle disease virus and avian influenza virus infection.

12 cl, 5 dwg, 1 tbl, 8 ex

Description

This invention relates to a recombinant vector of the Mononegavirales virus, containing an additional transcriptional unit comprising a foreign gene operably linked to the start sequence of the Mononegavirales virus in the initial part of the Mononegavirales virus and the end of the gene (GE) sequence of the end of the Mononegavirales virus in the “ downstream. " This invention also relates to a method for producing such a recombinant Mononegavirales virus vector, as well as to a vaccine containing such a recombinant Mononegavirales virus vector.

Live viruses capable of replicating in an infected host cell induce a strong and lasting immune response to the antigens they express. They effectively elicit both humoral and cell-mediated immune responses, and also stimulate the metabolic pathways of cytokines and chemokines. Therefore, living, attenuated viruses have certain advantages over vaccine compositions based on both inactivated and subunit immunogens, which mainly only stimulate the humoral component of the immune system.

Over the past decade, recombinant DNA technology has revolutionized the field of genetic engineering of the genomes of both DNA and RNA viruses. In particular, it has now become possible to introduce foreign genes into the virus genome in such a way that, after replication of the new vector virus, a foreign protein is expressed in the animal host, which can cause biological effects in the animal host. Essentially, recombinant vector viruses were used not only for the control and prevention of microbial infections, but also for the development of targeted therapy for diseases of non-microbial etiology, such as malignant neoplasms, as well as in gene therapy.

The creation of non-segmented, minus-filament RNA viruses (viruses of the Mononegavirales order) completely from cDNA clones using a method known as reverse genetics, first published in 1994 (Schnell et al., EMBO J 13 , 4195-4203, 1994), made it possible to use as vectors also viruses of the order of Mononegavirales (MV). Since then, studies have been published on the use of many viruses of order MV as viral vectors for the expression of foreign antigens derived from a pathogen in order to develop vaccines against this pathogen.

The order of Mononegavirales is divided into 4 main families: Paramyxoviridae, Rhabdoviridae, Filoviridae and Bornaviridae. Viruses belonging to these families have genomes that are single-stranded minus (-) sense RNA molecule, i.e. the polarity of the genome RNA is opposite to the polarity of messenger RNA (mRNA), which is designated as plus (+) sense. The classification of the main human and animal viruses of order MV is presented in the table below:

Table 1
Classification of viruses within the Mononegavirales order Family Kind View Rhabdoviridae Lyssavirus (rabies virus group) Rabies Virus (RV) Vesiculovirus (vesicular) Vesicular Stomatitis Virus (VSV) Novirhabdovirus Hematopoietic Tissue Infectious Virus (IHNV) Paramyxoviridae Respirovirus (respiratory) Sendai Virus (SeV) Human parainfluenza virus type 1 and type 3 (hPIV 1/3) Bovine Influenza Virus Type 3 (bPIV 3) Morbillivirus (measles virus) Measles virus (MV) Cattle plague virus Dog Plague Virus (CDV) Rubulavirus (rubulaviruses) Monkey virus type 5 (SV-5) Human parainfluenza virus type 2 (hPIV 2) Avulavirus
(bird virus) Newcastle disease virus (NDV) Pneumovirus
(pneumoviruses) Respiratory syncytial virus (hRSV) Bovine syncytial virus (bRSV) Filoviridae Ebola-like viruses Ebola virus Marburg virus

At present, the organization of the genome and the features of the cell cycle of viruses belonging to the MV order are well studied. The results of these studies have been published by many authors (Neumann et al., J. Gen. Virology 83 , 2635-2662, 2002; Whelan et al., Curr. Top. Microbiol. Immunol. 203 , 63-119, 2004; Conzelmann, K., Curr. Top. Microbiol. Immunol. 203 , 1-41, 2004). Despite the fact that viruses belonging to the Mononegavirales order may have different host cells and different morphological and biological properties, they have many common features, for example, such as the organization of the genome, as well as elements essential for their typical replication method and gene expression, which indicates the commonality of their origin. They are enveloped viruses that replicate in the cytoplasm of a cell and produce immature mRNAs.

The virus, belonging to the order Mononegavirales, consists of two main units: the ribonucleoprotein complex (RNP) and the membrane. For representatives of the viruses belonging to all the genera and families mentioned above, the sequences of the genomes were completely determined. The sizes of these genomes ranged from 9000 nucleotides to 19000 nucleotides, and they contained from 5 to 10 genes. The structure and organization of the genomes of viruses related to MV are very similar and are regulated by their special method of gene expression. All MV genomes contain three core genes encoding: nucleoprotein (N or NP), phosphoprotein (P), and RNA-dependent RNA polymerase (L). The viral envelope consists of a matrix protein (M) and one or more transmembrane glycoproteins (e.g., G, HN and F proteins), which play a role in the assembly / budding of viruses, as well as in the process of attachment and / or penetration of the virus into the cell. The composition of proteins, depending on the type of virus, changes due to auxiliary proteins, which either perform certain specific regulatory functions in transcription and viral replication, or are included in the reactions of the virus host cell (for example, C, V and NS proteins). The order of the MV virus genes is highly conserved: the N and P core genes are located in or near the 3'-terminal region, and the large (L) gene is located in the 5'-terminal region. Surface glycoprotein M genes, like other auxiliary genes, are located between the N, P and L genes.

In the RNP complex, genomic or antigenomic RNA is closely linked to protein N and is associated with an RNA-dependent RNA polymerase, which consists of proteins L and P. After infection of the cell, this RNP complex, and not just the RNA genome, serves as a matrix in two different RNA synthesis processes: transcription of subgenomic mRNAs and replication of full-length genomic RNA.

All tandemly organized genes are separated by structures called “gene compounds”. A gene compound includes a conserved “end of gene” (GE) sequence, a non-transcribed “intergenic region” (IGR) and a conserved “gene start” sequence (GS). These sequences are necessary and sufficient for gene transcription. During transcription, each gene is sequentially transcribed into mRNA using a viral RNA-dependent RNA polymerase, which starts the transcription process at the 3'end of genomic RNA in the first GS sequence. For each gene compound, transcription is interrupted as a result of RNA polymerase detachment in the GS sequence. Further transcription initiation occurs in the subsequent GS sequence, albeit with reduced efficiency. This results in a discontinuous process, which is also called a "stop - start" occurs trankriptsii intensity decrease in each gene of the compound in which the 3'-proximal genes in the genome of MV viruses transcribed more intensively than the following genes located in the final part. The modulated form of transcription of the MV virus genes, in which each gene is part of a single cistron or transcription unit, makes these viruses particularly suitable for the insertion or expression of foreign genes. Each transcriptional unit in the genome of the MV virus includes the following elements: 3'-GS-open reading frame (ORF) -GS-5 '.

At the 3'- and 5'-genomic ends, all MV virus genomes have a short non-transcribed region called the “leader” (about 40-50 nt) and the “trailer” (about 20-600 nt) , respectively. Leader and trailer sequences are important sequences that control genomic RNA replication, encapsulation and packaging of viruses .

Thanks to reverse genetics technology and the release of the infectious MV virus, it became possible to manipulate the RNA genome of this virus using its cDNA copies.

The RNP complex is the minimal replication initiation complex required for viral RNA synthesis. Infectious MV virus can be released by intracellular co-expression of (anti) genomic RNA and corresponding proteins from plasmids driven by (T7) RNA polymerase. Since the first publication of Schnell et al. in 1994, (Schnell et al., 1994 (see above)), many types of MV viruses were detected using the initial method (or a slight modification).

Newcastle disease and bird flu are serious bird diseases that can cause significant economic losses in poultry production worldwide. The Newcastle disease virus is an unsegmented virus that contains negative-stranded RNA of the MV order. The genome, which is 15 thousand bp in length, contains six genes that encode nucleoprotein (NP), phosphoprotein (PN) and V protein (P / V) matrix (M) protein, fusion protein (F), hemagglutinin protein β-neuraminidase (HN) and an RNA-dependent RNA polymerase or a large (L) protein. NDV genes are organized sequentially in the following order 3'-NP-P-M-F-HN-L-5 ', they are separated by intergenic regions of different lengths. Each gene is preceded by a gene start sequence (GS), followed by a non-coding region, an open reading frame encoding NDV proteins, a second non-coding region, and a gene end sequence. The length of the NDV genome is composed of six components, and this must be taken into account when introducing foreign genes.

