RU2215035C2 - Recombinant polioviral vector (variants), method for its preparing (variants), method for induction of immune response in individual (variants), method for preparing protein (variants), vaccine composition for therapy and prophylaxis of infectious diseases (variants) - Google Patents

Abstract

FIELD: genetic engineering, molecular biology, medicine, immunology. SUBSTANCE: replication-competent recombinant viruses, especially replication-competent recombinant polioviruses, comprise sequences of exogenous nucleic acids that encode exogenous polypeptide and sequence of nucleic acids that encodes artificial site of proteolytic cleavage for viral or cellular protease that processes (cleaves) proteolytically the protein precursor produced by virus and uses its. Recombinant precursor is cleft for usual proteins as its components releasing exogenous polypeptide. Replication-competent recombinant viruses are used as vaccines against cholera, gastroenteritis, influenza, hepatitis B, wooping cough and virus simplex and for induction of immune response also. Method for preparing recombinant poliovirus defines order of disposition of exogenous nucleotide sequence and nucleotide sequence encoding artificial site of proteolytic cleavage. Recombinant poliovirus produced by using recombinant polioviral vector in inserted into host-cell and protein is isolated from host-cell. Human cells are used as host-cells. Invention allows the development of vaccines alternative to existing now and to create novel vaccines that will allow to reduce morbidity and lethality from many diseases. EFFECT: improved induction and preparing method of vaccines, valuable medicinal properties of vaccines. 37 cl, 13 dwg, 2 tbl, 12 ex

Description

BACKGROUND OF THE INVENTION
There are currently many types of vaccines used to immunize individuals against diseases. While those that are available are usually safe and at least to some extent effective in creating an immune response, they all have limitations. In addition, against some diseases there are no effective vaccines. The development of alternatives to current vaccines and the creation of new vaccines to protect against diseases for which vaccines currently do not exist would be an important step in reducing the incidence and mortality of many diseases.

Summary of invention
The present invention relates to replication-competent recombinant viruses that contain exogenous nucleic acid sequences that encode an exogenous polypeptide or protein that is expressed as a component of a recombinant polyprotein precursor, which is then proteolytically cleaved by viral and / or cellular enzymes. As a result, the encoded exogenous polypeptide is released. Replication-competent viruses can be animal (non-human) viruses (for example, vertebrate and mammalian viruses), human viruses or plant viruses. The invention relates to replication-competent recombinant viruses, especially polioviruses in which the recombinant genome comprises an exogenous nucleic acid sequence (or sequence) encoding an exogenous polypeptide or polypeptides to be expressed and one or more of the nucleic acid sequences encoding an appropriate number artificial proteolytic cleavage sites, and which expresses the encoded polypeptide (s) and induces the production of ntitel specific for this polypeptide (s) in a mammal, which they are introduced. Appropriately selected replication-competent viruses are suitable for delivering an exogenous protein to an individual, for example, a vertebral, especially a mammal, and even more specifically, the person to whom they are introduced. In the case of plant viruses, for example, a replication-competent plant virus can be incorporated into seeds or into a plant at a different developmental stage, such that the encoded exogenous polypeptide is expressed and processed. Such an exogenous polypeptide can be used, for example, to protect plants from diseases or from insect attacks. Plants can be used to administer vaccines through oral administration.

 Replication-competent recombinant viruses of the present invention include a wide variety of virus types, such as picornaviruses (e.g., enteroviruses, polioviruses, lizard virus (FM DV), rhinoviruses, ecoviruses, hepatitis A viruses), Togaviruses (e.g. Synobis and rubella viruses) and Flaviviruses (e.g. yellow fever virus). The type of virus used is determined in part by the target exogenous antigen (s) to be expressed, the route of administration of the resulting recombinant viruses, and the nature of the desired immune response.

 In particular, the present invention relates to replication-competent recombinant polioviruses that differ from parent polioviruses in that they are modified so that they are not pathogenic and contain exogenous nucleic acid sequences and one or more of the artificial proteolytic cleavage sites, with so that during a viral infection they stably express the encoded product as a component of a recombinant polyprotein precursor. The precursor is proteolytically cleaved, releasing normal poliovirus proteins and an encoded exogenous protein. In addition, replication-competent recombinant viruses may contain a polylinker sequence (EcoR1, Not1, BssH2 and Xho1) to facilitate the insertion of target foreign nucleotide sequences. They also include a poly-amino acid region, for example, a poly-glycine region, which is usually adjacent to the inserted sequence, so that the structural flexibility of the region is increased and the efficiency of proteolytic cleavage is increased.

 The present invention relates to a new method for producing recombinant viruses, in which exogenous nucleic acid sequences are inserted into the viral genome. The method of the present invention uses the basic aspects of the virus life cycle, as a result of which the recombinant virus produces its normal components and replicates, and the amino acid sequence encoded by the exogenous nucleic acid sequence (s) (exogenous protein) is produced in significant quantities in infected cells. In this method, the original virus, the genome of which is encoded by a polyprotein precursor, which is proteolytically cleaved by viral or cellular enzymes to form one or more of the mature proteins, is modified as follows: The exogenous nucleic acid sequence that encodes the polypeptide to be produced and the nucleic acid sequence or sequences, which encode (encode) an artificial site or proteolytic cleavage sites for viral or cell The primary protease, which proteolytically cleaves the polyprotein precursor produced during the life cycle by the original virus, is introduced into the viral genome producing the recombinant virus. Sequences can be inserted into the viral genome anywhere, provided that their presence does not violate the viral sequence required for viral replication. So, for example, two sequences can be built at the native site at which the polyprotein is cleaved to obtain two native proteins (that is, at any site at which the native polyprotein is usually proteolytically cleaved). In cases where exogenous nucleic acid sequences are inserted at the end of the viral sequence encoding the protein, only one proteolytically cleavable sequence is required. If more than one exogenous nucleic acid sequence encoding the desired polypeptide is inserted into the viral genome, additional nucleotide sequences encoding the proteolytic cleavage site may be required. So, for example, if two or more polypeptide-coding nucleic acid sequences are inserted into the viral genome (inside the genome) and the encoded proteins need to be cleaved to form two or more separate proteins, then a sufficient number of nucleotide sequences encoding cleavage sites should also be inserted . So, for example, if two exogenous nucleotide sequences are inserted into the viral genome, each of which encodes a protein to be obtained, and two separate (individual) proteins are to be obtained, three or four nucleic acid sequences encoding proteolytic cleavage sites are required (i.e., three if two proteins are encoded by two nucleic acid sequences present in the viral genome without interruption of amino acid sequences, and four if two awns nucleic acids are separated by nucleic acid breaking in a sequence, the product which it encodes, should be removed for the "split" the two encoded proteins). In such cases, when the sequences are embedded inside (and not at the ends) of the viral genome, two proteolytic cleavage sequences are necessary in order to enable the release of both ends of the exogenous nucleic acid sequence; moreover, these sequences may be the same or different.

 In one embodiment of the present invention, two sequences are introduced into the genome of the parent virus between the first or unique start codon and the second codon at the 5 'end of the viral protein coding sequence so that the order of the recombinant genome becomes: 5' untranslated portion of the original virus is a unique start codon of the original virus - exogenous nucleic acid sequence encoding the product to be expressed - nucleic acid sequence encoding art the site of proteolytic cleavage, the second codon of the original virus, is the rest of the nucleic acid sequence of the original virus. In another embodiment, in which an exogenous nucleic acid sequence encoding an exogenous polypeptide is inserted into the viral genome, the sequence order in the resulting recombinant viral genome will be as follows: 5 'untranslated region of the original virus - unique starting codon of the original virus - initial codon (s) of the translated region the original virus - the nucleic acid sequence encoding the artificial site of proteolytic cleavage - the exogenous nucleic acid sequence t - a nucleic acid sequence encoding an artificial proteolytic cleavage site - remainder of the genome of the source virus. The encoded proteolytic cleavage sites may be the same or different.

 In one embodiment of the present invention, in which a recombinant poliovirus (which expresses an exogenous nucleotide sequence or sequences) is obtained, a nucleotide sequence encoding a protein (e.g., an antigen) to be expressed and a nucleotide sequence (s) encoding an artificial recognition sequence (or sequences ) for poliovirus 3C protease, integrate into the genome of the original poliovirus. In another embodiment, a nucleotide sequence (or sequences) encoding an artificial recognition sequence (s) for a poliovirus 2A protease is inserted into the viral genome along with one or more of the nucleotide sequences encoding the protein (s) to be expressed. In the case of DNA viruses, the genome is manipulated in its DNA form. In the case of virus RNA, the genome is manipulated in its cDNA form. Both are designated as the genome of the original virus. In that embodiment, when an exogenous nucleotide sequence encoding an exogenous polypeptide is inserted at the end of the poliovirus genome, a recombinant poliovirus genome of the following structure is obtained: 5 'untranslated portion of the original poliovirus - unique poliovirus start codon - exogenous nucleotide sequence - artificial 3C or 2A recognition polyovirus recognition site - the second codon of the poliovirus genome is the rest of the poliovirus genome. In that embodiment, when an exogenous nucleotide sequence encoding an exogenous polypeptide is inserted at the site inside the original poliovirus genome (i.e., at the internal site), the resulting recombinant poliovirus genome has the form: 5 untranslated region of the original polioviruses - unique poliovirus starting codon - polyoprotein coding region (plots) - a nucleotide sequence encoding an artificial recognition site of a 3C protease or 2A protease - an exogenous nucleotide sequence ( sequences) is the nucleotide sequence encoding the artificial recognition site of the 3C protease or 2A protease - the rest of the poliovirus. In cases where more than one protein or polypeptide is to be expressed, more than one exogenous nucleic acid sequence is inserted into a recombinant replication-competent poliovirus (i.e., a nucleotide sequence encoding each of the proteins or polypeptides to be expressed), together with an appropriate amount artificial proteolytic cleavage sites, as previously indicated.

 If the modified poliovirus genome is translated, a larger structure is obtained, but it is suitably cleaved into the usual regions constituting the poliovirus proteins due to the 3C and / or 2A proteases (proteases) present in the original poliovirus. The 3C and / or 2A protease (proteases) also accurately recognizes (recognizes) and cleaves (cleaves) the artificial recognition site (proteolytic cleavage), freeing the exogenous polypeptide encoded by the ecosogenic nucleotide sequence. As indicated, the recombinant polioviruses of the present invention express encoded exogenous protein (s) in detectable amounts. Source viruses continue to replicate and native proteins form in exactly the same way.

 Replication-competent recombinant viruses of the present invention, which are represented by recombinant polioviruses, can be used as vaccines against bacterial, viral, fungal (eg, yeast) infections, parasitic diseases, cancer or allergies. They can also be used to administer contraceptives (e.g., sperm antigen). Vaccine and contraceptive compositions that are or include replication-competent recombinant viruses, as indicated here, also form an object of the present invention.

 The recombinant poliovirus vaccines of the present invention are particularly advantageous if mucosal immunity is required to prevent infection. For example, effective mucosal immunity would be beneficial in preventing or reducing infection with HIV, rotaviruses, respiratory syncytial virus (RSV), hepatitis A virus, poliovirus, papilloma virus, measles virus and influenza viruses. Recombinant viruses are also suitable for creating immunity against bacterial diseases, such as those caused by Vibrio Cholerae and enerotoxigenic E. Coli. In addition, recombinant polioviruses can be used to create protection against infection by more than one organism by introducing more than one exogenous nucleotide sequence (for example, two or more of the nucleotide sequences, each of which encodes different antigens) into the genome of the original poliovirus. Alternatively, a mixture or cocktail of two or more recombinant viruses, each of which expresses a different antigen, can be used to immunize an individual against more than one organism or infectious agent. By using the emerging molecular biology approaches to develop improved or new vaccines, the definition of “ideal vaccine,” given by Task Force on Child Survivae, is a vaccine in which “a single administration at birth provides protection against many diseases,” can be promisingly implemented.

 Recombinant viruses can also be used to produce exogenous polypeptides in tissue culture that can be isolated or separated from the cells and viruses in which they are produced. They can be used to produce an exogenous polypeptide in vertebrate cells, in mammalian cells, including human cells, and in plant cells.

Brief Description of the Drawings
In FIG. 1 schematically illustrates a recombinant poliovirus of the present invention in which the poliovirus genome (SEQ IDs 24 and 25) is modified, and these modifications allow the insertion of exogenous nucleic acid sequences at the end of the poliovirus genome, and the expression of exogenous polypeptides is shown. The course of the proteolytic processing of viral 3C protease is also presented, when it cleaves both the native poliovirus polyprotein (PO) precursor and the exogenous protein insert.

 Figure 2 schematically shows the recombinant polioviruses of the present invention, with the insertion of exogenous nucleotide sequences at the end and inside the poliovirus genome, which leads to the expression of exogenous polypeptides. Also presented is a poly-glycine region adjacent to the integrated sequence, which increases the structural flexibility of this region.

 FIG. 3 is a photograph of plaque assay results, which is a phenotype of the parent poliovirus and recombinant poliovirus. In the presented HeLa plaque assay, cells were infected with the original poliovirus (A) or recombinant polioviruses containing: antigenic epitopes derived from rotavirus VP4 protein (B, C and D) or the complete coding sequence of subfragment B (CTB) of mature cholera toxin (E) .

 FIG. 4 presents assay results that show the expression and processing of recombinant polioviruses containing a toxin subunit of the Vibrio cholerae B sequence or VP4 rotavirus sequence.

 In FIG. 4A is a photograph of a Western blot obtained using extracts of HeLa cells infected with either parent poliovirus (line 1) or the recombinant poliovirus and cholera toxin B virus described in Example 2 (lane 2). The results show that the B subunit is expressed and suitably processed in the context of recombinant poliovirus (indicated by arrow).

 FIG. 4B is a photograph of extracts from the same HeLa cells infected with either the parent poliovirus (line 1) or the recombinant poliovirus cholera toxin B (CTB) (line 5) (example 2) and probed with rabbit antibodies that recognize the poliovirus structural proteins. The results show that it turns out more than the normal PI polyprotein precursor in the poliovirus recombinant, but there is a corresponding proteolytic cleavage, as a result of which a normal complement of poliovirus protein products is formed, and exogenous CTB protein obtained from CTB nucleotide sequences is also released. Lines 2-4 contain recombinant polioviruses containing antigenic epitopes (21-104 amino acids in length) derived from rotavirus VP4 protein.