Avian influenza (AI) is a disease in poultry characterized by both mild respiratory manifestations and severe illness with a high mortality rate. The causative agent of the disease is 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 antigenic properties of surface glycoproteins — hemagglutinin (HA) and neuraminidase (N), AI viruses were subdivided into subtypes. Currently, 16 subtypes of hemagglutinin (H1-H16) and nine subtypes of neuraminidase (N1-N9) are known. Antibodies to H and N are an important factor in the humoral response; they suppress infection or prevent disease.

Avian influenza and Newcastle disease viruses are divided into two distinct pathotypes according to their virulence. Symptoms caused by low pathogenic AIV (LPAIV) or lentogenic NDV are considered less significant. In contrast to this subtype, highly pathogenic avian influenza (HPAI) and Newcastle disease caused by highly virulent viruses (NDV: mesogenic and velogenic strains) are registered diseases.

To protect domestic chickens from highly virulent NDV strains, routine vaccination against NDV is carried out using ribogenic NDV strains, but vaccination against HPAI is not carried out in most countries, since HPAI is controlled by irradiation. However, vaccination can also be used to minimize losses and reduce the spread of the disease. Vaccine-induced immunity is species-specific. This means that the H5 subtype vaccine can protect against H5 AIV, but not from other H subtypes. Usually, influenza virus replication is localized in the respiratory tract , since the hemagglutenin of the LPAI viruses can only be cleaved using Clara tryptase , a serine protease localized in the respiratory tract. So far, all HPAI viruses have been subtypes of H5 and H7. These HPAI viruses contain multiple sequences of basic amino acids at the H cleavage site, so it can be cleaved by the furin and subtilisin-like enzymes found in different tissues into HA1 and HA2 subunits. Such viruses are able, therefore, to multiply in other organs.

Vaccines against the H5 and H7 subtypes can protect chickens and turkeys from clinical manifestations and death after HPAI infection. It has been experimentally proven that, in addition to the usual inactivated, oily, whole-virion AIV, vector virus, subunit protein and DNA vaccines are also effective means of immunization against AI. The creation of recombinant viruses with the aim of using them as vaccine vectors is an important consequence of the application of reverse genetics methods to various viruses. Different recombinant negative-strand RNA viruses expressing foreign proteins were constructed. In addition, hemagglutinin AIV has been introduced into various vector viruses, such as the infectious laryngotracheitis virus (ILTV) (Luschow et al., Vaccine 19 , 4249-59, 2001), the cattle plague virus (Rinderpest virus) (Walsh et al. , J. Virol. 74, 10165-75, 2000) and vesicular stomatitis virus (VSV) (Roberts et al., J. Virol. 247, 4704-11, 1998). NDV is also used for the expression of hemagglutinin AIV. The influenza A / WSN / 33 hemagglutinin gene was inserted between the P and M genes of the NDV strain of Hitchner B1. This recombinant protected mice from lethal infection, although the mice showed significant weight loss, they fully recovered within 10 days (Nakaya et al., J. Virol. 75 , 11868-73, 2001). Recombinant NDVs obtained later with the same insertions for foreign genes expressed H7 LPAI, however, only 40% of vaccinated chickens were protected against velogenous NDV and HPAI (Swaye et al., Avan Dis. 47 , 1047-50, 2003).

Influenza HA proteins are synthesized as precursor proteins (HA ₀ ). The HA ₀ precursor polypeptide is cleaved post-translationally at the conserved arginine residue (Arg) into two subunits: Ha1 and HA2. This cleavage contributes to the infectivity of the virus. (Apparently, the more efficient the HA precursor is cleaving, the more virulent the virus is). Sections of the compound HA ₁ -HA _{2 of} different influenza viruses were analyzed. It was found that in the genome of pathogenic strains there is a site that contains basic amino acids and is directly adjacent to the cleavage site (Senne et al., Avian Diseases, 40: 425-237, 1996; Steinhauser, Virology, 258, 1-20, 1999; Kawaoka and Webster, Proc. Natl. Acad. Sci. USA, 85.1988).

Highly pathogenic viruses are especially important in terms of the protection that an influenza vaccine should provide.

However, when these types of proteins, that is, proteins such as HA-proteins of pathogenic H5 influenza containing the main chain of amino acids in their sequence, were inserted into the Mononegavirales vector, certain problems arose.

First of all, the sequence of genes seems to be unstable in a certain respect. After several passages of the chicken embryo cell culture of the NDV vector into which the H5 gene (NDVH5) was inserted, spontaneous mutations occurred within the portion of the gene sequence that encodes the amino acid backbone.

In addition, the coding sequence for the chain of basic amino acids, apparently, contains a sequence that is recognized by the Mononegavirales virus as a gene end sequence (GE). A review of the GE sequences for Mononegavirales viruses is presented in Whelan (Curr. Top. Microbiol. Immunol., 283, 61-119, 2004). The GE sequences for Mononegavirales viruses are characterized by the usual conserved sequence: nUUUU.

It was found that portions of the coding sequence for a chain of basic amino acids, such as the one adjacent to the HA protein cleavage site of pathogenic avian influenza virus type H5, are similar to the GE sequence recognized by the Mononegavirales vector polymerases as the GE sequence. This can lead to a decrease in protein expression levels of full-length protein, which may affect the effectiveness of a vaccine based on such a vector.

The aim of the present invention was to provide a recombinant vector of the MV virus, which would exhibit a high level of expression of a protein encoded by a foreign gene inserted into the vector virus gene and / or which would show higher immunogenicity compared to existing vector MV viruses.

The inventors have found that this goal can be realized with the recombinant Mononegavirales virus of the invention.

The present invention provides a method for producing a recombinant Mononegavirales virus vector containing an additional transcriptional unit comprising a foreign gene operably linked to the start sequence of the Mononegavirales virus initial gene (GS) and the Mononegavirales virus end gene (GE) sequence, characterized in that the sequence of a foreign gene encodes a protein. This protein contains an extension of at least three basic amino acids, consisting of arginine (Arg) and / or lysine (Lys) residues and containing at least one lysine. The sequence of the nucleotides of the foreign gene is selected in such a way that it does not contain a sequence that can be recognized by the viral polymerase of the Mononegavirales virus as a gene end sequence (GE).

The sequence that can be recognized by the Mononegavirales virus polymerase as the end-of-gene sequence is the sequence that contains the minimal conserved sequence found in most Mononegavirales GE sequences denoted by a / uCUUUU (for negative polarity, which is the polarity of the Mononegavirales virus genome. For positive polarity (cDNA level) this motive is t / aGAAAA).

For the specific Mononegavirales vector, the known specific GE sequence was used (Whelan et al., (See above)). For example, for NDV GE-sequence is indicated in Whelan aaucUUUUUUu (negative polarity, which polarity for Mononegavirales virus genome). In the case of positive polarity (cDNA level) in the sequence, this extension would consist of adenine residues: gAAAAAA.

In the vector, in accordance with the invention, such a GE sequence should not be inside the coding sequence of a foreign protein.

There are several ways to achieve this goal that are open to a qualified person. The foreign sequence can be selected so that it by nature does not contain a sequence that can be recognized by the Mononegavirales virus polymerase as the gene end sequence (GE).

For example, when a sequence of a foreign gene encodes a viral protein, the foreign gene can be obtained from a strain of the virus, which, by nature, carries the gene encoding this protein, and its coding sequence does not include a potential GE sequence (whereas the same gene in another strain of this virus includes).

However, since the chances of obtaining such a variant of the gene sequence from the wild-type virus are very small, a more preferred strategy would be to induce mutations in the sequence of the foreign wild-type gene using directed mutagenesis to modify this sequence to such an extent that the potential GE sequence is no longer present .