 Figure 5 presents the results of SDS-PAGE analysis carried out to determine the structure and composition of recombinant variants. The nature of the migration of two viruses (initial and recombinant) is completely indistinguishable, which suggests that the recombinant viruses have a normal structure and composition of proteins. The identity of poliovirus capsid proteins is shown.

 Figure 6 presents the results of a Western blot analysis performed to determine the ability of recombinant polioviruses to elicit an immune response in a vaccinated host, compared with control values and false injections.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to recombinant replication-competent viruses, which include an exogenous nucleotide sequence that encodes an exogenous polyprotein that is obtained during the life cycle of the virus, and a nucleotide sequence that encodes an artificial proteolytic cleavage site for viral or cellular proteases that cleave the precursor polyprotein, produced by the original virus. Two types of sequences can be present anywhere in the original viral genome, provided that their presence does not violate the viral sequence required for viral replication.

 The present invention is based on a demonstration by applicants of the fact that an exogenous nucleotide sequence encoding an exogenous polypeptide can be built into the viral genome at the end of the viral genome, or locally within the viral genome (i.e., at the internal site) and can be obtained during the life the cycle of the virus as a polyprotein precursor that is cleaved by viral or cellular proteases that cleave the protein precursor typically produced by the parent virus (i.e., a virus in which inserted an exogenous nucleic acid sequence). In one embodiment, in which an exogenous nucleotide sequence encoding an exogenous polypeptide is inserted at the end of the viral genome, the sequence order in the resulting recombinant viral genome is as follows: 5 'untranslated region of the original virus - unique starting codon of the original virus - exogenous nucleotide sequence - nucleotide sequence encoding artificial site of proteolytic cleavage - the second codon of the original virus - the rest of the viral genome. A region of the recombinant genome, which is the 5 ′ untranslated region of the viral genome, can represent all, or part of the 5 ′ untranslated region, as it occurs in the original virus. In another embodiment, where an exogenous nucleotide sequence encoding an exogenous polypeptide is inserted into the viral genome, the sequence order in the resulting recombinant viral genome is as follows: 5 'untranslated region of the original virus - unique starting codon (s) of the original virus - original codon (s) of the translated region of the original virus - the nucleotide sequence encoding the artificial site of proteolytic cleavage - the exogenous nucleotide sequence - sequence n nucleotides encoding the artificial proteolytic cleavage site - remainder of the source of the viral genome.

 The encoded exogenous polypeptide is expressed in the context of the translation of a normal viral protein as a component of a recombinant or fusion precursor polypeptide (which includes an exogenous polypeptide, an artificial site or proteolytic cleavage sites, and a viral polyprotein). The recombinant polypeptide precursor is proteolytically cleaved by the action of a viral or cellular protease (protease), which cleaves the parent viral polyprotein of the precursor, which leads to the release of free exogenous protein from the viral proteins. A virus that is modified to include exogenous sequences is caused by the original virus, which may be a native virus (either pathogenic or, preferably, non-pathogenic), attenuated virus, vaccine strain, or recombinant virus. As used herein, the term polypeptide includes proteins or parts thereof (peptides), fusions of two or more of the proteins or peptides, and fusions of a protein and a peptide.

 In a specific embodiment, applicants have obtained a replication-competent recombinant poliovirus that comprises an exogenous nucleotide sequence encoding an exogenous protein to be expressed and a nucleotide sequence encoding an artificial proteolytic cleavage site for poliovirus 3C protease and / or protease 2A inserted at the end of the poliovirus genome, or on the site inside the poliovirus genome. They demonstrated that exogenous protein is expressed and released from poliovirus proteins through proteolytic cleavage. The resulting replication-competent recombinant polioviruses differ from the original viruses in that they comprise an exogenous nucleotide sequence (s) encoding an exogenous polypeptide or polypeptides and one or more of the artificial proteolytic cleavage sites and express an endogenous product during viral infection. The parent polioviruses may be native or wild-type polioviruses, or recombinant or genetically engineered polioviruses (in this case, the altered or mutated sequences do not encode an exogenous protein suitable for the purposes described herein for the polioviruses that make up the present invention).

 In one embodiment of the present invention, an exogenous nucleotide sequence encoding an exogenous polypeptide and a nucleotide sequence encoding artificial proteolytic cleavage sites are located at the end of the poliovirus genome, between the unique start codon and the second codon of the poliovirus genome, so that the sequence order in the recombinant genome is as follows: 5 'untranslated region of the poliovirus genome - unique poliovirus starting (first) codon - exogenous sequence Nost of nucleic acids - artificial protease recognition site - second codon of the poliovirus genome - remainder of the poliovirus genome. The expression of the recombinant poliovirus genome produces a recombinant polyprotein precursor or fusion polyprotein that includes an exogenous protein, an artificial proteolytic cleavage site, and a poliovirus polyprotein. Applicants have shown that proteolytic cleavage of a recombinant polyprotein precursor with a protease, for which an artificial cleavage site is integrated, leads to the production of components of a normal poliovirus protein and the release of exogenous protein. There is also viral replication, but exogenous protein is not included in the poliovirus virion.

 Two recombinant polioviruses of the present invention, in which an exogenous nucleotide sequence and a nucleotide sequence encoding an artificial proteolytic cleavage site, are inserted at the end of the poliovirus genome, are shown in FIGS. 1 and 2 (pMV 1.3). The recombinant poliovirus genome includes nucleic acid sequences (immediately after the 3 'unique starting codon) that encode the five amino acid residues present on the amino terminus of the polyprotein of the Mohoney poliovirus strain, type 1. The presence of these sequences is not necessary, but their presence may affect the efficiency of expression. As seen in FIG. 2, the recombinant poliovirus genome may also include a polyglycine region adjacent to the inserted (exogenous) sequences.

 Exogenous nucleic acid sequences encoding the protein or polypeptide to be expressed can be inserted into the viral genome: for example, into the multiviral genome. As can be seen in FIG. 2, there are a number of additional regions within the poliovirus genome where the exogenous nucleic acid sequence encoding the exogenous polypeptide and the nucleic acid sequences encoding the artificial proteolytic cleavage sites can be arranged to produce replication-competent recombinant polioviruses that express the encoded product. These sites within the poliovirus genome include the connection between the Vp1 coding region and the 2A coding region, the connection between the 2A coding region and the 2B coding region, and the connection between the 2C coding region and the 3A coding region. Polylinker / proteolytic fragments were inserted at these sites, and the resulting recombinant polioviruses were shown to be replication-competent. Using the methods and known methods described herein, an exogenous sequence or nucleic acid sequences can be inserted at these sites. The insertion of polylinker / proteolytic fragments by combining the Vpg coding region and the 3C coding region terminates viral replication.

 To facilitate the processing of exogenous sequences within the viral polyprotein, it is necessary to include the corresponding signals of proteolytic cleavage at both ends of the insert. Therefore, the sequence order in the poliovirus genome in which an exogenous nucleic acid sequence encoding an exogenous polypeptide and a nucleic acid sequence encoding artificial proteolytic cleavage sites is inserted, for example, between the Vp1 coding region and the 2A coding region, as follows: 5 'untranslated region of the poliovirus genome - poliovirus unique start codon - Vp0 coding region - Vp3 coding region - Vp1 coding region - nucleic sequence islot encoding artificial 3C protease recognition site or 2A protease recognition site - exogenous nucleic acid sequence - nucleic acid sequence encoding artificial 3C protease recognition site or 2A protease recognition site - remainder of the poliovirus genome.

 The determination that there are many sites of the poliovirus genome by which it is possible to embed sequences of exogenous nucleic acids to produce replication-competent recombinant polioviruses means, in a broad sense, that there is considerable flexibility and the possibility of variations in the design and production of recombinant viruses suitable, for example, as vaccines and protein. One or more of the exogenous nucleic acid sequences encoding an exogenous protein or polypeptide to be expressed and proteolytically cleaved can be constructed from one or more of the sites (at the end or inside) of the viral genome described herein. In addition, other sites through which insertions can be made without impairing the replication ability of the virus can be identified. It is possible that some exogenous nucleic acid sequences will be more stable, or more efficiently expressed and / or proteolytically cleaved, if they are integrated into a specific site of the viral genome. How true (if even true) can be estimated using the methods described here and, accordingly, the resulting recombinant viruses, for example, as is done with the example of polioviruses.

 Additional characteristics, such as polylinker sequences (e.g., EcoR1, Not1, BssH2, and Xho1) may be included in the design of replication-competent recombinant viruses to facilitate the incorporation of the target foreign sequence into the recombinant vector. Further, options such as a polyglycine fragment can be inserted next to the inserted sequence in such a way as to increase the structural flexibility of the site and potentially increase the efficiency of proteolytic cleavage.

 More than one nucleic acid sequence encoding an exogenous protein or polypeptide to be produced can be inserted into recombinant replication-competent viruses that result in the corresponding number of proteins or polypeptides. Two or more of the amino acid sequences, each of which can encode a different product, or can encode the same product (for example, if increased production of a protein or polypeptide is desired). Further, for a poliovirus, the proteolytic cleavage site (s) may be a cleavage site 3C, a cleavage site 2A, or both of them.

 Although the present invention is illustrated by the example of the preparation of a recombinant poliovirus, any virus in which proteolytic cleavage of the viral precursor protein occurs can be modified so that it produces a recombinant virus that expresses the exogenous protein and processes it accordingly. Thus, for example, recombinant picornaviruses (e.g., enteroviruses, polioviruses, FMDV, rhinoviruses, ecoviruses, hepatitis A virus) and recombinant flaviviruses (e.g. yellow fever virus) can be obtained and used in the same manner as described for recombinant polioviruses.

 The considered method for producing a replication-competent recombinant virus that expresses and proteolytically cleaves an exogenous protein is the following: A virus that produces a protein precursor in its natural life cycle (referred to as the original virus) that is proteolytically cleaved by a viral or cellular protease (s), modified using well-known genetic engineering methods (Sambrook. J. et. al. Molecular Cloning: A Laboratory Manual / 2d, ed /, Cold Spring Harbor Laboratory Precc (1989)) for embedding, at extremely at least two types of nucleic acid sequences in the viral genome: an exogenous nucleic acid sequence (a nucleic acid sequence obtained from a source different from the type of virus into which it is inserted) encoding an exogenous protein or polypeptide to be expressed and processed by a recombinant virus, and a nucleic acid sequence encoding an artificial recognition site for viral and / or cellular protease (proteases), which processes expressed recombinant protein with the release of viral and exogenous proteins. An additional type of nucleic acid sequence, for example, a poly-glycine fragment, can be inserted adjacent to an exogenous nucleic acid sequence to increase the structural flexibility of the site and potentially increase efficiency.

 The construction of a cDNA clone of a poliovirus vector illustrating the present invention is described in Example 1. One or more of the “fragments”, each of which includes exogenous nucleic acid sequences or sequences encoding an exogenous product (s), one or more artificial proteolytic recognition sites, and optionally, additional nucleic acid sequences, such as a poly-glycine fragment, can be inserted in this way. For example, one “fragment” that includes a nucleic acid sequence encoding an exogenous protein antigen against which an immune response needs to be developed and an artificial recognition site for proteolytic cleavage can be inserted at the 5 ′ end of the viral genome. In another embodiment, two or more such “fragments” or one “fragment” that include nucleic acid sequences encoding more than one protein or polypeptide and an appropriate number of proteolytic cleavage sites (for example, to achieve expression and release of two or more different protein antigens or two copies of one protein agent), can be integrated into the viral genome. Nucleic acid sequences encoding the proteins or polypeptides to be expressed may be in tandem of "fragments" (that is, without a discontinuing sequence) or may be separated by nucleic acids that do not encode the protein or peptide to be expressed. One or more of the "fragments" can be embedded in some or all sites of the viral genome. The resulting recombinant polyprotein precursor comprises one or more of exogenous proteins or peptides and one or more of proteolytic cleavage sites. Processing a recombinant polyprotein precursor results in the release of an exogenous product or products.

 A recombinant virus, such as the recombinant poliovirus described herein, can be used to induce an immune response against an antigen and an individual, and thereby provide protection against infection or infection with an exogenous pathogen (bacterial, viral, fungal, parasitic) where the antigen is found. Therefore, they are useful as vaccines to provide protection against such pathogens. As described in Example 2, a recombinant poliovirus was constructed that includes nucleotide sequences encoding the antigenic epitopes of rotavirus Vp4 protein. As also indicated in Example 2, a recombinant poliovirus was constructed which includes the entire coding region from the cholera toxin subunit B. Recombinant polioviruses that include nucleic acid sequences encoding influenza A antigens or coregulated pilus (tcp A) Vibrio choleral toxin have also been obtained. The phenotype of these recombinants and the original poliovirus is shown in Fig.3. Recombinant poliovirus containing the complete coding region from the cholera toxin subunit B is expressed in HeLa cells and expression and processing are determined. The results show that the B subunit is expressed and accordingly processed in the context of the recombinant virus (figa). The obtained results also show that a longer proteolytic VP1 polyprotein is formed in recombinant polioviruses, but a corresponding proteolytic cleavage occurs, which leads to the formation of a normal complement of the poliovirus protein and the free sequence of the cholera toxin subunit B (Fig. 4B). Recombinant poliovirus containing antigenic epitopes (21-104 amino acids) derived from rotavirus VP4 protein were also expressed in HeLa cells. Lanes 2-4 in FIG. 4B contain recombinant polioviruses containing these epitopes.

 Example 3 describes an assessment of the immunogenicity of recombinant polioviruses prepared as described herein. Transgenic mice that express the human poliovirus receptor are infected intramuscularly with a recombinant poliovirus that expresses the complete cholera toxin B subunit (CTB) and is based on the Mahoney or Sabin vectors. The control mice were either given an imaginary injection, or they were infected with a recombinant virus that expresses Vibrio cholerae pilin. The results of the Western blot analysis shows (Fig.6) that mice immunized with CTB recombinant Mohoney-based poliovirus clearly contain IgG antibodies that are reactive with PTS monomer and pentamer. In control mice, as expected, such specific antibodies were absent. In addition, mice immunized with PTS with Sabin-based recombinant poliovirus do not produce, in this single case, reactive PTS-specific antibodies. The reason for this is not clear, but it may be due to the replicative characteristics of recombinant polioviruses and can be eliminated by increasing the dose of the recombinant virus vaccine.

 A recombinant poliovirus containing a nucleic acid sequence encoding other antigens against which it is desirable to develop an immune response can be obtained by a method similar to that described for the proteins of the toxin B subunit of cholera and rotavirus VP4. Recombinant polioviruses are especially useful without preventing disease (or reducing the severity in which they occur), which may require mucosal immunity to prevent infection. For example, recombinant polioviruses can be especially useful in creating protection against infection with HIV, rotaviruses, RSV, hepatitis A virus and influenza virus, or to reduce the severity of the disease.

The surface of the mucous membrane in the human body is more than 400 m ² and represents a huge contact surface of the immune system with the environment. The total number of lymphoid cells associated with the surfaces of the mucous membrane exceeds all other lymphoid cells taken together, and these cells are responsible for the synthesis of at least 60% of the total amount of immunoglobulin produced during the day (Childers, NK et al., Annu , Rev. Microbiol 43: 503-536 (1989)). The presence of specific IgA antibodies in secrets such as tears, saliva and milk, in the absence of local exposure to the antigen, led to the creation of the concept of a common immune system of the mucous membrane. The offspring of immune cells sensitized in intestinal lymphoreticular tissues (GAL T) appears to migrate to the mucous surface at a distance and protects it. Despite the degree and importance of the immune system of the mucous membrane, relatively little is known about the determinants of cellular and humoral immunity on the surface of the mucous membrane. The experimental evaluation of this information is limited as a result of the relatively inaccessibility of the corresponding lymphocytes, as well as the difficulties in delivering well-characterized antigens to reproducible target sites. Recombinant polioviruses should help deliver well-characterized antigens in a reproducible manner and can remove some of these experimental barriers. A number of viral and bacterial diseases of significant importance cannot be prevented by vaccination at present. These include diarrheal diseases caused by rotaviruses, respiratory diseases resulting from RSV infection, and bacterial gastroenteritis caused by V. cholerae or enterotoxigenic E. coli. Mucosal immunity is created after a natural infection by these pathogens, which gives significant or complete resistance and resistance to reinfection. The recombinant polioviruses of the present invention can be used to deliver appropriate protective antigens to the mucosal immune system and thereby provide a new immunization strategy.

 Poliovirus vaccines have been used intensively and have proven to be very safe and effective. Biologically active, molecular clones of the polioviruses used include Type I poliovirus (Mohoney strain) (Racainello, V. et al., Proc. Natl. Acad. Ssi. USA 78: 4887-4891 (1981)) and Sabin type 1 poliovirus vaccine strains, 2 and 3 (Omata, T. et al. Gene 33: 1-10 (1984); Toyoda. H. et al. J. Mol. Bio. 174: 561-585 (1984)). Derivatives of polioviruses can also be obtained, which are apparently less likely to turn into a virulent form, or they can be made avirulent from a virulent form due to site-directed mutagenesis due to insertions, deletions and / or modifications of the nucleotide sequences.

 Recombinant poliovirus vaccines can successfully replace current poliovirus vaccines for oral administration, which requires the preparation of a mixture of three different attenuated strains (PV1, PV2 and PV3), each of which has varying levels of antigenic potential, and there is some risk of reversal in the wild type of pathogenic form. PV1 is considered both the safest and most antigenic component, while PV3, as you know, suffers from a fairly high frequency of reversal to the pathogenic type. Enteroviruses: polioviruses, Coxsackie viruses, ecoviruses and the latest Melnik enteroviruses, in Virology (2d ed), D.N. Fields, D.M. Knipe et al. (ed) Raven Press Ltd. NY 1990, pp. 549-604: Nkowane, B.M. et al., Vaccine-Associated-Paralytic Poliomyelitis United States: 1973-1984 J. AMA 257: 1335-1340 (1987); Miller, Population genetics as applied to poliovirus vaccines, Am. J. Pub. Health 52: 472-483 (1962). Recombinant PV1-based polioviruses that incorporate the components PV2 and PV3 may be safer vaccines than current ones. In addition, with PV1-based recombinant polioviruses, it may be possible to achieve effective immunity against all poliovirus strains by maximizing the attenuation of antigenically potential recombinant viruses containing antigenic determinants from all three poliovirus serotypes.

 Particular advantages in using recombinant polioviruses as vaccines are that they are genetically stable and reproducibly carry embedded information as the poliovirus genome spreads from cell to cell upon replication. As a result, immunization should be effective with a limited number of doses of recombinant poliovirus. In addition, since the introduced antigens are expressed inside the infected cells, both cellular and humoral immunity are stimulated. Although exogenous nucleic acid sequences carry recombinant polioviruses and are expressed during the replication cycle, exogenous proteins are not included in the particles of the mature virus. Thus, the structure of both virion and hosts of recombinant polioviruses does not change under the influence of exogenous proteins. A number of important variables limit the effectiveness of available vaccines, especially if they are used in developing countries. Although some of these causes are practical or economic in nature, others have a biological basis. Immunization with measles vaccines is a good example of a biological barrier that the present invention can help overcome. Measles causes the deaths of 2 million children annually in developing countries. Bloom., B. R., Nature 342: 115-120 (1989). Although effective vaccines exist to prevent measles virus infection, the presence of antibodies from the mother that neutralize the vaccine significantly reduces its effectiveness in young children. Murphy, B.R. and R.M. Chanock, in: Virology (2d ed.), B.N. Fields et al. (ed), Raven Press, NY pp. 469-502 (1990); Preblud, S.R. and S.L. Katz, Vaccine, S.A., Plotkin and E.A. Mortimer, W.B. Saunders Publishing, Philadelphia (1988).

 In developing countries, the epidemiology of measles is such that many children are already sick before they are effectively vaccinated. Recombinant polioviruses carrying antigens derived from measles virus can help overcome this barrier. Poliovirus vaccines are administered shortly after birth, and their effectiveness is not significantly affected by the presence of antibodies received from the mother. The polio-measles recombinant virus will express measles antigens in infected cells, providing an immune response. However, due to the fact that measles antigens do not enter the particles of the recombinant virus, recombination of the vaccine vector should not occur.

 There are additional areas in which the vaccines of the present invention should be useful. So, for example, it has been established that diarrhetic diseases cause 5 to 10 million deaths annually. Institute of Medicine, New Vaccine Development: Establishing Properties, National Academy Press, Washington, D.D. (1985). Rotaviruses are one of the most important etiological agents for severe cases of diarrhea in small children, and they are believed to cause approximately one million deaths annually in this part of the population. Kapikian, A. Z. and R.M. Chanock, Jn: Virology (3d ed.), B.N. Field et al. (Ed), Raven Press, NY, pp. 1352-1404 (1990). Although significant progress has been made in determining the host's immune response to rotavirus infection, vaccination attempts have been limited by the genetic complexity of the virus and the limited effectiveness of the available attenuated vaccines. Similarly, it has been described that the antigens of V, cholerae are apparently targets of a protective immune response. However, possible cholera vaccines were limited by either undesirable side effects or inadequate immunogenicity. Recombinant polioviruses carrying immunogenic, but non-pathogenic, protective determinants from rotaviruses and V. cholerae can be considered as possible vaccines.

 Respiratory diseases caused by infectious antigens result in an estimated 10 million deaths per year, with RSV being the main viral pathogen. Mclntosh, K. and R.M. Chanock: In: Virology (2d ed), B.N. Field et al. (Ed), Raven Press, N.Y. pp. 1045-1074 (1990). Attempts to create an RSV vaccine have been unsuccessful to date, but significant progress has been made in identifying protective antigens. In order for RSV vaccines to be effective, they must create immunity in the mucous membrane and must be administered shortly after birth, and in doing so they must neutralize the effect of the antibodies received from the mother. Recombinant RSV polioviruses can potentially meet all of these requirements.

 The prevention of hepatitis B virus diseases is also an object of the vaccines of the present invention.

 Hepatitis B virus belongs to a family of viruses called Hepadnaviridae. This virus contains a small cyclic DNA fragment, which is partially single-stranded. Infectious virion also contains DNA polymerase, which makes the DNA genome completely double-stranded. The hepatitis B virus (HBV) replication cycle involves the formation of intermediate RNA.

 Hepatitis B virus is widespread worldwide. It is people, apparently, who are the main custodians of the virus, although the surface antigen of the virus has been found in some species of primates. According to existing estimates, in the USA there are 22 cases of hepatitis per 100,000 population, but they believe that this estimate should be increased by a factor of 10. Of these cases, approximately 45% refers to hepatitis B. Thus, we can assume that about 1-1.25 million people in the United States are chronically infected with hepatitis B.

 The most effective way to transmit hepatitis B virus is through parenteral transmission. The virus was also found in other secretions of the infected body, but the rest of the types of transmission of the virus have not yet been accurately established.

 HBV is also involved in the development of primary hepatocellular carcinoma in chronically infected viruses. This disease is most commonly found in Africa, China, South Asia, Alaska and the coast of Greenland. Hepatocellular carcinoma often corresponds to the general geographical distribution of HBV infection.

 Prevention of a disease caused by Bordetella pertussis is a further object of the invention. This bacterium causes pertussis or persistent cough, a severe and potentially fatal respiratory tract infection. The currently used pertussis vaccines contain chemically inactivated whole B. pertussis cells. Non-cellular pertussis vaccines have been developed and are based on material obtained chemically and physically from B. pertussis cultures.

In addition, vaccines have been described that are obtained by purifying individual specific pertussis antigens, which are then combined to produce vaccines (published European patent application 484621). One of the antigens included in such a vaccine is a 69 kD external membrane protein (Shahin, RD et al., Abstracts of the 89 ^th Annual Meeting of the American for Microbiology, p. 51 (1989). This antigen can be obtained from in accordance with the method of the present invention.

 The prevention and treatment of diseases caused by Herres viruses (Herres cviridae) is another objective of the present invention. Herpes viruses are large DNA viruses that create a latent infection that can exist throughout the life of the host. After a latent period, the virus can be reactivated by stimuli such as immunosuppression or irradiation.

 Simplex virus type 1 virus causes lesions in the mouth such as vesicle lichen. Simplex type 2 nerres is the cause of genital herpes, which can be further complicated by cervical carcinoma. These Nerres viruses are widespread among the population, and there is currently no registered vaccine against these viruses.

 The vaccines against Hepres viruses currently under development use such glycoproteins, which are viral proteins essential for the penetration of the virus into cells. Of particular interest are glycoproteins designated gD Herres Simplex types 1 and 2. DNA sequences of genes encoding gD-1 and gD-2 are presented in US Pat. Nos. 4,818,694 and 4,891,315. Any of these glycoproteins can be prepared in accordance with the present invention. Of interest are also glycoproteins designated gB Herpes Simplex types 1 and 2. DNA sequences of genes encoding gB-1 and gB-2 are represented by Stuve L.L. et al. J. Virology 61: 326-335 (1987) and Bzik, D.J. et al., Virology 155: 322-333 (1986). Any of these glycoproteins can be obtained in accordance with the present invention.

 Rotavirus diseases are a further object of the present invention. Currently, rotaviruses are considered by WHO as the main cause of childhood gastroenteritis, and the fight against this disease is given high priority by obtaining suitable vaccines (Bull W.H.O., 61: 251-254 (1983)).

 The rotavirus genome consists of 11 double-stranded RNA segments. These genes encode the production of at least 6 structural proteins of the virus that are located inside the double envelope of complete viral particles. Three of these proteins are the outer shell glycoproteins.

 A necessary approximation in the development of such a vaccine is the creation using recombinant DNA technology of several of these glycoproetins of the outer shell of rotaviruses. One of these glycoproteins is designated VP7. The DNA sequence of the gene encoding VP7 represented by Both G.W. et al., Proc. , Natl. Acad Sci U.S.A. 80: 3091-3096 (1983). This glycoprotein can be obtained by the method of the present invention.

 Any DNA sequence that encodes a peptide or protein of an exogenous organism that, when expressed, produces protective immunity against such an organism, or against conditions or ailments caused by this antigen, can be considered as exogenous nucleic acids for the purposes of the present invention. Nucleic acid sequences encoding one or more of exogenous polypeptides (e.g., antigens or epitopes) can be included in the vaccines of the present invention. If more than one exogenous antigen or epitope is encoded by exogenous nucleic acid sequences, they can be antigens or epitopes of a single pathogen or antigens, or epitopes from more than one (different) pathogens. In a preferred embodiment, such an organism is a pathogenic microorganism. So, for example, such an exogenous epitope can be found on bacteria, parasites, viruses or fungi that are agents that cause diseases or ailments. In addition, allergen epitopes, sperm cells, and cancer cells can be used. Such bacteria, parasites, viruses or fungi include, but are not limited to those listed below.

 Replication-competent recombinant viruses are suitable as vaccines against a wide range of pathogens. So, for example, you can get DNA or RNA from any of the organisms listed below and use to produce recombinant viruses (see table. 1a).

 Potentially suitable antigens for vaccine compositions can be determined using various criteria, for example, by the participation of antigens in neutralizing the infectious ability of pathogens (Norrby, E., 1985, Summary In: Vaccines 85, Larnet RA ,, RM Chanock and F. Brown / ed / ), Cold spring Harbor Laboratory Cold Spring Harbor NY, pp.388-389), by type or group of specificity, by recognizing a patient's antiserum or immune cells, and / or by demonstrating the protective properties of antigen or antigen-specific immune cells. In addition, encoded epitopes should preferably exhibit very little (or not at all) antigenic variation during or among different isolators of the same pathogen.

 Peptides or proteins that are known to contain antigenic determinants can be incorporated into recombinant viruses. If specific antigens are known, identification should be made and their immunoreactive sequences should be characterized. One way to accomplish this task is to use monoclonal antibodies produced on the surface of other pathogen molecules. Peptide sequences that can be recognized by antibodies are specific epitopes. Alternatively, small conjugates of small synthetic peptides with carrier molecules can be tested for the creation of monoclonal antibodies that bind to sites corresponding to the peptide on an inactive molecule (see, for example, Wilson et al., Cell 37: 767 (1984)). Other methods known to those skilled in the art that can be used to identify and characterize antigenic determinants are also within the scope of the invention.