It is also part of the invention to provide a resulting recombinant Mononegavirales virus vector in which a foreign gene encodes a protein containing an extension of at least three essential amino acids, consisting of arginine (Arg) and / or lysine (Lys) residues and containing at least at least one lysine; a foreign gene which does not contain a sequence recognized by the Mononegavirales virus polymerase virus as a gene end sequence (GE).

The part of the foreign gene that needs to be modified, especially if the foreign protein is the HA protein of the pathogenic avian influenza H5 virus, can be part of the sequence of the wild-type foreign gene that encodes a chain of at least three basic amino acids.

An extension section of basic amino acids includes at least three basic amino acids selected from the group consisting of lysine (one letter of code: K) and arginine (one letter of code: R), in which at least one amino acid the remainder is lysine. This would include a KKK sequence, a combination of 2K with 1R (KKR, KRK, RKK) and a combination of 1K and 2R (KRR, RKR and RRK), as well as longer extension sites from the basic amino acids, including the specific 3 amino acid sequences mentioned here. As can be seen from Steinhauser et al. (see above), elongation regions from basic amino acids directly adjacent to cleavage sites in HA proteins of pathogenic influenza viruses may be longer and may include sequences such as RRKKR or RRRKK.

The codons encoding lysine are aaa and aag, while the codons encoding arginine contain aga and agg. It was found that (especially in the coding sequence for the HA protein of the pathogenic avian influenza H5 virus), the coding sequence that encodes the backbone of amino acids directly adjacent to the protein cleavage site may contain the t / aGAAAA GE motif.

To modify the GE sequence inside the wild-type foreign gene, at least one mutation must be introduced in which the nucleotide “A” of the adenine containing region is located inside the GE sequence, which has the usual motif (t / aGAAAA), originally present in the sequence of a foreign wild-type gene, is replaced by another nucleotide. In fact, if a mutation is inserted in the “aaaa” region, which is part of a sequence recognized as a GE sequence, at least one of the adenine nucleotides must be replaced by another nucleotide (for example, guanine (“g”)).

When changing the sequence using site-directed mutagenesis, these changes are mostly silent (this means that mutations (mutations) do not lead to mutations at the amino acid level). However, mutations at the nucleotide acid level are also acceptable, which lead to conservative mutations at the amino acid level (meaning that one basic amino acid is replaced by another basic amino acid, for example, L becomes R or R becomes L). For example, if in that part of the wild-type gene sequence encoding the main amino acid chain, where at least one lysine residue is encoded using the “aaa” codon, then this codon in the modified foreign gene inserted in the vector of the present invention can be mutated in “aag”. Or, where in the wild-type sequence a particular arginine residue, forming part of the main chain of amino acids, is encoded with “aga”, this codon in this gene can be mutated into “agg”, since, according to the invention, it is made part of the vector.

At least one codon “aaa” (encoding lysine) in the wild-type sequence can be mutated to create a modified foreign gene sequence that is incorporated into the vector of the present invention. Such a mutation may include the substitution of the codon “aaa” with the codon “aga”. More preferred are vectors in which, for example, an extension region of the basic amino acids consists of at least two lysine amino acids in a row, and in which at least one of these lysines is encoded by the “aag” codon, although they may be introduced two or more mutations. For example, if the coding sequence contains two codons “aaa” in a row, both can be replaced by codons “aag”.

In order for the resulting vector to be more stable, it is desirable to introduce at least two mutations into the foreign gene. In those regions where the foreign gene contains two “aaa” codons in a row, both of them are more likely to undergo mutations, and can be replaced by “aag” codons.

Good results were obtained when working with vectors in which a part of a foreign gene encoding an extension region of basic amino acids contained one mutation in each of three or four consecutive codons encoding a basic amino acid. In the case where the main chain includes only three amino acids, three mutations can be caused , but when four or more basic amino acids are present, the number of mutations caused can be greater.

For example, where the wild-type sequence contains the RRKK amino acid sequence encoded by the aga aga aaa aaa nucleotide sequence (a sequence of positive polarity in which the motif of the potential GE sequence is underlined), this coding sequence can be mutated into ag g ag g aa g aa g which still encodes RRKK.

Preferably, the Mononegavirales virus vector is a Newcastle disease virus (NDV) vector and a foreign gene of the avian influenza pathogen H5 HA protein.

Good results were obtained when working with the NDV vector into which the modified HA gene of the pathogenic avian influenza virus H5 (NDVH5m) was incorporated.

The HA gene was incorporated in the intergenic region between the NDV fusion genes (F) and hemagglutinin-neuramidase (HN) NDV vector. The HA gene was a modified H5 gene in which the wild-type sequence “aga aga aaa aaa” was changed to the sequence “ag g ag g aa g aag” in accordance with the method of the present invention.

This vector produced significantly more full-length HA transcripts, expressed a significantly higher level of HA, and also incorporated more proteins in its envelope.

In addition, NDVH5m stably expressed the modified HA gene for over 10 passages in chicken embryo cell culture. Immunization of chickens with NDVH5m induced NDV- and AIVH5-specific antibodies and, after infection with a lethal dose of velogenic NDV or highly pathogenic AIV, protected chickens from the corresponding clinical disease. Note that the spread of the influenza virus was not observed. Moreover, immunization with NDVH5m allowed serological recognition of animals vaccinated and infected with a field strain of the AIV virus, based on a study of antibodies to the nucleoprotein (NP) AIV. Therefore, recombinant NDVH5m is suitable as a bivalent vaccine against NDV and AIV and can be used as a marker vaccine to control bird flu (AI).

Methods for producing a recombinant vector of the MV virus containing an additional transcriptional unit including a foreign gene are well known in the art.

More specifically, in the method of the invention, the isolated nucleic acid molecule and the MV virus genome were used in their cDNA form (positive polarity). This made it easy to manipulate and integrate the desired nucleic acid molecules into the viral genome.

In general, a foreign gene could be inserted into different parts of the genome between two genes, for example, in intergenic regions (IGR), 3'- and 5'-non-coding regions of a gene, as well as a 3'-proximal promoter (in front of N / NP genes ) or the 5'-distal end (after L-genes) of the genome.

A foreign gene could be successfully inserted before the N / NP gene, between NP-P, P-M, M-G / F, G / F-HN, HN-L and after the L gene.

The easiest way is to use an existing restriction enzyme recognition (RE) recognition sequence at one of these sites by cutting the enzyme and inserting a suitable transcription cassette. Since naturally existing restriction enzyme recognition sequences are not always localized to the desired site, RE recognition sites can be introduced into the genome in a conventional manner using site-directed or PCR mutagenesis.

The structure of the embeddable transcriptional cassette depends on the embedment site. For example, in the case where the transcription cassette is integrated into the IGR, the cassette may include the following elements: 3 'RE recognition site-GS-non-coding region-ORF (foreign gene) non-coding region-GE-RE recognition site 5' .

Alternatively, in the case where the transcription cassette is inserted into the 5'-non-coding region of the natural MV virus gene, the cassette may consist of: 3 'RE recognition site-GE-IGR-GS-non-coding region-ORF (foreign gene) - non-coding region - RE recognition site 5 '.

Similarly, when the transcription cassette is inserted into the 3'-non-coding region of the natural MV virus gene, the cassette may consist of: 3'RE recognition site-non-coding region-ORF (foreign gene) -non-coding region-GE-IGR-GS- RE recognition site 5 '.

Obtaining such transcriptional cassettes and introducing them into the genome of the MV virus are routine molecular biological procedures, such as those described in the references and Examples. In particular, methods such as site-directed mutagenesis and PCR mutagenesis can be used for this purpose (Peeters et al., 1999, see above; Current Protocols in Molecular Biology, eds .; FM Ausubel et al., Wiley NY, 1995 edition, pages 8.5.1.-8.5.9; and Kunkel et al., Methods in Enzymology Vol. 154, 376-382, 1987).

In particular, the recombinant vector of the MV virus according to the present invention can be obtained using the well-known “reverse genetics” method, which allows genetic modification of non-segmented, minus-stranded RNA viruses of order MV (indicated, for example, in Conzelmann, KK reviews , Current Topics Microbiol. Immunol. 203 , 1-41, 2004; and Walpita et al., FEMS Microbiol. Letters 244 , 9-18, 2005).

In this method, the corresponding cell is co-transfected with a vector containing a cDNA molecule including a nucleotide sequence that encodes a full-length genome or, more preferably, an antigen (positive polarity) of the MV virus, and one or more vectors containing cDNA molecules, including nucleotide sequences that encode the necessary supporting proteins, under suitable conditions for transcription and co-expression of the (anti) MV genome and supporting proteins and production of re ombinantnogo vector MV. In this method, the aforementioned nucleic acid molecule encoding the (anti) genome of the full length MV virus contains an additional transcriptional unit, as explained above.

By vector is meant a replicon, such as a plasmid, phage or cosmid, to which another segment of DNA can be attached, so as to replicate the attached segment of DNA and its transcription and / or expression in a cell transfected with this vector.

Preferably, the vector for transcription of the full length genome is a plasmid that includes a cDNA sequence encoding the (anti) MV virus genome flanked by the T7 polymerase promoter at its 5 ′ end and a ribozyme sequence (hepatitis delta) at its 3 ′ end, although T3 or SP6 RNA polymerase promoters may also be used.

For intracellular expression of the corresponding support proteins, plasmids are used predominantly containing cDNA sequences encoding these proteins under the control of the corresponding expression control sequences, for example, the T7 polymerase promoter.

In a particularly preferred embodiment of the method for producing a recombinant vector of the MV virus, according to this invention, expression plasmids are used that encode the N (or NP), P and L proteins of the MV viruses.

The number or proportions of transfected supporting plasmids used in this reverse genetics method vary widely. The proportions for the supporting N: P: L plasmids can vary from 20: 10: 1 to 1: 1: 2, and effective transfection techniques are known for each virus.

An exact copy of genomic RNA is formed in the transfected cell using the combined action of the T7 RNA polymerase promoter and the ribozyme sequence, and then this RNA is packaged and replicated using viral structural proteins supplied by cotransfected expression plasmids.

Preferably, the T7 polymerase is provided by a recombinant vaccinia virus that infects the transfected cell, in particular the vaccinia virus vTF7-3, in addition, other recombinant smallpox vectors such as avianpox virus can be used to express T7 RNA polymerase fpEFLT7pol or other viral vectors.

Separation of the released virus from the vaccinia virus can be carried out by a simple physical method, such as filtration. Sendai virus or NDV can be released by inoculating supernatan of transfected cells into chicken embryos.

For transfection of transcription and expression vectors that constantly express (T7) RNA polymerase and / or one or more necessary support proteins, it is even preferable to use cell lines.

For example, measles virus accumulation can be achieved in the human embryonic kidney cell line, 293-3-46, which expresses both T7 RNA polymerase and supporting measles virus proteins N and P (Radecke et al., EMBO J. 14 , 5773 -5784, 1995). Another very useful cell line that can advantageously be used in the present invention is the BSR cell line expressing T7 RNA polymerase, i.e. the BSR-T7 / 5 cell line (Buchholz et al., J. Virol. 73 , 251-259 , 1999).

In addition, more detailed information regarding reverse genetics technology used in the present invention for the production of MV virus is given in a review by Conzelmann K.K. (see above) and in Example 1.

Since the recombinant vectors of the MV virus can stably express foreign genes, the vectors have been investigated for the purpose of prophylactic and therapeutic applications.

In accordance with the present invention, in a recombinant vector of the MV virus, the foreign gene may vary depending on the specific form of the vector of the MV virus, as well as the use of the vector virus.

A foreign gene can encode an antigen of a (other) microbiological pathogen (for example, a virus, a parasite bacteria), it is especially important that a foreign gene encodes a pathogen antigen that is capable of eliciting a protective immune response.

Examples of heterologous gene sequences that can be inserted into the viral vectors of this invention are influenza glycoprotein genes, especially H5 and H7 avian influenza hemagglutinin genes, infectious bursitis virus genes (IBDV), especially VP2, infectious bronchitis virus (IBV) genes, feline leukemia virus, canine distemper virus, equine infectious anemia virus, rabies virus, Ehrlichia, especially Ehrlichia canis, respiratory syncytial viruses, parainfluenza viruses, human metapneumoviruses and virus and measles.

Alternatively, a foreign gene may encode a polypeptide immunomodulator that is capable of enhancing or modulating the immune response to a viral infection, for example, by coexpressing a cytokine such as interleukin (e.g. IL-2, IL-12, IFN-y, TNF-α or GM-CSF).

The MV order includes viruses that can replicate in the human and / or animal body (for example, rabies virus or Newcastle disease virus can replicate in both organisms). Therefore, a foreign gene can be selected from a large number of microbiological pathogens of humans and animals.

Despite the fact that in the present invention all MV viruses can be used as vector viruses, in the present invention, the Rhabdoviridae family virus is preferred as the recombinant vector of the MV virus, more preferably the genus Lyssavirus or Novirhabdovirus, and even more preferably the type of rabies virus or IHNV, respectively.

Also, as a recombinant MV virus, a virus of the Paramyxoviridae family is proposed, more preferably the genera are: Respovirus, especially hPIV3 or bPIV3 species; Morbillivirus, especially a type of CDV; Pneumovirus, especially a species of RSV; and Avulavirus, especially the NDV species.

In a most preferred embodiment of the invention, the Newcastle disease virus (NDV) is used as the recombinant vector of the MV virus. It is known that NDV is able to replicate in organisms of both humans and animals, in particular birds, mainly chickens. Therefore, according to the present invention, the recombinant NDV vector may contain a foreign gene encoding a pathogen antigen, in particular a respiratory pathogen, or an immunomodulator capable of eliciting an appropriate immune response in humans or in any of these animals.

Reverse genetics methods for the genetic manipulation of NDV were presented by Peeters et al. (J. Virology 73 , 5001-5009, 1999), Römer-Oberdörfer et al. (J. Gen. Virol. 80 , 2987-2995, 1999) and in a review by Conzelmann, KK (see above). In addition, it is also known that NDV can be used as a vector for the expression of foreign genes, for example, to elicit an immune response in animals infected with the NDV vector (Nakaya et al., 2001, see above; Swayne et al., Avian Dis. 47 1047-50, 2003).

A foreign gene can be introduced into the NDV genome at various positions, as noted above in general terms for MV viruses. In particular, in the recombinant NDV vector according to this invention, a foreign gene (as part of the corresponding transcription unit) can be inserted between the following genes: NP-P, PM, MF, F-HN, HN-L and in the 3'-proximal and 5'-distal loci (Zhao et al., 2003, see above; Nakaya et al., 2001, see above), preferably in the 3'-proximal, PM, MF and F-HN regions, region F- HN is most preferred.

In a particular embodiment of the invention, in a recombinant NDV vector, an additional transcriptional unit is located between the F-HN genes.

The recombinant NDV vector according to the present invention can be used to induce an immune response in birds, in particular chickens, against other pathogens. Therefore, the recombinant NDV vector preferably contains a foreign gene that encodes a protective antigen of avian pathogen, in particular: influenza virus, Marek's disease virus (MDV), infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), infectious bursitis virus (IBDV) , chicken anemia virus (CAV), reovirus, bird retrovirus, poultry adenovirus, turkey rhinotracheitis virus (TRTV), E. coli, species Eimeria, Cryptosporidia, mycoplasmas such as M. gallinarum, M. synoviae and M. meleagridis, species Salmonella, Campylobacter, Ornithobacterium (ORT) or Pasteurella sp.

More preferred is a recombinant NDV vector containing a foreign gene that encodes the antigen AIV, MDV, ILTV, IBV, TRTV, E. coli, ORT or Mycoplasma.

In particular, the recombinant mutant of the NDV vector contains the hemagglutinin (HA) gene of influenza virus, preferably avian influenza virus (AlV), more preferably highly pathogenic AIV, in particular H5 or H7 AIV.