 The exogenous proteins in the vaccine compositions of the present invention may also contain an epitope of an exogenous organism. If an exogenous polypeptide is expressed in a vertebrate host, it induces an immune response that protects against conditions or ailments caused by an antigen containing this epitope. For example, in this embodiment of the invention, exogenous proteins that encode the epitope (s) or protein (s) of snake venom, bee venom, hormone, sperm (for contraception), an allergy-causing antigen or any other antigen against which It is desirable to induce an immune response. In another embodiment, a tumor-specific antigen can be expressed as a recombinant exogenous protein to induce a protective immune response against cancer.

 The gene sequence encoding an exogenous protein that needs to be expressed due to the recombinant virus according to the method of the present invention can be isolated by methods known to specialists (but not limited to) for purification of the genomic DNA of the microorganism, due to cDNA synthesis from the microorganism RNA, due to the methods recombinant DNA (Maniatis et al., Molecular Cloning, A Laboratory Manual, 1982, Cold, Spring Harbor Laboratory, Cold Spring Harbor. NY.) or chemical synthesis.

 If the recombinant viruses of the present invention are used as vaccines, they are administered to patients by known methods. They are usually administered in the same way as (currently available) vaccines and / or by methods that mimic the methods of infection by pathogens of interest. So, for example, recombinant poliovirus vaccines can be administered orally, and others, for example, recombinant measles or flavivirus vaccines, can be administered subcutaneously. They can be administered in vaccine compositions containing, in addition to the replication-competent recombinant virus, a physiologically acceptable carrier. The composition may also include an immunostimulating agent or adjuvant, a flavoring agent or a stabilizer.

 The recombinant viruses of the present invention, especially recombinant polioviruses, can also be used as a system for producing exogenous polyprotein in host cells, for example, a mammal, especially a human, or in other types of cells (for example, mammalian cells modified to contain a poliovirus receptor). The exogenous protein is then isolated from the cells in which it is produced using known methods.

 In any application (for example, immunization or tissue culture), the exogenous nucleic acid sequence that is inserted into the virus can be obtained from a natural source, from a source obtained using genetic engineering methods, or chemically synthesized. An exogenous nucleic acid sequence introduced into a virus can encode a complete antigen against which it is desirable to develop an immune response, or epitopes of an antigen or part thereof.

 The size of the nucleic acid sequence that can be inserted into the viral genome is apparently not limited. The maximum size of exogenous nucleic acid sequences can be determined, for example, by evaluating their effect on virus replication, as indicated here. Exogenous nucleic acid sequences encoding at least 150 additional amino acids (i.e., 450 nucleotides) were inserted into the poliovirus genome without a noticeable change in virus replication efficiency. The inserted sequences are expected to present antigenic structures in the host immune system, determined by both the primary sequence and the conformation of the structure.

 A plant virus can also be used as a source virus, into which exogenous nucleic acid sequences are inserted in the described manner. So, for example, a plant virus with a DNA genome (for example, cauliflower mosaic virus) or a plant virus with an RNA genome (for example, tobacco mosaic virus, turnip yellow mosaic virus) can be used. They can be modified by embedding an exogenous nucleic acid sequence (or sequences) that encodes an exogenous polypeptide (e.g., an insect or disease toxin) to be incorporated into a plant. The resulting replication-competent recombinant plant virus is introduced into plants (for example, seeds or plants at another stage of development), where an exogenous polypeptide is produced. So, for example, an exogenous nucleic acid sequence encoding a toxin, such as a scorpion or spider toxin, can be embedded in such a way that they are expressed in plants that are protected from the insects that feed on them. In addition, plants in which exogenous nucleic acid sequences are expressed can be orally administered as a source of encoded protein or polypeptide.

 Similarly, the parent virus can be an animal virus that is modified to include an exogenous nucleic acid sequence that must be expressed in an animal (for example, to protect or immunize an animal against a pathogen or to provide a growth acceleration factor).

 The invention will now be illustrated by the following examples, which should not be construed as limiting.

Examples
Example 1
Construction of poliovirus vector cDNA clone
All manipulations with molecular clones are carried out in accordance with standard recombinant DNA techniques (Ausubel FM et al. Current Protocols In Molecular Biology, Greene Publishing - Willey Interscience, New York, 1987). Used biologically active molecular clones of poliovirus include poliovirus type 1 (Mahoney strain) (Racaniello, V. et al. Proc. Natl. Acad. Sci USA 78: 4887-4891 (1981) and vaccine strains of Sabin poliovirus types 1, 2 and 3 ( Omata, T. et al. Gene 32: 1-10 (1984); Toyoda, H. et al. J. Mol. Bio. 174: 561-585 (1984). These molecular clones were modified by the incorporation of T7 RNA polymerase promoter at the 5 'end of the viral genome to facilitate in vitro transcription by T7 RNA polymerase of infectious poliovirus RNA. Au subel, FM. et al. Current Protocols In Molecular Biology, Greene Publishing-Wiley Interscience, NY (1987). Poliovirus gene we were further modified to include a synthetic polylinker containing a variety of restriction enzyme recognition sites (for example, EcoR1 and Xho1). The sequences integrated into the poliovirus genome include these sequences encoding recognition and cleavage sites for the poliovirus 3C protease (3) AXXQG) (Palmenberg, AC, Ann. Rev. Microbio 44: 603-623 (1990)). These modifications are carried out in accordance with a variant polymerase chain reaction (PCR), known as elongation with overlap (Horton, PM et al. Biotechniques 8: 528-535 (1990)). It is illustrated by poliovirus type 1.

 In short, two independent PCR reactions are carried out using oligonucleotides 1 and 2 and 4 (see table 1b) to amplify a portion of the poliovirus genome from positions 1-740 and 740-1540, respectively.

 PCR products containing the fragment to be inserted are purified by agarose gel electrophoresis, and the second PCR amplification is carried out with oligonucleotide primers 1 and 4. The DNA fragment from 1582 base pairs (bp) from this amplification represents an insert of 42 nucleotides (containing new restriction enzyme sites and sequences coding for proteolytic cleavage signals) and sequences 1-740 and 740-1540. (An insert of 42 nucleotides is inserted between bases 740 and 741). This fragment is digested with Sal 1 and Aat 2, purified by gel electrophoresis, and ligated with Sal 1 - Aat 2, digested with the poliovirus clone pSR - XPA (previously described plasmid containing the full length poliovirus cDNA clone (Andino, R. et al. Cell 63: 369-380 (1990)). The resulting plasmid (named pM V 2.2) is linearized by digesting with Cla and in vitro synthesis of viral RNA by T7 polymerase is carried out in accordance with the usual scheme (Ausubel, F.M. et al. Current Procols in Molecular Biology - see earlier).

 Replication-competent poliovirus is isolated by transfection of in vitro synthesized poliovirus RNA in HeLa cells according to the method of Luthman H. et al. Nucl. Acid Res. II: 1295-1308 (1983). Isolated virus is characterized in accordance with standard methods in accordance with the ability to replicate, in structural composition and nucleotide sequence (Ansubel FM et al. - see earlier).

Exogenous genetic sequences derived from various viral and bacterial pathogens have been expressed in these recombinant polioviruses. The general strategy used in this case is illustrated by the example of the complete subunit of the cholera toxin B. Oligonucleotides having the following sequences (selected to allow frame insertion into the poliovirus vector) are used to amplify the complete subunit of toxin B cholera encoding the region contained in plasmid pjM17 (Rearson, DN et al. Proc. Natl. Acad. Sci USA 79: 2978-2980 (1982)):
5'-TSA GGA ATT SAS ACC TSA AAA TAT T-3 '(last 1D 5),
5'-TCA GST CGA GGG ATT TGC CAT ACT AAT-3 '(last 1D 6).

 These are primers 5 and 6, presented in table. 2.

 The resulting 312 bp DNA was digested with EcoR1 and Xho1 (sites introduced by PCR primers) and ligated with EcoR1 and Xho1 digested pM V 2.2. The resulting plasmid is designated rCH-52, and the virus obtained after RNA transfection, rCH-52. A similar approximation can be used to embed sequences derived from plasmid clones of toxin-double regulation of fimbriae Vibrio cholerae (tcpA) (Sun, D. et al. Jnfect. Jmmunol 59: 114-118 (1991)), rotavirus VR4 gene (Gorziglla, M . et al., Proc. Natl. Acad. Sci. USA 87: 7155-7159 (1990)) and the influenza A virus hemagglutinin gene (Wiley D. C. et al. Ann. Rev. Biochem. 56: 365-394 ( 1987)). For tcpA, the integrated nucleic acid sequence corresponds to the carboxy terminus of the amino acids (157-199) of the protein. For influenza A, the integrated nucleic acid sequence corresponds to amino acids 134-284.

The immunological characteristics of recombinant polioviruses to confirm the appropriate expression and processing of poliovirus components, as well as the inserted sequences, were obtained using Western blot analysis (Ausubel F.M. et al., See earlier). Preservation of the composition and structure of the native virion is checked by conventional centrifugation with a sucrose gradient (15-30%) of metabolically labeled ( ³⁵ S) lysates of infected cells. The gradient fractions are then analyzed in standard SDS-PAGE analysis (Ausubel F.M. et al., See previously).

Example 2
Design and evaluation of recombinant poliovirus containing subunit B of the toxin Vibrio cholerae.

 Recombinant polioviruses were constructed containing either the complete coding region of the cholera toxin subunit B (103 amino acids), or several sections of the rotavirus VP4 capsid protein (21-104 amino acids long).

DNA fragments encoding the peptides of the B or VP4 subunit were amplified and their ends modified by the inclusion of appropriate restriction sites for cloning purposes. The cholera toxin subunit B expressed in the absence of toxin subunit A is not toxic, but can provide antigenic stimuli to enhance the protective immune response. DNA fragments were inserted into the poliovirus vector cDVK clone, and RNA was transcribed from the resulting plasmid. Recombinant cDNA clones of subunit B of the cholera toxin and rotavirus VP4 were separately transfected into HeLa cells. Recombinant poliovirus plaques were obtained after incubation at 37 ^o C for 3 days (figure 2). Figure 2 shows the morphology of the starting and recombinant poliovirus plaques. Extracts obtained from HeLa cells infected with either wild-type poliovirus (lane 1) or recombinant cholera toxin B poliovirus (lane 2) were electrophoretically processed on SDS-PAGE gels and analyzed by Western blot (Figure 3A). Western blot was manifested by rabbit antiserum specific for the intact cholera toxin (A and B subunits). As can be seen, the B subunit is expressed and is accordingly processed in the context of recombinant polioviruses (indicated by arrow).

 Extracts from the same HeLa cells infected with either the original poliovirus (Fig. 3B, lane 1) or the cholera-polio toxin B recombinant virus (Fig. 3B, lane 5) were probed with rabbit antibodies recognizing poliovirus structural proteins. As can be seen, more than normal PI polyprotein was obtained in poliovirus recombinants due to the presence of an exogenous polypeptide.

 Appropriate proteolytic processing ensures the creation of a normal complement of poliovirus protein products, as well as the release of exogenous cholera toxin sequences. Immunological characterization of recombinant viruses was carried out using Western blot analysis (Ausubel, F.M. et al., See earlier). The preservation of the native virion structure is confirmed by conventional density gradient centrifugation and Western blot analysis (Ausubel, F.M. et al., See earlier).

Example 3
Assessment of the immunogenicity of recombinant polioviruses
In an experimental model, transgenic mice that express the human poliovirus receptor (Ren. Et al., Cell 63: 353-362 (1990), kindly provided by Vincent Racaniello) are used to study initial immunogenicity. Transgenic mice are infected by intramuscular injection of recombinant polioviruses (50 μl 2 · 10 ⁸ pfu / ml) that express the complete cholera toxin subunit B (CTB) in the context of Mahoney or Sabin / MoSB and MsSB vectors, respectively. Control mice were either infected with a recombinant virus that ecopresses Fimbria Vibrio cholerae (TcpA) (MoPi) or mimicked the injection. Mice were infected twice at 30-day intervals. After 43 days, 10 μg of purified CTB (Calbiochem) in Freund's incomplete adjuvant was intraperitoneally administered to the vaccinated mice. After 5 days, blood was taken from the mice, and their serum was tested for the presence of JgG reactive antibodies with purified PTS. Antibodies reactive with PTS were detected by Western blot analysis as follows. 5 ng of CTB is subjected to SDS-PAGE separation in one wide strip of 10% polyacrylamide gel and transferred to nitrocellulose. Serum (1: 100 dilution) from immunized animals is introduced into independent bands of a multi-slot apparatus (Mini - Protean 11, multi - Serecn BioRad). Bound antibodies are detected using affinity purified rabbit anti-mouse JgG conjugated with peroxidase in accordance with standard methods (ECL, Amercham).

 The results of this analysis are presented in FIG. 6. Serum from 5 out of 5 mice immunized with CTB recombinant poliovirus, MoSB (lanes 3-7), clearly contains JgG antibodies reactive with CTB monomer and pentamer. The serum of those mice that were mimicked by injection (lanes 1 and 2), or of an irrelevant control (MoRi, lanes 12-14), as expected, did not contain such specific antibodies. In addition, mice immunized with Sabin-based recombinant PTS expressing poliovirus do not show PTS-specific antibody reactivity (MsSB, lanes 8-11). Based on these preliminary results, a number of conclusions can be drawn. First, immunization with recombinant polioviruses can either directly create or specifically primate the corresponding antibody response in immunized animals. The absence of PTS-specific antibodies in the serum of control mice, which were similarly immunized with purified PTS; and the JgG isotype of CTB reactive antibodies indicate that the anamnestic reaction was caused by vaccination. The reason for this is unclear, but it may be due to the related replicative characteristics of recombinant polioviruses, and can be eliminated by increasing the dose of the recombinant viral vaccine. Both wild-type and recombinant Sabin polioviruses replicate worse than their wild-type Mahoney counterparts. Further, the transgenic mice used in these experiments do not support the efficient replication of Sabin vaccine strain polioviruses. Thus, in vivo limited replication can prevent the creation of effective immunization with Sabin-based recombinants.

Example 4
Recombinant Virus Safety Assessment
In parallel with studies of the immunogenicity of recombinant polioviruses, preliminary safety assessments of these viruses were carried out. For this purpose, equal titers (5 • 10 ⁶ PFU) of either the original Mahoney (pathogenic) or Sabin (attenuated) strains or their recombinant derivatives that express PTS were introduced into the spinal column of mice. Although all the mice that were inoculated with the Mahoney strain were paralyzed, none of the mice that were injected with recombinants experienced paralysis. This result suggests that the insertion of PTS sequences into the poliovirus genome weakens rather than enhances the pathogenicity of the resulting recombinant virus. A more detailed study of this phenomenon is currently underway.