In principle, the HA gene of all strains of bird flu can be used in this invention. The nucleotide sequences of many HA genes have already been decoded and can be obtained from the nucleotide sequence data banks, such as GenBank or the NCBI database.

As noted above, the hemagglutenin (HA) gene, a recently isolated highly pathogenic H5N2 subtype AIV A / chicken / ltaly / 8/98, can be successfully used as a foreign gene in the present invention. This gene is back transcribed, cloned in the pcDNA3 eukaryotic expression vector (Invitrogen) and sequenced (Lüschow et al., Vaccine, vol. 19, p. 4249-4259, 2001, and GenBank, access No. AJ305306). From the obtained expression plasmid pCD-HA5, the HA gene can be obtained by amplification using specific primers that generate artificial RE recognition sites that allow the integration of the HA gene into the NDV genomic sequences.

As noted above, in the future, the HA gene of the highly pathogenic H7N1 subtype AIV A / chicken / ltaly / 445/99 can be used in this invention as a foreign gene. The HA gene is back transcribed and amplified by PCR. The product, 1711 np in length, was cloned into a Smal-digested pUC18 vector (Amersham) and sequenced (Veits et al., J. Gen. Virol. 84, 3343-3352, 2003; and GenBank, access No. AJ580353).

In a particularly useful recombinant vector of the MV virus according to this invention, the MV vector virus is attenuated, that is, the vector virus is not pathogenic for the target animal or shows a significant decrease in virulence compared to the wild-type virus. Many MV viruses used here as viral vectors have long been registered as safe live attenuated vaccines, these include measles and NDV viruses, while other viruses such as SeV and VSV are considered non-pathogenic to humans. In addition, there are conventional methods for the production and screening of attenuated viruses that detect limited replication or infection. Such methods include serial cultivation of the virus in a heterologous substrate and chemical mutagenesis.

The recombinant NDV vector according to this invention can be obtained from any conventional ND vaccine strain. Examples of such suitable NDV strains belonging to the commercially available ND vaccines are: Clone-30 ^{®, La Sota, Hitchner B1,} NDW, C2 and AV4, Clone-30 ^® - strain is preferred.

The inventors also found that the recombinant vector of the MV virus according to the invention is able to induce a protective immune response in animals.

Therefore, in yet another embodiment of this invention, a microbiological pathogen vaccine is provided that contains a recombinant vector of the MV virus, as explained above, in live or inactivated form, and a pharmaceutically acceptable carrier or diluent.

The vaccine according to the invention can be manufactured by conventional methods, such as those commonly used to make commercially available live or inactivated MV virus vaccines.

Briefly, the sensitive substrate is inoculated with the recombinant vector of the MV virus and the virus is propagated until it is replicated to the desired titer, after which the virus-containing material is collected. Then the collected material is prepared as a pharmaceutical preparation with immunizing properties.

Any substrate capable of supporting replication of the recombinant vector of the MV virus can be used in this invention. Depending on the MV virus, host cells of both prokaryotic and eukaryotic origin can be used as a substrate. Appropriate host cells may be vertebrate cells, such as primacy cells. Examples of such cells are: human cell lines HEK, WI-38, MRC-5 or H-239, monkey cell line Vero, rodent cell line CHO, BHK, dog cell line MDCK, or bird cells CEF or CEK.

The most suitable substrate on which, according to this invention, the recombinant NDV vector can multiply, are chicken SPF embryos. Chicken embryos can be inoculated, for example, with 0.2 ml NDV, while the allantoic fluid contains at least 10 ²⁰ EID ₅₀ per egg. More preferably, 9-11 day old chicken embryos are inoculated with approximately 10 ⁵⁰ EID ₅₀ and then incubated at 37 ⁰ C for 2-4 days. After 2-4 days, the ND virus product can be collected, preferably by collecting allantoic fluid. Then the liquid can be centrifuged for 10 minutes. at 2500 g followed by filtration of the supernatan through a filter (100 μm).

The vaccine according to this invention contains a recombinant vector of the virus MV together with a pharmacologically acceptable carrier or diluent commonly used for such compounds.

A vaccine containing a live virus can be manufactured and marketed in suspension or in lyophilized form. Carriers include stabilizers, preservatives and buffers. Diluents include water, aqueous buffer, and polyols.

In another aspect of the present invention, the use of a vaccine containing a recombinant vector of the virus MV in inactivated form is also provided. The main advantages of an inactivated vaccine are its safety and the fact that it can induce high levels of protective antibodies that last for a long time.

To inactivate viruses collected after the breeding step, inhibition of viral reproduction is necessary. This can be achieved using well-known physical and chemical methods.

If necessary, the vaccine according to the invention may contain adjuvant. Examples of suitable compounds and mixtures with adjuvant activity for this purpose include: hydroxide, phosphate or alumina, oil-in-water emulsions or water-in-oil emulsions, for example, based on mineral oil such as Bayol F® or Marcol 52®, or vegetable oil, such as vitamin E acetate and saponins.

According to the invention, the vaccine can be administered in the form of any known effective form depending on the type of virus vector MV. Suitable methods for administering the vaccine may be parenteral, intranasal, oral vaccination, as well as spray vaccination.

The vaccine of the NDV vector according to the invention is advantageously administered in the form of a low-cost mass route of administration of the vaccine commonly used to vaccinate NDV. NDV vaccines can be administered through drinking water, as well as applied as a spray.

The vaccine according to this invention contains as an active ingredient an effective dose of a recombinant vector of the MV virus, that is, a certain amount of immunizing MV virus material, which will cause immunity in vaccinated birds against infection by a virulent microbiological organism. In this case, immunity is defined as the induction of a significantly higher level of protection against mortality and clinical symptoms in the population of vaccinated people and animals compared with the unvaccinated group. In particular, according to the invention, the vaccine protects a large percentage of vaccinated people and animals from the manifestation of clinical symptoms of the disease and mortality.

Typically, a live vaccine is administered at a dose of 10 ^2.0 -10 ^8.0 for tissue culture / dose for infection of the embryo (TC / ElD ₅₀ ), preferably at a dose in the range of 10 ^4.0 -10 ^7.0 TC / ElD ₅₀ . Inactivated vaccines may contain the antigenic equivalent of 10 ⁴⁰ -10 ⁹⁰ .

The present invention also includes a combination of vaccines containing, in addition to the recombinant vector of the MV virus, according to this invention, a vaccine strain capable of inducing protection against an additional pathogen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. one.

The structure of recombinant NDV expressing AIV H5. Transcription control signals are marked with: a gray triangle for transcription start and a gray rectangle for transcription stop sequences. NH ORF NDV, was replaced by H5 ORF AIV, and then the HN gene was inserted as described in the materials and methods (stages A-C). Recombinant NDVH5 (stage D) contains an authentic HA sequence, whereas recombinant NDVH5m carries HA in which the sequence of the cleavage site was changed using a silent mutation (stage E-F).

FIG. 2.

Northern blot analysis of transcripts produced by NDV recombinants. Total RNA of CEK cells infected with NDV Clone 30 (NDV), NDVH5, NDVH5m or AIV A / chicken / Italy / 8/98 (H5N2), and uninfected cells (NI) were isolated after 8 hours of infection. After separation of RNA in a denaturing agarose gel, it was transferred onto nylon membranes and hybridized with ³² P-labeled gene-specific antisense cDNAs NDV-F (left), AIV-H5 (middle) and NDV-HN (right). Six mg of the corresponding RNA was used for Northern blot hybridization, with the exception of the AIV lane in the middle of the blot where only 2 mg was taken to avoid excessive AIV signal. The sizes of the RNA marker were indicated in thousand bp left.

FIG. 3.

Western blot analysis of proteins produced in infected cells or in recombinants expressing HA AIV incorporated into NDV. Lysates of infected cells (A) or purified virions (B) of NDV Clone 30, NDVH5, NDVH5m or AIV (H5N2) were also incubated with antiserum specific for the H5 subtype AIV (α-AIV, top panel) or with NDV-specific antiserum ( α-NDV; bottom panel). Binding was detected by chemoluminescence after incubation with secondary antibodies conjugated to peroxidase.

The localization of marker proteins is indicated on the left, and the unsplit (HA ₀ ) and cleaved forms (HA1, HA2) of AIV hemagglutinin are indicated on the right.

Additional viral proteins corresponding to NP / NA and M were also detected in infected AIV cells or virions.

FIG. four.

HA-specific antibodies determined using the HI test (A, gray columns) or IF test (A, white columns). Mortality rates and clinical indices in chickens immunized once (B, C) or twice (D) NDVH5m (H5m), and control animals (C) after infection with velogenic NDV Herts (B) or pathogenic H5N2 AIV (C, D) . For 10 days, animals were monitored daily for clinical signs; they were classified as healthy (0), sick (1), seriously ill (2), or dead (3). The clinical index was calculated, equal to the average value of the number of all chickens per group for this period.

FIG. 5.

Serological tests using the NP-ELISA method. Chicken serum was tested by indirect NP-ELISA at a dilution of 1: 500. An increased H / N ratio is graphically shown for chick serum immunized with rNDV / AIVH5-BMUT taken 21 days after immunization (p.i.) and 21 days after subsequent AIV infection (p.c.). A value of 2.0 is indicated on the graph by a line of dots.

EXAMPLES

Example 1 The structure of vectors containing the unmodified (wild-type) H5 gene and the modified H5 gene, and protein expression.

Using reverse genetics technology, a Newcastle disease virus (NDV) was engineered to express the avian influenza virus (AIV) hemagglutinin (HA) subtype H5. A cloned copy of the full length of the genome of the lentogenic strain NDV Clon 30 was used to embed an open reading frame encoding the HA highly pathogenic (HP) isolate AIV A / chicken / Italy / 8/98 (H5N2, Genbank, accession number AJ305306) in the intergenic region between the NDV genes, encoding a fusion protein (F) and hemagglutinin-neurominidase (HN).

After three passages of chicken embryo cell culture, the titer of the NDVH5 recombinant was the same as the titer of the parental Clone 30 virus (10 ⁹ TCID ₅₀ / ml), and the presence of the integrated H5 gene was confirmed by RT-PCR. To verify the correct transcription of a foreign gene, Northern blot analysis was performed (Fig. 2). Although the H5 ORF in the recombinant NDV and AIV (H5N2) are identical, the H5 ORF cloned in NDV flanks approximately 270 nucleotides derived from non-coding sequences of the HN gene. Therefore, the size difference between NDVH5 and AIV mRNA is in good agreement with the expected size of approximately 2 thousand bp and 1.7 thousand bp mRNA, respectively (Fig. 2, middle). However, a second transcript with a length of about 1 thousand bp appears in NDV (Fig. 2, middle, lane 3).

As Northern blot analysis showed, recombinant NDVH5m produced only the H5 transcript of the expected size of 2 thousand bp (Fig. 2, middle, lane 4), confirming that the short transcript in NDVH5 ends near the HA cleavage site and most likely represents only the HA ₁ region.

As a result of the modification, NDVH5m produced 1.8 times more full length HA (HA ₀ ) than NDVH5. Full-length HA transcripts in infected AIV cells were 3.4 and 6 times greater than in cells infected with NDVH5m and NDVH5, respectively. In contrast to the sequence of the H5 cleavage site NDVH5, which changed after 5 passages, the NDVH5 sequence in this region remained stable during the analyzed 10 passages (Table 1). Hybridization with F and HN NDV probes confirmed the correct transcription of the flanking NDV genes, since no differences in the sizes of the F and HN mRNAs of the parent NDV Clone 30 were detected (Fig. 2). In addition, there was only a slight decrease in the transcription rate of the HN gene as a result of embedding HN ORF (Fig. 2, right).

To confirm the specific expression of H5 AIV by NDV recombinants, the infected CEF cells underwent an indirect IF test. Incubation with NDV-specific antiserum revealed clear fluorescence in cells infected with Clone 30, NDVH5 and NDVH5m NDV, which was not observed in cells infected with AIV-H5N2. On the other hand, an antiserum specific for the H5 subtype of AIV showed the presence of specific H5 expression in cells infected with AIV, NDVH5 and NDVH5m, whereas in cells infected with parental NDV, such expression was not observed. Despite the fact that the intensity of H5 expression in infected AIV cells is higher than in the case of infection with both recombinants, it was found that the level of H5 expression in cells infected with NDV recombinants is significant.

These results are consistent with data confirming that to obtain comparable hybridization signals on RNA gels, it was necessary to apply only 3 times the amount of NDVH or NDVH5 RNA, this is shown in Figure 2 (middle). This fact indicates that AIV produced approximately 3 times more transcripts and, apparently, the same amount of protein. It is interesting that, apparently, H5 expression in NDVH5m infected cells is more efficient than in NDVH5 infected cells, they could be attributed to silent mutations that cancel the premature end of H5 transcription.

Example 2. AIV H5 protein is incorporated into the shell of NDV particles.

Previous studies have shown that the incorporation of foreign proteins into the envelope of viruses could passively in the absence of specific incorporation signals (Kretzschmar, E. Et al., 1997, J. Of Virology, vol. 71, p. 5982-5989). Since the effectiveness of such passive incorporation mainly depends on the level of protein expression, we determined whether the HA NDV protein expressed by recombinant viruses was incorporated into the recombinant membrane and whether the protein was incorporated (Fig. 3). In cells infected with both recombinants, an antiserum specific for the H5 AIV subtype revealed three proteins with molecular sizes of approximately 70, 50, and 25 kDa, which appear to be the uncleaved HA ₀ protein and the cleaved HA ₁ and HA ₂ proteins (FIG. . 3A). This shows that HA expressed by recombinants is available for proteolytic enzymes. The total HA protein produced in cells infected with the NDVH5 virus was significantly less than in the case of NDVH5m, and the HA2 protein in cells infected with NDVH5m was barely noticeable. As expected, no reactivity was detected in cells infected with Clone 30 NDV, whereas in cells infected with AIV-H5N2, all relevant types of HA protein were detected. Since an increase in AI-specific serum was observed against the entire virus, other types of protein were also detected in cells infected with AIV, presumably corresponding to NP / NA and M (Fig. 3). Interestingly, Western blot analysis of purified virions showed that the HA protein was successfully incorporated into the envelope of both recombinants (Figure 3B). Despite the fact that a larger amount of NDVH5 virus protein was subjected to Western blot analysis, the amount of HA2 protein incorporated into NDVH5 was significantly lower than the amount of HA2 protein in NDVH5 virions. This result indicates that NDVH5 produces and incorporates much less HA2 protein, which is likely due to the suitability of less protein as a result of premature termination at the cleavage site.

Example 3. Recombinant NDV carrying HA AIV protein is safe for chickens.

One of the sensitive methods for measuring the degree of virulence of a given NDV isolate is to determine the pathogenicity of the virus for 1 day old chickens after intracerebral inoculation (CEC (1992) Official Journal of the European Community L 260, 1-20). Viruses of greatest virulence will give indices that approach a maximum value of 2.0, while lentogenic strains will give values close to 0.0. Since hemagglutenin AIV is an important determinant of virulence, intracerebral pathogenicity indices (ICPI) for NDVH5 and NDVH5m were determined to evaluate whether HPAI virus H5 expression changes the virulence of NDV. ICPI values were 0.0 with a maximum value of 2 for both recombinants. It follows that the expression of H5 AIV, in addition to its own proteins, did not significantly affect the virulence of NDV. In comparison, a live virus NDV vaccine is only suitable if the ICPI of the lentogenic vaccine strain is below 0.5.

Example 4. Recombinant NDV expressing HA AIV protein protects chickens from infection with NDV and AIV.

Since there was a higher level of H5 protein expression during NDVH5m infection, only this recombinant was tested in animal experiments. Recombinant NDVH5m was administered to 25 three-week-old chickens at a dose of 10 ⁶ EID ₅₀ for oculonasal administration.