Example 5
Construction of additional replication-competent polioviruses
Recombinant polioviruses, schematically presented in figure 2, were constructed in the same way, using the methods and materials described here (see examples 1 and 2) and known to specialists. To use the potential for expression of exogenous coding sequences at sites other than 5 'recombinant poliovirus protein, polylinker / proteolytically cleaving fragments were inserted into a number of additional sites within the viral genome. These sites include the connection between V p1 and 2A (pMoV2.I and pMoV2.2), the connection between 2A and 2B (pMoV 2.5), the connection between 2C and 3A (pMoV 3.1) and the connection Vpg and 3C (pMoV 3.5). To facilitate the processing of exogenous sequences within the viral polyprotein, appropriate proteolytic cleavage signals were integrated at both ends of the insert. With this embedding method, three of the new insert sites allow the creation of replication-competent recombinant viruses that stably contain the desired insert. The resulting viruses shown in FIG. 2, designated pMoV 2.1, pMoV 2.2, pMoV 2.5 and pMoV 3.1, are all replication-competent and in fact exhibit almost wild-type plaque morphology and replication kinetics. The only recombinant modification that leads to termination of virus replication is the position of the inserted sequence between Vpg and 3C. The ability to insert exogenous antigenic sequences into at least four possible positions in the poliovirus genome provides flexibility in the preparation of recombinant poliovirus vaccines.

 As shown in FIG. 2, vectors that include a 3C cleavage site and vectors that contain a 2A cleavage site were constructed. Vectors that use a poliovirus 3C protease to release exogenous sequences from a polypeptide precursor contain a Q-G processing site. Similar recombinant viruses that use the poliovirus 2A protease to carry out the appropriate processing of the recombinant polyprotein precursor contain a specific 2A processing site provided by the Y - G pair and surrounding amino acids. The viruses pMoV 2.1 and pMoV 2.2 in FIG. 2 represent such vectors based on 2A and, as previously indicated, show growth similar to wild-type viruses. The poliovirus vectors (FIG. 2) were designed to contain a more extensive polylinker sequence (EcoR1, Not1, BssH2 and Xho1) than those present in the recombinant poliovirus shown in FIG. 1, to facilitate the incorporation of desired exogenous nucleic acid sequences into a recombinant vector. In addition, variants have also been successfully obtained that include a poly-glycine region adjacent to the insertion sequence so as to provide structural flexibility to the region and potentially increase the efficiency of proteolytic cleavage. In general, these vectors enhance the capabilities of the core strategy and provide a variety of alternative approaches for creating vaccine vectors.

 An exogenous nucleic acid sequence (or sequences), each of which encodes a protein or polypeptide to be expressed, and a nucleic acid sequence, or sequences, each of which encodes a proteolytic cleavage site (e.g., 3C proteolytic site or 2A proteolytic site), can be introduced into the original poliovirus genome at any of the sites using known methods and methods described in the present invention. The ability of the resulting recombinant polioviruses to produce encoded protein (s) or polypeptide (s) can be evaluated as previously described. The immunogenicity of recombinant polioviruses and their safety can also be assessed by the methods described previously (see examples 3 and 4).

Example 6
Recombinant Poliovirus Construction Strategy
Examples 1-5 used embedding in a poliovirus genome framed synthetic polylinkers containing restriction enzyme recognition sites. Example 6 describes a strategy for constructing recombinant polioviruses that do not require embedding of exogenous restriction sites or changing the environment of the poliovirus genome around the embedding site for an exogenous gene. Next, Example 6 describes a strategy for embedding exogenous nucleic acid sequences in a frame with a polyprotein open reading frame. Examples 7-9 further describe the incorporation of specific exogenous nucleic acid sequences into this recombinant poliovirus in such a way as to obtain a vector for expression of exogenous polypeptides.

 In this example 6, the Racaniello and Baltimore system is used, which allows genetic manipulation of poliovirus (Racaniellо, V.R. and Baltimore, D. Science 214: 916-919 (1981)). Plasmids containing full-length cDNA copies of polyogenomic RNA have been found to produce infectious viruses, followed by transfection into appropriate host cells in culture. This system has been used to study many aspects of poliovirus replication, genetic stability, and attenuation of vaccine strains.

 More specifically, the poliovirus vector used is derived from plasmid pLED 3.2. Plasmid pLED 3.2 consists of plasmid pBR 322, in which a full-length cDNA copy of the Sabin type 3 poliovirus genome is inserted. Sabin type 3 virus is an attenuated strain currently used for oral polio vaccines. Poliovirus cDNA is cloned after the RNA of the palimerase promoter of the T7 bacteriophage. The presence of this promoter allows in vitro transcription using T7 RNA polymerase to obtain the full length of RNA transcripts that produce poliovirus with subsequent transfection into tissue culture cells. Samples of plasmid pLED 3.2 were deposited in the American type culture collection under accession number ATCC 75062 (12301 Parklawn Drive, Pockwille, Maryland 20852, USA). August 20, 1991

 The strategy of embedding exogenous nucleic acid sequences in a frame with an open reading frame of a poliovirus polyprotein involves the use of overlapping polymerase chain reaction (PCP) (Horton, R. M. et al., Gene 77: 61-68 (1989)). This method allows the fusion of gene sequences without the need for embedding restriction sites. Exogenous genes are connected in a frame with an open reading frame of a polyprotein without changing the flanking poliovirus sequence.

 This strategy involves two PCR cycles: in the first, overlapping flanking sequences are introduced into the gene to be cloned; in the second, two PCR fragments are used as template DNA; leading to exact fusion due to overlapping sequences.

 Expression clones are constructed using overlapping PCR extensions to fuse the exogenous gene at the beginning of the poliovirus polyprotein coding region (base 743). In addition, the sequence encoding the poliovirus protease 3C recognition site is inserted in the 3 'direction to the exogenous gene. The specific primers used in these examples are presented below in table. 2.

In the first PCR cycle, the reaction mixture contains approximately 2 nG of template DNA, 100 pmol of each primer, 200 μmol of each dNTP, 10 mM KCl, 10 mM Tris-HCl, pH 8.3, 3.75 mM MaCl ₂ and 5 units. AmpliTag DNA polymerase, fragment Stoffel / Perkm / Elmer / Cetus, Norfalk, CT /. The reaction mixture is placed in 100 μl volumes and topcoated with 40 μl of light mineral oil. The conditions of the RCR cycle are as follows: 95 ^{° C. for} 2 minutes, ice 2 minutes, then 95 ^{° C. for} 1 minute, 50 ^{° C. for} 1 minute, 72 ^{° C. for} 2 minutes for 30 cycles. The resulting PCR fragments are purified using a Promega Magic PCR kit (Promega, Madison, WY) or agarose gel electrophoresis (Sambrook. J. et al., Molecular Cloning: A Laboratory Manual 12 nd. Ed. Cold. Spring Harbor Press, Cold Spring Harbor, NY (1989)).

 Plots of pLED 3.2, which are located at the edges of base 743, are also amplified in the first round of PCR reactions in order to provide a second template DNA to achieve fusion. Additional overlapping sequences are not added to these fragments; this allows the use of these fragments to construct a number of different fusions, each of which contains different exogenous genes. The primers used for the plot in the opposite direction (5 ') from the base 743 are primers C and D (see table. 2). The primers that are used for the plot in the forward direction (3 ') from the base 743 are primers E and F (see table. 2). The matrix for these reactions is pLED 3.2, linearized with Xba1. PCR reactions are carried out as described previously. As a result of these reactions, fragments are obtained from 472 and 524 base pairs for the 5 'and 3' sections of pLED 3.2, respectively. The resulting PCR fragments were purified on 0.8% low melting point agarose (Seaplaque FMC (Rockland, ME)), and then used as templates for subsequent reactions.

 The second round of PCR reactions involves mixing the two PCR fragments (just described) and amplification in the presence of external primers to obtain the fusion product. In the constructs that will be described in Examples 7-11, the exogenous gene is fused to the 5 ′ or 3 ′ fragment in separate PCR reactions. The fusion products are then cloned into pLED 3.2 using the unique restriction sites provided in this plasmid.

The PCR reaction mixture of the second round contains I μl of each matrix, 100 pmol of each primer, 200 μmol of each dN TP 10 mm KCl, 10 mm Tris-HCl, pH 8.3, 3.75 MgCl ₂ and 5 units. AmpliTag DNA polymerase, Staffel fragment (Perkin - Elmer / Cetus). The reaction mixture was distributed in 100 μl volumes and 40 μl of light mineral oil was added. The conditions of the PCR cycle are as follows for the second round: 94 ^° C for 5 minutes, 72 ^° C for 10 minutes, then 94 ^° C for 1 minute, 50 ^° C for 1 minute, 72 ^° C for 2 minutes for 30 cycles. Fusion fragments were isolated on 0.8% low melting point agarose, digested with the appropriate restriction enzymes, and subcloned into pLED 3.2.

Example 7
Construction of a recombinant poliovirus containing hepatitis B virus surface antigen
The poliovirus vector obtained from the plasmid pLED 3.2 according to the method of Example 6 is used to construct a viral vector for the expression of the S gene encoding the hepatitis B surface (S) antigen. The antigen has been found to be protective for hepatitis B. S antigen is a protein of 226 amino acids long. In this example, the hepatitis B gene is derived from the ayw subtype of a human virus, specifically from cloned genomic DNA. The hepatitis B virus gene S from the ayw subtype has a length of 675 base pairs (Galiber F. et al., Nature, 281: 646-650 (1979)).

Primers A and B (see Table 2) are used in the first round of PCR to amplify the gene and insert overlapping pLED 3.2 sequences into the PCR product. The underlined sequences in these two primers correspond to the sequence in pLED 3.2, with the rest of the sequence corresponding to the S gene. In primer AS, the gene sequence corresponds to the initiation codon for the S antigen at 157 base pairs in the genome of the hepatitis B virus. Antisense primer B encodes the carboxyl end of the S antigen plus recognition site for the polio 3C protease. These primers are used with cloned hepatitis B DNA as a template to generate PCR, a fragment of 717 base pairs. The first round of PCR, the cycle is carried out under the following conditions: 94 ^° C for 2 minutes, ice 2 minutes, then 94 ^° C for 1 minute, 26 ^° C for 1 minute, 70 ^° C for 2 minutes for 10 cycles, 94 ^° C for 1 minute, 45 ^o With 1 minute, 70 ^o With 2 minutes for 20 cycles (as you can see, they differ from the conditions of example 6).

 In the second PCR reaction, the fragments previously described are used to create fusions between pLED 3.2. flanking regions and S gene. Two PCR reactions are carried out with either a 5 ′ pLED 3.2 flanking region or a 3 ′ pLED 3.2 flanking region plus the S gene PCR fragment as template. The sense primer for 5 ′ fusion is primer C, and it primates at the 5 ′ end of pLED 3.2 of the flanking region. Antisense primer B primates at the 3 'end of the S gene. For 3 ′ fusion, the sense primer is the S gene sense primer (A) with the 3 ′ LED 3.2 antisense primer (F). The resulting PCR fragments are 1175 and 1222 base pairs for 5 ′ and 3 ′ fusions, respectively. Then the fusion fragments are purified by agarose electrophoresis.

 Fusion fragments obtained in the second round of PCR reactions are then digested with restriction enzymes to finally construct a poliovirus vector containing the S gene. 5 'LED 3.2 - S gene fusion digest m1u1 and BstX1 (a unique site within the S gene). The 3 'LED 3.2 - S gene fusion is digested with Avr 11 and BstX1. plasmid pLED 3.2 is digested with a base pair of 278 M1u1 and a base pair of 1249 using Avr1 to remove a fragment of 971 base pairs. After purification by agarose gel electrophoresis, the resulting digests were mixed and ligated together using standard techniques (Sambrook et al, see earlier) to obtain pLED 3.2 / HBV. The DNA sequence of this full length fusion construct is determined using Applied Biosystems (Foster City, CA), a 370A DNA sequencer.

 Then transcription and transfection reactions are carried out. RNA transcripts derived from the T7 promoter are transfected into V ero cells to create infectious polioviruses. For these experiments, approximately 5 μg pLED 3.2 / HBV was prepared to create RNA transcripts by digesting the plasmid Pvu1. Pvu1 digestion results in two fragments, the larger fragment containing the T7 promoter, followed by the full length polyogen containing S gene fusion. The product of the digestion reaction is extracted with phenol and precipitated with ethanol. The precipitated DNA was resuspended in 20 μl of water. Full-length RNA transcripts are synthesized in vitro from Pvu1-digested DNA using T7 RNA polymerase (Moss, E.G., et al., J. Virol, 63: 1884-1890 (1989)).

 The transcription product is analyzed by agarose gel electrophoresis.

Confluent 25 cm ² monolayers of V cells are transfected using the DEAE transfection scheme (Van der Werf Proc. Natl. Acad. Sci. USA 83: 2330-2334 (1986)). Approximately 25 μg of RNA is mixed with DEAE-dextran (0.5 mg / ml) and then poured onto V ego cells. After 30 minutes of incubation at room temperature, the inoculum is removed and the cells are washed. Fresh modified Earl support medium is added and the cells are incubated at 33.5 ^° C. After 5 days of incubation, a complete cytopathic effect is observed (cell lysis in a monolayer), and the recombinant poliovirus is collected from the culture medium.

 Viral portions containing a recombinant poliovirus obtained from the pLED 3.2 / HBV construct are titrated in plaque assays on V cells. In addition, RNA was extracted from the recombinant virus and analyzed by transcription and PCR using the Gene Amp PCR kit of thermostable rTrh RNA reverse transcriptase (Perkin - E1mer - Cetus). The results demonstrate that the S gene is stably stored in recombinant pLED 3.2 / HBV virus when maintained in culture.

 Various methods are used to analyze the expression of S antigen from the poliovirus vector. These methods, which are standard procedures, include immunoperoxidase staining of infected cells, immunoprecipitation of viral and foreign proteins, Western blot and Dot blot (Coligan, J.E. et al. Current Protocolsin Jmmunology, Johu Wiley and Sons (1992) )

 Immunoperoxidase staining analysis is used to determine the proteins in the virus V-infected cells. V his cells are infected with a virus and fixed with ethanol or methanol at various intervals after infection. The fixed cell monolayer is incubated with an antiserum against either poliovirus proteins or S antigen. The plates are washed and then incubated with a secondary antibody (e.g., goat anti-rabbit IgG) conjugated to horseradish perocoidase (HRP). The plates are washed again, and then an HRP substrate, 3,3'-diaminobenzidine tetrahydrochloride (Sigma, San Luis, MO) is added. Cells that express proteins that cross-react with antibodies are stained.