During the observed period, all animals remained healthy without any adverse reactions or clinical signs of the disease. As the HI test showed, H5-specific AIV antibodies began to be detected on day 14 in 28% serum, and this value increased to 92% on day 21 after vaccination (Fig. 4A). However, using the indirect IF test, even earlier signs of immunity against AI were detected in 96% serum on day 7 after immunization (Fig. 4A).

To determine the protective effect of a single vaccination, groups of 5, 10 vaccinated animals and the corresponding number of control animals were subjected to the first round of lethal infections against NDV and AIV, respectively. As expected, 100% of the vaccinated animals were protected against lethal cyclic NDV infection, while all control animals showed typical signs of ND and all died within 4 days (Fig. 4B). Infection with highly pathogenic AIV-H5N2 caused severe disease in non-immunized chickens with a mortality rate of 100%. However, all animals from the group of immunized NDVH5m survived infection with a lethal dose of highly pathogenic AIV. Seven out of ten tsalyal remained completely healthy, and three animals showed very weak respiratory symptoms. However, the resulting clinical index of 0.05 was negligible compared with the clinical index of 2.61 for the control group (Fig. 4C). To determine the effect of booster vaccination in AI-protected chickens, the remaining ten chickens received secondary immunization 42 days after the first vaccination and became infected two weeks after that. All animals were fully protected from clinical disease after receiving a lethal dose of homologous HPAI virus, while all control animals developed severe illness and death within 4 days, and the clinical index was 2.66 (Fig. 4D).

Example 5. NDV-AI vaccine reduces the spread of AIV.

In order for the AI vaccine to be successfully used, it must prevent the spread of the virus, be safe and effective in preventing the disease. To determine the extent of the virus, tracheal and cloacal smears were subjected to a real-time RT-PCR quantitative assay at various time intervals after infection. On day 2 after infection, viral RNA was detected in all non-immunized but infected chickens of both control groups. The values of the threshold cycle (CT) of smears swayed in the control group of the first and second AIV infections varied between 32.2-38.1 and 29.2-35.5, respectively. Since all control animals died within 4 days after infection, no further test was performed in this group. For comparison, no vRNA was detected in most immunized animals (49 out of 60 smears).

Example 6. NP-ELISA detects a circulating AIV.

The greatest concern associated with routine vaccination of birds is the likelihood that the vaccine will allow the virus to circulate undetectably among birds. Since the recombinant NDV vaccine contains only the HA gene, an ELISA method based on the highly immunogenic nucleoprotein gene was used to analyze serum taken from vaccinated animals before infection and at different times after infection. Before infection, antibodies against NP AIV were absent in the serum of all animals, however, in 90% and 100% of chickens at 7 and 21 days after AIV infection, respectively, NP seroconversion was detected (Fig. 5). This shows that the NP-based ELISA test is not only able to differentiate vaccinated animals from infected animals, it is also able to detect any circulating virus among vaccinated birds.

Example 7. Materials and methods for Examples 1-6.

Viruses and cells.

Recombinant NDV based on the Clone 30 vaccine strain has been previously described (Römer-Oberdörfer, A., Mundt, E., Mebatsion, T., Buchholz, UJ & Mettenleiter, TC (1999), J. Gen. Virol. 80 (Pt 11 ), 2987-2995). Influenza virus isolate A / chicken / Italy / 8/98 (H5N2) was kindly provided by I. Capua. The velogenic NDV strain Herts 33/56 and the NDV Clone 30 vaccine (Nobilis ^® ) were obtained from Intervet Int, BV, Boxmeer, The Netherlands. Viruses were propagated in 10-day-old pathogen-free chicken embryos (SPF). BSR-T7 / 5 cells stably expressing phage T7 RNA polymerase (Buchholz, UJ, Finke, S. & Conzelmann, KK (1999) J. Virol. 73, 251-259) were used to obtain infectious NDV from cDNA.

Protein expression and viral replication studies were performed using primary chicken embryo fibroblasts (CEF), primary chicken embryonic kidney cells (CEK), or quail muscle cells (QM9-R).

The structure of recombinant viruses expressing the H5 AIV gene.

The H5 AIV gene was inserted using the plasmid pflNDV-1 expressing the full length Clone 30 antigenic RNA (Römer-Oberdörfer, A., Mundt, E., Mebatsion, T., Buchholz, UJ & Mettenleiter, TC (1999), J. Gen Virol. 80 (Pt 11), 2987-2995). First, the Notl / BsiWl fragment (nt 4953-8852) of the Clone 30 genome was cloned into the pUC18 plasmid (step A, Fig. 1). Then, the newly created Ncol and Aflll sites were embedded (step B) using primers MP1 (5'-gacaacagtcctcaa ccatgg accgcgccg-3 ', SEQ ID NO: 1) and MP2 (5'-ctggctagttgagtcaatt cttaag gagttggaaagatggc-3', : 2). Open reading frame (ORF) H5 AIV, which was amplified from plasmid pCD-H5 (Lüschow, D., Werner, O., Mettenleiter, TC & Fuchs, W. (2001) Vaccine 19, 4249-4259) using specific primers, containing artificial restriction sites Ncol and Aflll (PH5F2: 5'-ccttccatggagaaaatagtgcttc-3 ', SEQ ID NO: 3, and PH5R2: 5'-cctccttaagtataattgactcaattaaatgcaaattctgcactgcaatgatcc-3', SEQ ID NO: 4: after digestion with Ncol and AfIII (step C). In addition, new SgfI and SnaBI sites were introduced into the intergenic region in front of the L gene (step C) using MP3 primers (5'-caaaacagctcatgg tacgta atacgggtaggacatgg-3 ', SEQ ID NO: 5) and MP4 (5'-gaaaaaactaccgg gcgatcgc tgaccaaaggacgatatacggg-3 ', SEQ ID NO: 6). Similar sites flanking the HN gene (step D) were created using MP3 and MP5 primers (5'-gaaaaaactaccg gcgatcgc tgaccaaaggacgatatacggg-3 ', SEQ ID NO: 7) to clone the HN gene after H5 ORF (step E). The H5 cleavage sequence, similar to the NDV transcription termination sequence, was modified by silent mutation using the MPH5F2 primer (5'-ggaatgtccctgaaagaaggaggaagaagagaggactatttggggc-3 ', SEQ ID NO: 8), as shown in step F of FIG. 1. Finally, the NotI / BsiWI fragment of pflNDV-1 was replaced by a similar fragment obtained from steps E or F to create full-sized NDVH or NDVHm clones, respectively (Fig. 1). The length of the resulting clones (17196 nucleotides) is divided into six, which represents the “rule of six”. All mutagenesis reactions described herein were made using the Quik Change II XL site-directed mutagenesis kit (Stratagene).

Transfection and virus production.

To obtain recombinant NDV expressing H5 AI, full-length clones along with plasmids expressing NP, P, and L proteins were transfected into BSR-T7 cells using Lipofectamine 2000 (Invitrogen) at a DNA: Lipofectamine 2000 ratio of 1: 1.5. Virus propagation and confirmation of the production of an infectious virus was carried out mainly by the method described previously (Römer-Oberdörfer, 1999, see above; Engel-Herbert, I., Werner, O., Teifke, JP, Mebatsion, T., Mettenleiter, TC & Römer-Oberdörfer, A. (2003) J. Virol. Methods 108, 19-28).

Northern blot analysis.

CEK cells were infected with NDV Clone 30, NDVH5, NDVH5m or AIV-H5N2 at a multiplicity of infection (MOI) of 10 per cell and incubated for 8 hours at 37 ° C. The total RNA of infected and uninfected cells was prepared (Chomczynski, P. & Sacchi, N. (1987) Analytical Biochemostry 162, 156-159), separated in denaturing agarose gels and hybridized with radiolabeled cDNA as described previously (Fuchs, W. & Mettenleiter, TC (1996) J. Virol. 77, (Pt 9), 2221-2229). Plasmids containing the open reading frames of AIV-H5N2 hemagglutinin, NDV Clone 30 F and HN were used for in vitro transcription of ³² P-labeled cDNAs (Sp6 / T7 transcription kit, Roche).