 Monolayer V of his cells is infected with a recombinant virus. Cell lysates can be examined by immunoprecipitation or Western blotting for viral encoded proteins. For immunoprecipitation, cell lysates are incubated with an antiserum against poliovirus or exogenous protein. Protein A Sepharose (Sigma, San Luis. MO) is then added to the mixture, and incubation is continued. The sepharose beads are centrifuged, washed and eluted with SDS by dissociation buffer. Precipitated proteins can be analyzed by SDS-acrylamide gel electrophoresis. For Western blotting, cell sheets are separated into SDS-PAGE, transferred to nitrocellulose and incubated with antiserum to proteins of interest. Blots are washed, incubated with a secondary antibody (e.g., goat anti-rabbit IgG) conjugated to HRP. The blots are washed again, and then the HPP substrate, 4-chloro-1-naphthol (Bio Rad Richmond CA) plus hydrogen peroxide, is added to identify cross-reacting proteins.

 In this example 7 and the following examples, you can use the method described in example 3 earlier, using transgenic mice to assess the immunogenicity of recombinant polioviruses and exogenous polypeptides.

Example 8
Construction of a recombinant poliovirus containing genes for the pre-S region of the hepatitis B virus
The hepatitis B virus envelope contains three proteins, all of which are encoded inside the S region of the hepatitis B virus genome. Each antigen is encoded by different start sites within the open reading frame S of the gene region. The main protein is the S antigen, which contains 226 amino acids. The other two proteins are medium and large protein, which are encoded by the pre-S1 and pre-S2 genes, respectively (Tiollais, P. et al, Nature, 317: 489-495 (1985)). It is believed that the inclusion of pre-S1 and / or pre-S2 antigens in the vaccine, which may also contain the S antigen, can increase the effectiveness of hepatitis B virus vaccines.

 The strategy for constructing poliovirus vectors containing pre-S1 and pre-S2 genes is similar to the strategy described in Example 7 for the S gene. Primers are designed to amplify each gene and to insert poliovirus flanking sequences on both sides of the gene. The primers are presented in table. 2, PCR is more reactive than the medium and cycling conditions are presented earlier.

 Primers G and H are used to amplify the pre-S1 region from hepatitis B DNA. Primer G embeds the poliovirus sequence (underlined in Table 2) at the 5 'end of the pre-S1 gene, with the pre-S1 sequence starting at base 2580 in the virus genome hepatitis B. The antisense primer H is located in the pre-S region of the genome (common for both pre-S1 and pre-S2) from base 3152-6 and includes the ecoR1 site at base I. PCR amplification of the cloned genomic DNA of hepatitis B with primers G and H leads to a fragment of 338 base pairs. This fragment is used as template DNA, along with the 5 'LED 3.2 fragment (from primers C and D described in Example 6) in the second PCR reaction. Amplification with primers C and H leads to a fused 5 'LED 3.2 - pre-S1 fragment of 770 base pairs. To construct the 3 'end of the pre-S1 gene, the PCR fragment 3' LED 3.2-pre-S2 is used (see the next paragraph), since this section is the same in both prestructures. 5 'LED 3.2. - the pre-S1 fragment is digested with M1u1 and EcoR1. The 3'LED 3.2 pre-S2 fragment is digested with ECOR1 and Avr11. The resulting fragments were purified by agarose gel electrophoresis and ligated into pLED 3.2, which was digested with M1u11 and Avr11. The resulting construct is designated pLED 3.2 / pre-S1.

 According to the method of Example 7, the DNA sequence of this full length of the poly-fused construct is determined using an Applied Biosistem 370A DNA sequencer.

 Then transcription and transfection reactions are carried out. RNA transcripts created from the T7 promoter are transfected into its V cells to create poliovirus. For these experiments, approximately 5 μg pLED 3.2 / pre-1 was prepared to create RNA transcripts by digesting the plasmid Pvu1. Pvu1 digestion results in two fragments, with the larger fragment containing the T7 promoter followed by the full length polyogen containing pre-1 gene fusion. The product obtained after digestion is extracted with phenol and precipitated with ethanol. The precipitated DNA was resuspended in 20 μl of water. Full-length RNA transcripts are synthesized in vitro from Pvu1 - digested DNA using T7 RNA polymerase. Transcription products are analyzed by agarose gel electrophoresis.

Confluent 25 cm ² V cell monolayers are transfected with transcripts using the DEAE transfection scheme. Approximately 25 μg of RNA is mixed with DEAE-dextran (0.5 mg / ml), and then poured over its V cells. After 30 minutes of incubation at room temperature, the inoculum was removed and the cells were washed. Fresh modified Earl support medium is added and the cells are incubated at 33.5 ^° C. The cultures are incubated until a complete cytopathic effect is observed, and recombinant polioviruses are collected from the culture medium.

 A viral product containing recombinant polioviruses obtained from the pLED 3.2 / pre-S1 construct is titrated using plaque assay on its V cells. In addition, RNA was extracted from the recombinant virus and analyzed by reverse transcription and PCR using Gene Amp Thermostable rTh reverse transcriptase RNA, PCR kit (Perkin-E1mer / Cetus). The results show that the npe-S1 gene is stably preserved in the recombinant pLED 2.3 / pre-S1 virus when introduced into the culture.

 Various methods are used to analyze the expression of the pre-S1 antigen from the poliovirus vector. These methods include immunoprecipitation of viral or foreign proteins, Western blotting and dot blotting.

 The expression clone for pre-S2 uses primer Y as a sense primer, and primer B as an antisense primer (see Table 2). These primers amplify the complete pre-S2 gene and insert LED 3.2 sequences at the 5 'and 3' ends. As a result of the PCR reaction, a fragment of 867 base pairs is created. After purification, this fragment is used as a matrix for the second round of PCR reactions with either 5 'LED 3.2 or 3' LED 3.2 fragments, which leads to the formation of fusion products, designated 5 'LED 3.2 / pre-2 and 3' LED 3.2 / pre- 2. In the second round of PCR, C and B for 5 ′ fusion and Y and F for 3 ′ fusion are used as primers; Xba1 and Xba1 are digested with the 5 ′ LED 3.2 / -pre-S2 fragment. The 3 'LED 3.2 / pre-2 fragment was digested with Xba1 and Avr11. The resulting fragments were purified by agarose gel electrophoresis and ligated into pLED 3.2, which was digested with M1u1 and Avr11. The resulting construct is designated pLED 3.2 / pre-S2.

 By the method of Example 7, the DNA sequence of this full length of the poly-fusion construct is determined using the Applied Biosistems DNA sequencer. RNA transcripts obtained from the T7 promoter are transfected into its V cells to create an infectious poliovirus. For these experiments, approximately 5 μg of pLED 3.2 / pre-S2 is prepared to create transcripts by digesting the plasmid Pvu 1. Pvu1 digestion results in two fragments, the larger fragment containing the T7 promoter, followed by the full length polyogen containing npe-S2 gene fusion. The product of the digestion reaction is extracted with phenol and precipitated with ethanol. The precipitated DNA was resuspended in 20 μl of water. In vitro, full-length RNA transcripts are synthesized from DNA digested with Pvu1 using T7 RNA polymerase. Transcription products are analyzed by agarose gel electrophoresis.

Confluent 25 cm ² monolayers of V cells are transfected with transcripts using a DEAE transfection scheme. Approximately 25 μg of RNA is mixed with DEAE-dextran (0.5 mg / ml) and then poured on top of its V cells. After 30 minutes of incubation at room temperature, the inoculum is removed and the cells are washed. Fresh modified Earl medium was added and the cells were incubated at 33.5 ^° C. The cultures were incubated until a complete cytopathic effect was observed, and recombinant polioviruses were collected from the culture medium.

 The resulting viruses containing recombinant polioviruses obtained from the pLED 3.2 / pre-S2 construct are titrated in plaque assays on its V cells. In addition, RNA was extracted from the recombinant virus and analyzed by reverse transcription and PCR using a Gene Amp PCR kit of thermostable rTh reverse transcriptase RNAs (Perkin-Elmer / Cetus), the results show that the pre-S2 gene is stably stored in recombinant pLED 3.2 / pre-S2 virus when introduced into the culture.

 Various methods are used to analyze the expression of pre-S2 antigen from the poliovirus vector. These methods include immunoperoxidase staining of infected cells, immunoprecipitation of viral and foreign proteins, Western blotting and dot blotting.

 To determine the proteins in virus V infected cells, immunoperoxidase staining analysis is used for both pre-S1 and pre-S2. V ero cells are infected with the virus and fixed with ethanol or methanol at various points in time after infection. Fixed cell monolayers are incubated with antiserum against either poliovirus proteins or against pre-S1 or pre-S2 antigens, as appropriate. The plates are washed and then incubated with a secondary antibody (e.g., goat anti-rabbit IgG) conjugated to HPR. The plates are washed again, and then an NRP substrate, 3,3'-diaminobenzidine tetrachloride (Sigma) is added. Cells that express proteins that cross-react with antibodies are stained.

 Monolayers V of his cells are infected with a recombinant virus. Cell lysates can be examined by immunoprecipitation or Western blotting for the presence of virus-encoded proteins. For immunoprecipitation, cell lysates are incubated with an antiserum against poliovirus or exogenous protein. Protein A is then added to Sepharose (Sigma) to the mixture, and incubation continues. The sepharose beads are centrifuged, washed and eluted with SDS by dissociation buffer. Precipitated proteins can be analyzed by SDS-polyacrylamide gel electrophoresis. For western blotting, cell lysates were separated using SDS-PAGE, transferred to nitrocellulose and incubated with antiserum. Blots are washed, incubated with a secondary antibody (e.g., goat anti-rabbit JgG) conjugated to HRP. Blots are washed again, and then HPP substrate, 4-chloro-1-naphthol (Bio Rad, Richmond, CA) plus hydrogen peroxide are added to identify cross-reacting proteins.

Example 9
Construction of a recombinant poliovirus containing rotavirus VR7 gene.

The expression clone designated pLED 3.2 / VP7 was constructed, the gene encoding the rotaviruono VP7 antigen being inserted into the poliovirus genome. The clone contains the full length gene for VR7, whose length is approximately 1 Kb. This clone is constructed using overlap PCR extension, and the gene is inserted at the beginning of the poliovirus open reading frame (base 743). For the first round of PCR, primers P and Q are used (Table 2). Matrix DNA is a plasmid clone containing the full-length rotavirus VR7 gene. The PCR product from this amplification has a length of 1012 base pairs. The conditions of the PCR cycle for the first round of reactions differ from the standard conditions specified in example 6, and are: 94 ^o With 2 minutes, ice 2 minutes, then 94 ^o With 1 minute, 26 ^o With 1 minute, 70 ^o With 2 minutes in for 10 cycles; 94 ^o With 1 minute, 45 ^o With 1 minute, 70 ^o With 2 minutes for 20 cycles.

 The second round of PCR reactions is carried out as described previously. A fragment of the first round of PCR is mixed with either 5 ′ LED 3.2 or 3 ′ LED 3.2 and amplified with primers C and Q for 5 ′ fusion, or with primers P and F for 3 ′ fusion. The resulting fusion products are digested with M1u1 and Bge11 for 5 ′ fusion or Bge11 and Avr11 for 3 ′ fusion. These digests are then subcloned into pLED 3.2 to obtain pLED 3.2 / P7.

 The DNA sequence of this full length fusion construct is determined using an Applied Biosystems DNA sequencer (Foster City, CA) 370A.

 Then transcription and transfection reactions are carried out. RNA transcripts obtained from the T7 promoter are transfected into its V cells to create an infectious poliovirus. For these experiments, approximately 5 μg pLED 3.2 / V P7 was prepared to create RNA transcripts by digestion with the Pvu1 plasmid. Pvu1 digestion results in two fragments, and after a larger fragment containing the T7 promoter, the full length follows a polyogen containing V P7 gene fusion. The product of the digestion reaction is extracted with phenol and precipitated with ethanol. Precipitated DNA is resuspended in 20 μl of water. Full-length RNA transcripts are synthesized in vitro from Pvu1-digested DNA using T7 RNA polymerase (Moss, E. G., et al., J. Virol., 63: 1884-1890 (1989)). Transcription products are analyzed by agarose gel electrophoresis.

Confluent 25 cm ² monolayers of V cells are transfected with transcripts using a DEAE transfection scheme (Vander Werf. S. Proc. Natl. Acad. Sci. USA., 83: 2330-2334 (1986)). Approximately 25 μg of RNA is mixed with DEAE-dextran (0.5 mg / ml) and then poured over V of its cells. After 30 minutes of incubation at room temperature, the inoculum is removed and the cells are washed, fresh, modified Earl support medium is added, and the cells are incubated at 33.5 ^° C. The cultures are incubated until a complete cytopathic effect is observed, and recombinant polioviruses are harvested from the culture medium.

 Viruses containing a recombinant poliovirus obtained from the pLED 3.2 / VR7 construct are titrated in plaque assays on its V cells. In addition, RNA was extracted from the recombinant virus and analyzed by reverse transcription and PCR using the Gene Amp PCR kit. thermostable rTh reverse transcriptase RNA (Aperkin / Elmer / Cetus). The results show that the VR7 gene is stably preserved in the recombinant pLED 3.2 / VR7 virus during cultivation.

 Various methods are used to analyze the expression of VR7 from a poliovirus vector. These methods, which are standard procedures, include immunoperoxidase staining of infected cells, immunoprecipitation of viral and foreign proteins, Western blotting and dot blotting (Coligan J. E., et al. Current Protocols in Jmmunology, johu Wiley and Sons (1992) )

 An immunoperoxidase staining assay is used to determine the proteins in virus V-infected cells. V cells are infected with the virus and fixed with ethanol or methanol at various points after infection. Fixed cell monolayers are incubated with antiserum against either poliovirus proteins or against VR7. The plates are washed and then incubated with a secondary antibody (e.g., goat, anti-rabbit IgG) conjugated to horseradish peroxidase (HRP). The plates are washed again, and then add NRP substrate, 3.3'-diaminobenzidine tetrahydrochloride (Sigma, San Luis, MO). Cells that express proteins that cross-react with antibodies are stained.