Western blot analysis.

CEK cells were infected with NDV Clone 30, NDVH5, NDVH5m or AIV-H5N2 at an MOI of 5 and incubated for 30 hours at 37 ° C. Lysates of infected cells or virions purified using a continuous sucrose density gradient (30-60%) were separated using SDS-PAGE and transferred to nitrocellulose filters (Trans-Blot SD cell, Bio-Rad). Blots were incubated with anti-NDV polyclonal rabbit antiserum or anti-AIV subtype H5 polyclonal chicken antiserum (Intervet Int. BV, Boxmeer, NL). The binding of peroxidase-conjugated species-specific secondary antibodies (Dianova) was detected by chemoluminescence using a kit with a chemoluminescent substrate SuperSignal West Pico (manufactured by Pierce) on x-ray films (Hyperfilm MP, Amersham).

Animal experiments.

The safety of NDV recombinants was assessed by determining the intracerebral pathogenicity index (ICPI) in 1 day old chickens according to the method described in the Council of the European Community Directive (CEK (1992) Official Journal of the European Community L 260, 1-20). Vaccination experiments were conducted on 25 three-week-old SPF chickens, which were administered 10 ⁶ ELD ₅₀ NDVH5m oculonasally. To determine the protective capabilities of the recombinant, three weeks after a single vaccination, five vaccinated and five additional control animals were infected intramuscularly with 10 ^5.3 ELD ₅₀ NDV strain Herts 33/56. Moreover, 10 vaccinated animals and 9 additional control animals were infected oculonasally 10 ^7.7 ELD ₅₀ highly pathogenic AIV H5N2. Six months after the first vaccination, the remaining ten chickens were subjected to secondary immunization, then two weeks later they were infected with the same AI together with five additional control animals. After immunizations and infections, all birds were examined daily for 10 days to identify clinical signs.

Real-time analysis of virus spread using RT-PCR.

To study the spread of AIV at 2, 4, 8, and 14 days after infection, oropharyngeal and cloacal smears were taken for analysis using RT-PCR in real time. RNA from oropharyngeal and cloacal smears was prepared using Tecan-Automat using the Nucleo Spin kit (Macherey-Nagel) or manually using the viral RNA isolation kit (Qiagen). To detect the spread of AIV after infection, for influenza A virus, real-time RT-PCR was used based on amplification of the M gene (18). RNA extraction and inhibition factors during RT-PCR were tested using a heterologous internal control system (19). Double analysis was performed on the cycler MX3000p (Stratagene) using the one-step kit for RT-PCR (Superscript ^TM III One-Step RT-PCR system c Platinum® Taq DNA Polymerase (Invitrogen )). The temperature profile was as follows: 30 min 50 ° C, 2 min 94 ° C, followed by 42 cycles of 30 sec at 94 ° C, 30 sec at 57 ° C and 30 sec at 68 ° C.

HI and NP-ELISA.

To determine the presence of NDV and AIVH5 antibodies on days 0, 7, 14, and 21, blood samples were taken, which were then examined using the hemagglutination inhibition test (HI) according to the method described in the Council of the European Union Directive (CEK (1992) Official Journal of the European Community L 260, 1-20; CEK (1992) Official Journal of the European Community L 167, 1-1620b 21). To detect the presence of AIV-H5 antibodies after immunization, sera for indirect immunofluorescence (IF) were additionally used by incubating the sera at a dilution of 1: 100 with AIV infected with CEF. Antibodies against AIV nucleoprotein (NP) were studied using indirect enzyme-linked immunosorbent assays (ELISAs) based on nucleoprotein (NP-ELISA). For this purpose, a purified recombinant glutathione-S-transferase-NP fusion protein obtained using baculovirus covering the entire coding region of the NP AIV gene was used as antigen. Sera diluted 1: 300 in PBS containing 0.05% Tween 20 were examined in duplicate. Binding of secondary POD-conjugated goat-anti-chicken IgG (H + L) (ROCKLAND) antibodies was detected by color reaction using o-phenylenediamine, and absorption was measured at 492 nm.

Example 8. The structure of NDV vectors containing the modified H7 gene.

In experiments, mainly similar to those described above, an NDV vector was constructed that carried the modified H7 gene. The insert was obtained from HP H7N1 AIV isolate: A / chicken / Italy / 445/99, the sequence of which is published in GenBank under accession number AJ 580353; see also: Veits, J et al. 2003 (J. of Gen. Virol., Vol. 84, p. 3343). The H7 gene is inserted into the NDV vector between the F and HN genes and is flanked by non-coding sequences from the HN gene.

The potential sequence of the end of the gene is located after the site of the H7 HA gene cleavage site. The initial sequence in nucleotides 1195-1206: ata gaa aaa act encoding IEKT amino acids was mutated into: ata ga g aa g act still encoding IEKT.

As a result, stable and improved expression was observed (compared to the expression of the unmodified H7 gene) and the presentation of the modified H7 sequence using the NDV vector.

Claims (12)

1. A method of obtaining a recombinant vector based on the Newcastle disease virus containing an additional transcriptional unit comprising a gene encoding the HA protein of the pathogenic avian influenza virus H5 operably linked to the start sequence of the gene (GS) of the initial part of the Newcastle disease virus and the sequence of the end of the gene (GE) the final part of the Newcastle disease virus, characterized in that the protein contains an extension section consisting of at least three basic amino acids, where inenia consists of arginine (Arg) and / or lysine (Lys) residues and contains at least one lysine, the nucleotide sequence of the indicated gene encoding the HA protein being selected so that it does not contain a sequence that can be recognized by viral polymerase Newcastle disease virus as a gene end sequence (GE). 2. The method according to claim 1, characterized in that to create a modified sequence of the gene encoding the HA protein of the pathogenic avian H5 virus, part of the wild-type sequence of the gene encoding the HA protein of the pathogenic avian H5 virus of the bird coding for the specified extension section, consisting of of at least three essential amino acids, modified by site-directed mutagenesis to eliminate any sequence that can be recognized by viral polymerase of the Newcastle disease virus as a consequence gene end-of-life (GE). 3. The method according to claim 2, characterized in that within the t / aGAAAA sequence, originally presented in the wild-type sequence of the gene encoding the HA protein of the pathogenic avian influenza virus H5, at least one mutation is introduced so that the “A” extension section including adenine is replaced by another nucleotide. 4. The method according to claim 3, characterized in that this mutation (s) are silent. 5. The method according to claim 3 or 4, characterized in that at least one codon aaa is replaced by the codon aag. 6. The method according to claims 3 and 4, characterized in that at least two point mutations are introduced. 7. The method according to claims 3 and 4, characterized in that at least two aaa codons are replaced by aag codon. 8. The method according to claims 3 and 4, characterized in that the coding sequence aga aga aaa aaa is replaced by the coding sequence agg agg aag aag. 9. Recombinant expression vector for Newcastle disease virus containing an additional transcriptional unit, including a gene encoding the HA protein of the pathogenic avian influenza virus H5, operably linked to the start sequence of the gene (GS) of the initial part of the Newcastle disease virus and the sequence of the end of the gene (GE) of the final part of the virus Newcastle disease, characterized in that said protein contains an extension portion consisting of at least three basic amino acids, wherein said extension portion comprises from residues of arginine (Arg) and / or lysine (Lys) and contains at least one lysine, and the specified gene does not contain a sequence that can be recognized by the viral polymerase of the Newcastle disease virus as a gene end sequence. 10. The vector according to claim 9, characterized in that the extension section of the basic amino acids contains at least two lysines in a row, and in which at least one of these lysines is encoded by the aag codon. 11. The vector according to claim 9 or 10, characterized in that the extension section of the basic amino acids contains the amino acid sequence Arg Lys Lys encoded by the aggaagaag nucleotide sequence. 12. A vaccine for protecting birds from viral diseases, containing the recombinant Newcastle disease virus vector according to any one of claims 9 to 11 and a pharmaceutically acceptable carrier or diluent.

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