 Monolayers V of his cells are infected with a recombinant virus. Cell lysates can be examined by immunoprecipitation or western blotting for the presence of virus-encoded proteins. For immunoprecipitation, cell lysates are incubated with an antiserum against polioviruses or an exogenous protein, then Protein A Sepharose (Sigma) is added to the mixture and incubation continues. The sepharose beads are centrifuged, washed and eluted with SDS dissociation buffer. Precipitated proteins can be analyzed by SDS-polyacrylamide gel electrophoresis. For western blotting, cell lysates are separated by SDS-PAGE, transferred to nitrocellulose and incubated with antiserum to obtain proteins of interest. Blots are washed, incubated with a secondary antibody (e.g., goat anti-rabbit JgG) conjugated to HRP. The blots are washed again, and an HPR substrate, 4-chloro-1-naphthol (Bio Rad, Richmond, CA) is added to identify cross-reacting proteins.

Example 10
Construction of poliovirus polylinker cassette vectors
Exogenous genes can also be inserted into the poliovirus genome by constructing two poliovirus cloning vectors, similar to those described in Examples 1-5, in that these vectors embed unique cloning sites of restriction enzymes, each at a different location within the poliovirus genome. Such a multilinker cassette vector technique provides a simpler cloning procedure than the method of example 6, although the latter limits the addition of additional amino acids that are not in the original exogenous polypeptide.

 The first vector (vector 2) contains a polylinker cassette in a frame with a poliovirus protein, immediately followed by the AUG initiating codon at base 743. The following restriction sites are then introduced: No1, Hpa1 and Pac1. They follow immediately after the sequence encoding the polyo 3C protease recognition site. This sequence is then adapted by adding or deleting 1-2 additional bases to maintain a correct open reading frame in this area.

 Overlapping PCR extensions are used to embed the restriction sites during vector construction. To construct vector 1, primers J and K are synthesized (Table 2) for introducing a polylinker cassette at base 743. In the first round of PCR reactions, pLED 3.2 is used as template DNA, primers C and J for the 5 'region, and primers K and F for 3 'plot. The expected sizes for the amplified fragments are 512 and 551 base pairs for 5 'and 3' sections, respectively. These fragments are used directly from the PCR reaction, and they serve as a matrix for the second round of PCR with primers C and F. A fragment of 1016 base pairs is obtained which contains a polylinker sequence and a 3C protease recognition site in the middle of the fragment. This fragment is purified, M1u1 and Avr11 are digested and subcloned into pLED 3.2, which was digested with the same restriction enzymes.

 Proteolytic processing of poliovirus polyprotein occurs at many stages during virus replication. The initial proteolytic cleavage is carried out due to 2A protease, which leads to the formation of proteins designated P1 and P2-P3. Each of them is further cleaved with a 3C protease to produce individual viral proteins. During proteolytic processing of polyprotein, partial cleavage products have been shown to have functions that differ from those of the final cleavage products (Harris, K. S. et al., Semin. Virol., 1: 323-333 (1990)). The incorporation of an exogenous gene into a polyprotein at sites encoding functional precursors does not create viable viruses. The second cassette vector is designed to incorporate restriction sites through the connection between P1 and P2 (base 3377).

 The restriction sites No1, Hpa1 and Pac1 are inserted. For these restriction sites, there is a 2A 5 ′ protease recognition site: the 3C protease recognition site is inserted in the 3 ′ direction to these sites. The first round of PCR reactions uses pLED 3.2 as template DNA, primers L and M for the 5 ′ region, and primers N and O for the 3 ′ region (Table 2).

 The expected sizes for the amplified fragments are 563 and 692 bp for 5 'and 3', respectively. These fragments are used directly from the PCR reaction, and they serve as a matrix for the second round of PCR with primers L and O. The resulting fragment is 1096 bp long and contains a 2A protease recognition site, a polylinker sequence and a 3C protease recognition site in the middle. This fragment is then digested with BstE11 and subcloned into pLED3.2 digested with the same enzyme.

 Cassette vectors are used as expression vectors for exogenous genes. Exogenous genes are cloned into cassette vectors, either using existing compatible restriction sites, or by adding such reotriction sites to the ends of the gene using PCR. In the latter case, the exogenous gene is amplified by PCR using primers corresponding to the 5 'and 3' ends of the gene. If these primers are synthesized, a restriction site is added to the 5 ′ end of the sense primer, and a second restriction site is added to the 5 ′ end of the antisense primer. After amplification of the gene in a standard PCR reaction, the resulting PCR fragment will contain a gene flanked by two restriction sites. Both the vector and the PCR fragment are both digested with two restriction enzymes and then ligated together, resulting in an expression clone of interest.

 It should be noted that primers can be designed so that they match any region of the exogenous gene of interest, so that partial genes can be included in the vector. Moreover, the digestion of the vector Hp1 leads to the obtuse ends of the fragments. Therefore, any fragment with blunt ends of an exogenous gene can be cloned into a vector.

Example 11
Construction of a recombinant poliovirus containing B. pertussis 69 kD external membrane protein gene.

 The poliovirus cassette vectors of Example 10 were used to express 69 kD of the B. pertussis outer membrane protein. The mature protein is encoded within a gene region of approximately 1.8 kD. This region, or shorter sections of the open reading frame, can be amplified by PCR with primers of specific sequences. These primers are designed to contain a Not1 site from the 5 ′ end of the antisense primer. Primers must be selected so that the open reading frame and the recognition site of the poliovirus 3C protease are framed. After amplification using PCR, the resulting fragment was purified by agarose gel electrophoresis or using Magic PCR kit (Promega), Not1 and Prac1 were digested and ligated into a vector that was restricted with the same enzymes. The resulting plasmid is designated pLED 3.2 / 69 kD. The DNA sequence of this full length poly-fusion construct is determined using the 370A Applied Biosystem DNA sequencer (Foster City, CA).

 Next, transcription and transfection reactions are carried out. RNA transcripts obtained from the T7 promoter are transfected into its V cells to create an infectious poliovirus. Approximately 5 μg pLED 3.2 / 69 kD was prepared for these experiments to generate RNA transcripts by digestion with the Pvu1 plasmid. Pvu1 digestion results in two fragments, the larger fragment containing the T7 promoter followed by the full length polyogen containing 69 kD gene fusion. The product of the digestion reaction is extracted with phenol and precipitated with ethanol. The precipitated DNA is re-suspended in 20 μl of water. Full-length RNA transcripts are synthesized in vitro from Pvu1-digested DNA using T7 RNA polymerase (Moss, E. G. et al. J Virol 63: 1884-1890 (1989)). Transcription products are analyzed electrophoretically on an agarose gel.

Confluent 25 cm ² monolayers of V cells are transfected using transcripts using a DEAE transfection scheme (Van der Werf, S. Proce Natl. Acade Sci., USA, 83: 2330-2334 (1986)). Approximately 25 μg of RNA is mixed with DEAE-dextran (0.5 μg / ml), and then poured onto V cells. After 30 minutes of incubation at room temperature, the inoculum was removed and the cells were washed, fresh Earle support medium was added and the cells were incubated at 33.5 ^° C. The cultures were incubated until a complete cytopathic effect was observed, and recombinant polioviruses were collected from the culture medium.

 The original viruses containing recombinant polioviruses obtained from the pLED 3.2 / 69 kD construct are titrated in plaque assays on its V cells. In addition, RNA was extracted from the recombinant virus and analyzed by reverse transcription and PCR using the Gene Amp PCR kit of thermostable rTkh reverse transcriptase RNA (Perkin - Elmer / Cetus). The result shows that the 69 kD gene is stably preserved in the recombinant pLED 3.2 / 69 kD virus during cultivation.

 Various methods are used to analyze the expression of 69 kD of the outer membrane protein from the poliovirus vector. These methods, which are standard procedures, include immunoperoxidase staining of infected cells, immunoprecipitation of viral and foreign proteins, western blotting and dot blotting (Coligan, J. E. et al., Current Protocols in Jmmunology, Jonn Wiley and Sons (1992)).

 An immunoperoxidase staining assay is used to determine the proteins in V cells infected with the virus, V cells are infected with the virus and fixed with ethanol or methanol at various points in time after infection. Fixed cell monolayers are incubated with antiserum against either poliovirus proteins or 69 kD of an external membrane protein. The plates are washed and then incubated with a secondary antibody (e.g., goat anti-rabbit JgG) conjugated to horseradish peroxidase (HRP). The plates are washed again and then HRP substrate, 3,3'-diaminobenzidine tetrahydrochloride (Sigma, San Luis, MO) is added. Cells that express proteins that cross-react with antibodies are stained.

 V ego cell monolayers are infected with a recombinant virus. Cell lysates can be examined by immunoprecipitation or western blotting for viral encoded proteins. For immunoprecipitation, cell lysates are incubated with an antiserum against poliovirus or exogenous protein. Protein A Sepharose (Sigma) is added to the mixture and continued to incubate. The sepharose beads are centrifuged, washed and eluted with SDS by dissociation buffer. Precipitated proteins are analyzed by SDS-polyacrylamide gel electrophoresis. For western blotting, cell lysates are isolated by SDS-PAGE, transferred to nitrocellulose and incubated with antiserum for the proteins of interest. Blots are washed, incubated with a secondary antibody (e.g., goat anti-rabbit JgG) conjugated to HRP. Blots are washed again, and then the NRP substrate, 4-chloro-1-naphthol (Bio Rad, Richmond, CA) plus hydrogen peroxide, is added to identify cross-reactive proteins.

Example 12
Construction of a recombinant poliovirus containing the Herpes Simplex glycoprotein D gene.

 The poliovirus cassette vectors of Example 10 are used to express Herpes Simplex virus D glycoprotein D. This protein is encoded by a gene of approximately 1.2 kD. This region or shorter regions of the open reading frame can be amplified by PCR with primers of specific sequences. These primers are designed to contain a Not1 site from the 5 ′ end of the sense primer, and a Pac1 site from the 5 ′ end of the antisense primer. These primers should be selected such that the open reading frame and the poliovirus 3C protease recognition site are framed. After amplification by PCR, the resulting fragment was purified by agarose gel electrophoresis, or using Magic PCR kit (Promega), Not1 or Pac1 was digested and ligated into a vector that was restricted with the same enzymes.

 The resulting plasmid is designated pLED 3.2 / gD. The DNA sequence of this full length poly-fused construct is determined using a DNA sequencer 370A Applied Bioystems (Foster City, CA).

 Then transcription and transfection reactions are carried out. RNA transcripts obtained from the T7 promoter are transfected into its V cells to create an infectious poliovirus. For these experiments, approximately 5 μg pLED 3.2 / gD was prepared to create RNA transcripts by digestion with the Pvu1 plasmid. Pvu1 digestion results in two fragments, and after a larger fragment containing the T7 promoter, the full length follows a polyogen containing gD gene fusion. The product of the digestion reaction is extracted with phenol and precipitated with ethanol. The precipitated DNA was resuspended in 20 μl of water. Full-length RNA transcripts are synthesized in vitro from Pvu1-digested DNA using T7 RNA polymerase (Moss E.G., et al., J. Virol., 63: 1884-1890 (1989)). Transcription products are analyzed by agarose gel electrophoresis.

Confluent 25 cm ² monolayer V of his cells is transfected with transcripts using a DEAE transfection scheme (Vander Werf 83: 2330-2334 (1986)). Approximately 25 μg of RNA is mixed with DEAE-dextran (0.5 mg / ml), and then poured over its V cells. After incubation for 30 minutes at room temperature, the inoculum was removed and the cells were washed. Fresh modified Earl support medium was added and the cells were incubated at 33.5 ^° C. The cultures were incubated until a complete cytopathic effect was observed, and recombinant poliovirus was collected from the culture medium.

 Viral material containing a recombinant poliovirus obtained from the pLED 3.2 / gD construct is titrated in a plaque assay on its cells. In addition, RNA was extracted from the recombinant virus and analyzed by reverse transcription and PCR using the Gene Amp PCR kit with thermostable rTh reverse transcriptase RNA (Perkin-Elmer / Cetus). The results show that the gD gene is stably preserved in the recombinant pLED 3.2 / gD virus during cultivation.

 Various methods are used to analyze gD expression from a poliovirus vector. These methods, which are standard procedures, include immunoperoxidase staining of infected cells, immunoprecipitation of viral and foreign proteins, western blotting and dot blotting (Coligan, J.E., et al., Eds. Current. Protocols in Jmmunology john Wiley and Sons (1992)).

 Immunoperoxidase staining analysis is used to determine the protein in virus-infected V cells. V cells are infected with viruses and fixed with ethanol or methanol at various points in time after ignoring. Fixed cell monolayers are incubated with antiserum against either poliovirus proteins or gD. The plates are washed and then incubated with a secondary antibody (such as goat or anti-rabbit IgG) conjugated to horseradish peroxidase (HRP). The plates were washed again and HRP substrate, 3,3'-diaminobenzidine tetrahydrochloride (Sigma) was added. Cells that express proteins that cross-react with antibodies are stained.

 Monolayers of V ego cells are infected with a recombinant virus. Cell lysates can be examined by immunoprecipitation or by Western blotting for viral encoded proteins; for immunoprecipitation, cell lysates are incubated with antiserum against poliovirus or exogenous protein. Protein A sepharose (Sigma) is then added to the mixture, and incubation continues. The sepharose beads are centrifuged, washed and eluted with SDS by dissociation buffer. Precipitated proteins can be analyzed by SDS-polyacrylamide gel electrophoresis. For western blotting, cell lysates are separated by SDS-PAGE, transferred to nitrocellulose and incubated with antiserum for the proteins of interest. Blots are washed, incubated with a secondary antibody (such as goat anti-rabbit IgG) conjugated to HRP. The blots are washed again and then the NRP substrate, 4-chloro-1-naphthol (Bio Rad, Richmond, CA) plus hydrogen peroxide is added to identify cross-reacting proteins.

Equivalents
Specialists can find out, or using routine experimentation, determine the equivalents of specific variants of the invention. These equivalents are included in the scope indicated by the claims.

Claims (36)

1. Recombinant poliovirus vector designed to stimulate an immune response against a pathogenic microorganism, or virus, or target cell, having the formula
C-B-A-B-C,
where A is an exogenous nucleotide sequence encoding an exogenous polypeptide to be expressed selected from the group consisting of the β subunit of the cholera vibrio toxin, the fimbria cholera vibrio toxin regulated, the VP4 rotavirus capsid protein, the influenza virus hemagglutinin, the hepatitis B virus antigen Hepatitis B virus S1 antigen, hepatitis B virus pre-S2 antigen, 69 kDa B. pertussis outer membrane protein, herpes simplex virus D glycoprotein D and rotavirus VP7 antigen;
B is a nucleotide sequence encoding an artificial proteolytic cleavage site for a protease that proteolytically processes a protein precursor produced by the original poliovirus modified to produce a recombinant poliovirus, such as poliovirus proteolytic cleavage site 2A and / or 3C;
C represents the genome of the original poliovirus, as shown in the pMOVl sequence. 3, pMOV2.1, pMOV2.5 or pMOV3.1 in FIG. 2B modified to produce a recombinant poliovirus,
where A and B are embedded in C at the junction between Vpl and 2A, or between 2A and 2B, or between 2C and 3A of the original poliovirus genome.  2. The recombinant poliovirus vector according to claim 1, characterized in that the exogenous nucleotide sequence A and the nucleotide sequence encoding the artificial site of proteolytic cleavage B are present in the recombinant genome in the following order: 5'-untranslated region of the original virus - a unique starting codon (codons ) the source virus - the initiating codon (s) of the translated portion of the source virus - the nucleotide sequence encoding the artificial site of proteolytic cleavage Ogen nucleotide sequence - nucleic acid sequence encoding an artificial proteolytic cleavage site - remainder of the source of the viral genome.  3. The recombinant poliovirus vector according to claim 2, characterized in that the genome of the original poliovirus is the genome of the Mahoney poliovirus or its derivative.  4. The recombinant poliovirus vector according to claim 1, characterized in that it is a poliovirus, where the exogenous nucleotide sequence A and nucleotide sequences encoding the artificial proteolytic cleavage sites for the poliovirus 3C protease or poliovirus 2A protease B are present in the recombinant poliovirus genome in the following order: 5'-untranslated region of the poliovirus genome - unique poliovirus start codon - encoding poliovirus protein region (s) - artificial site cognition 3S- or 2A protease - exogenous nucleic acid sequence - artificial recognition site 3S- or 2A protease - the rest of the poliovirus.  5. The recombinant poliovirus vector according to claim 4, characterized in that the genome of the original poliovirus is the genome of the Sabin poliovirus or its derivative.  6. The recombinant poliovirus vector according to claim 5, characterized in that the Sabin poliovirus is selected from the group consisting of Sabin poliovirus type 1, poliovirus Sabin type 2 and poliovirus Sabin type 3. 7. Recombinant poliovirus vector designed to stimulate an immune response against a pathogenic microorganism, or virus, or target cell, having the formula
C-A-B-C,
where A is an exogenous nucleotide sequence encoding an exogenous polypeptide to be expressed selected from the group consisting of the β subunit of the cholera vibrio toxin, the fimbria cholera vibrio toxin regulated, the VP4 rotavirus capsid protein, the influenza virus hemagglutinin, the hepatitis B virus antigen Hepatitis B virus S1 antigen, hepatitis B virus pre-S2 antigen, 69 kDa B. pertussis outer membrane protein, herpes simplex virus D glycoprotein D and rotavirus VP7 antigen;
B is a nucleotide sequence encoding an artificial proteolytic cleavage site for prostheses that proteolytically processes a protein precursor produced by a parent poliovirus modified to produce a recombinant poliovirus, such as poliovirus proteolytic cleavage site 2A and / or 3C;
C represents the genome of the original poliovirus modified to produce a recombinant poliovirus, where A and B are inserted into C at a location in the genome of the original poliovirus so that they do not violate the viral sequence necessary for replication of the poliovirus,
where the recombinant poliovirus vector has the sequence shown in FIG. 1A.  8. The recombinant poliovirus vector according to claim 7, characterized in that the exogenous nucleotide sequence A and the nucleotide sequence encoding the artificial proteolytic cleavage site for the poliovirus 3C protease or poliovirus 2A protease B are present in the recombinant poliovirus genome in the following order: 5 ' - non-translated portion of the poliovirus genome - a unique poliovirus start codon - exogenous sequence A - a sequence encoding an artificial proteolytic site In the first digestion - the second codon of the original poliovirus genome - remainder of the poliovirus genome of the original.  9. The recombinant poliovirus vector according to claim 8, characterized in that the genome of the original poliovirus is the genome of the Sabin poliovirus or its derivative.  10. The recombinant poliovirus vector according to claim 9, characterized in that the Sabin poliovirus is selected from the group consisting of Sabin poliovirus type 1, poliovirus Sabin type 2 and poliovirus Sabin type 3.  11. The recombinant poliovirus vector according to claim 8, characterized in that the genome of the original poliovirus is the genome of the Mahoney poliovirus or its derivative.  12. The method of producing the recombinant poliovirus vector according to claim 1, capable of stimulating an immune response against a pathogenic microorganism, or virus, or target cell, characterized in that it is carried out a) obtaining a poliovirus, which in its natural life cycle produces a polyprotein precursor that is proteolytically is being processed; b) inserting into the poliovirus genome a) (1) an exogenous nucleotide sequence encoding the polypeptide to be expressed, and (2) a nucleotide sequence encoding an artificial proteolytic cleavage site for a protease that proteolytically processes the protein precursor produced by the poliovirus obtained in stage a), where b (1) and b (2) are inserted into the genome of poliovirus a) in such a place that they do not violate the viral sequence necessary for the replication of poliovirus.  13. The method according to p. 12, characterized in that the nucleotide sequence b (1) encodes a polypeptide antigen selected from the group consisting of the β subunit of the cholera vibrio toxin, the toxin regulated cholera vibrio toxin, the rotavirus capsid protein VP4, influenza virus hemagglutinin, S hepatitis B virus antigen, pre-S1 hepatitis B virus antigen, hepatitis B pre-S2 antigen, B. pertussis outer membrane protein with a mass of 69 kDa, herpes simplex virus D glycoprotein and rotavirus antigen VP7.  14. The method according to p. 12, characterized in that the exogenous nucleotide sequence b (1) and the nucleotide sequence encoding the artificial site of proteolytic cleavage for poliovirus 3C protease or poliovirus 2A protease b (2) are present in the recombinant poliovirus genome in the following order: 5'-untranslated region of the original poliovirus genome - unique poliovirus start codon - exogenous nucleotide sequence b (1) - nucleotide sequence encoding an artificial site of prot the eolytic cleavage b (2) - the second codon of the original poliovirus genome - the rest of the original poliovirus genome.  15. The method according to p. 14, characterized in that the nucleotide sequence b (1) encodes a polypeptide antigen selected from the group consisting of the β subunit of the cholera vibrio toxin, the toxin regulated cholera vibrio toxin, the rotavirus capsid protein VP4, the influenza virus hemagglutinin, S hepatitis B virus antigen, pre-S1 hepatitis B virus antigen, hepatitis B pre-S2 antigen, B. pertussis outer membrane protein with a mass of 69 kDa, herpes simplex virus D glycoprotein and rotavirus antigen VP7.  16. The method according to p. 12, characterized in that the exogenous nucleotide sequence b (1) and the nucleotide sequence encoding the artificial site of proteolytic cleavage b (2) are located in the poliovirus genome selected from the group consisting of 1) a compound between Vpl and 2A; 2) connections between 2A and 2B and 3) connections between 2C and 3A.  17. The method according to p. 16, wherein the nucleotide sequence b (1) encodes a polypeptide antigen selected from the group consisting of the β subunit of the cholera vibrio toxin, the fimbria cholera vibrio toxin regulated toxin, the VP4 rotavirus capsid protein, the influenza virus hemagglutinin, S hepatitis B virus antigen, pre-S1 hepatitis B virus antigen, hepatitis B pre-S2 antigen, B. pertussis outer membrane protein with a mass of 69 kDa, herpes simplex virus D glycoprotein and rotavirus antigen VP7.  18. The method according to p. 16, wherein the nucleotide sequence b (2), which encodes an artificial proteolytic cleavage site, encodes a proteolytic cleavage site for poliovirus 3C protease or a proteolytic cleavage site for poliovirus 2A protease.  19. The method for producing the recombinant poliovirus vector according to claim 7, characterized in that the exogenous nucleotide sequence and the nucleotide sequence encoding the artificial site of proteolytic cleavage are present in the recombinant genome in the following order: 5'-untranslated portion of the poliovirus genome - a unique starting codon of the poliovirus - exogenous nucleotide sequence - nucleotide sequence encoding an artificial site of proteolytic cleavage - second codon gender Iovirus - the rest of the poliovirus genome.  20. The method according to p. 19, wherein the exogenous nucleotide sequence encodes a polypeptide antigen selected from the group consisting of the β subunit of the cholera vibrio toxin, the toxin regulated cholera vibrio toxin, the rotavirus capsid protein VP4, the influenza virus hemagglutinin, the S virus antigen hepatitis B, pre-S1 hepatitis B virus antigen, pre-S2 hepatitis B virus antigen, B. pertussis outer membrane protein of 69 kDa mass, herpes simplex virus D glycoprotein and rotavirus antigen VP7.  21. The method according to p. 19, characterized in that the nucleotide sequence encoding an artificial site of proteolytic cleavage, encodes an artificial site of proteolytic cleavage for poliovirus 3C protease or an artificial site of proteolytic cleavage for poliovirus 2A protease.  22. The method according to p. 21, characterized in that the poliovirus is Sabin poliovirus or Mahoney poliovirus.  23 The method according to p. 19, characterized in that the exogenous nucleotide sequence and the nucleotide sequence encoding the artificial proteolytic cleavage site for poliovirus 3C protease or poliovirus 2A protease are present in the recombinant poliovirus genome in the following order: 5'-untranslated portion of the original poliovirus genome - a unique poliovirus start codon - an exogenous nucleotide sequence - a nucleotide sequence encoding an artificial proteolytic site cleavage - the second codon of the original poliovirus genome - the rest of the original poliovirus genome.  24. The method according to p. 23, wherein the exogenous nucleotide sequence encodes a polypeptide antigen selected from the group consisting of the β subunit of the cholera vibrio toxin, the toxin regulated cholera vibrio toxin, the rotavirus capsid protein VP4, the influenza virus hemagglutinin, the S virus antigen hepatitis B, pre-S1 hepatitis B virus antigen, pre-S2 hepatitis B virus antigen, B. pertussis outer membrane protein of 69 kDa mass, herpes simplex virus D glycoprotein and rotavirus antigen VP7.  25. The method according to p. 19, characterized in that the exogenous nucleotide sequence and the nucleotide sequence encoding the artificial site of proteolytic cleavage are located in the poliovirus genome selected from the group consisting of 1) the connection between Vpl and 2A; 2) connections between 2A and 2B and 3) connections between 2C and 3A.  26. The method according to p. 25, wherein the exogenous nucleotide sequence encodes a polypeptide antigen selected from the group consisting of the β subunit of the cholera vibrio toxin, the toxin regulated cholera vibrio toxin, rotavirus capsid protein VP4, influenza virus hemagglutinin, S-antigen hepatitis B, pre-S1 hepatitis B virus antigen, pre-S2 hepatitis B virus antigen, B. pertussis outer membrane protein of 69 kDa mass, herpes simplex virus D glycoprotein and rotavirus antigen VP7.  27. The method of claim 25, wherein the nucleotide sequence that encodes an artificial proteolytic cleavage site encodes a proteolytic cleavage site for poliovirus 3C protease or a proteolytic cleavage site for poliovirus 2A protease.  28. A method of inducing an immune response in an individual against a pathogen, characterized in that the method comprises administering to the individual a recombinant poliovirus produced using the recombinant poliovirus vector according to claim 1, wherein the exogenous nucleotide sequence A encodes a pathogen antigen in an amount sufficient to induce an immune response from the individual.  29. A method of inducing an immune response in an individual against a pathogen, characterized in that the method comprises administering to the individual a recombinant poliovirus produced using the recombinant poliovirus vector according to claim 7, wherein the exogenous nucleotide sequence A encodes a pathogen antigen in an amount sufficient to induce an immune response from the individual.  30. The method according to p. 29, characterized in that the method comprises administering to the individual a recombinant poliovirus produced using the recombinant poliovirus vector according to claim 8, wherein the exogenous nucleotide sequence A encodes a pathogen antigen in an amount sufficient to induce an immune response in an individual .  31. A method for producing a protein, characterized in that a) the incorporation of a recombinant poliovirus produced by the recombinant poliovirus vector according to claim 1 is carried out in a suitable host cell; b) maintaining the product under conditions that allow the virus to replicate according to claim 1; and c) isolating the protein from the host cell.  32. The method according to p. 31, characterized in that the host cells are human cells.  33. A method for producing a protein, characterized in that a) the incorporation of a recombinant poliovirus produced by the recombinant poliovirus vector according to claim 7 is carried out in a suitable host cell; b) maintaining the product under conditions that allow the virus to replicate according to claim 7; and c) isolating the protein from the host cell.  34. A vaccine composition for the treatment or prevention of infectious diseases, such as cholera, gastroenteritis, influenza, hepatitis B, pertussis and herpes simplex, characterized in that it contains a recombinant poliovirus produced using the recombinant poliovirus vector according to claim 1, and is physiologically acceptable carrier.  35. A vaccine composition for the treatment or prevention of infectious diseases, such as cholera, gastroenteritis, influenza, hepatitis B, pertussis and herpes simplex, characterized in that it contains the recombinant poliovirus produced using the recombinant poliovirus vector according to claim 7, and is physiologically acceptable carrier.  36. The recombinant poliovirus vector described in paragraph 1 or 7, to obtain a recombinant poliovirus applicable in the treatment of infectious diseases such as cholera, gastroenteritis, influenza, hepatitis B, pertussis and herpes simplex.  37. The recombinant poliovirus vector, characterized in paragraph 1 or 7, to obtain a recombinant poliovirus applicable in the induction of an immune response in an individual against a pathogen such as cholera vibrio, rotavirus, influenza virus, hepatitis B virus, Bordetella pertussis, herpes simplex virus moreover, the exogenous nucleotide sequence A encodes an antigen of the specified pathogen.

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