MXPA00004707A - Alphavirus vectors - Google Patents

Abstract

A modified alphavirus expression vector is provided wherein at least one optimal heterologous splice site is introduced to the alphavirus replicon to prevent aberrant splicing of the alphavirus, which may be Semliki Forest virus following administration of the vector to a host.

Description

VECTORS OF ALFAVIRUS FIELD OF THE INVENTION The present invention relates to the field of DNA vaccines and is particularly related to modified alphavirus vectors that are used in these vaccines.

BACKGROUND OF THE INVENTION The Semliki Forest virus (SFV) is a member of the genus Alphavirus in the Togaviridae family. The mature virus particle contains a single copy of a ssRNA genome with a positive polarity that is capped at the 5 'end and polyadenylated at the 3' end. It works like a messenger RNA and naked RNA can initiate an infection by entering the cells. During infection / transfection, two thirds of the genome is translated into a polyprotein that is processed to form four non-structural proteins (nsP1 to 4) by self-cleavage. Once the ns proteins have been synthesized, they are responsible for replicating the more (42S) chain genome in less full-length chains (reference 14). These less strings later serve as templates for the synthesis of new chain genomes plus (42S) and subgenomic mRNA 26S (reference 1 - throughout this application several references will be cited in parentheses in order to more fully describe the state of the technique to which the invention belongs The complete bibliographic information of each citation is found at the end of the specification The expositions of these references are incorporated here as reference for the same). This subgenomic mRNA, which is collinear with the last third of the genome, codes for the structural proteins of SFV. In 1991, Liljestrom and Garoff (ref 2) designed a series of expression vectors based on the SFV cDNA replicon. These vectors had the deletion of genes of structural protein of the virus to find the form towards heterologous inserts, but the non-structural coding regions were preserved for the production of the replicase complex nsPl up to 4. The short sequence elements 5 'and 3' required for RNA replication were also preserved. A polylinker site was inserted 3 'from the 26S promoter, followed by translation stop sites in all three frames. A Spel site was inserted just after the 3 'end of the SFV cDNA for linearization of the plasmid, for use in in vitro transcription reactions. The injection of SFV RNA encoding a heterologous protein has been shown to result in the expression of the foreign protein and the induction of antibodies in several studies (references 3, 4). The use of SFV RNA inoculation to express foreign proteins for the purpose of immunization would have several advantages associated with the immunization of plasmid DNA. For example, SFV RNA coding for a viral antigen could be introduced in the presence of antibodies to that virus, without loss of potency to neutralization by antibodies to the virus. Also, because the protein is expressed in vivo, the protein must have the same conformation as the protein expressed by the virus itself. Therefore, existing concerns about conformational changes that may occur during the purification of the protein, leading to a loss of immunogenicity, protective epitopes and possible immunopotentiation, could be prevented by immunization with plasmid DNA. In WO95 / 27044, the disclosure of which is incorporated herein by reference, the use of alphavirus cDNA vectors based on the cDNA complementary to the alphavirus RNA sequence is described. Once transcribed from the cDNA under the transcriptional control of a heterologous promoter, the alphavirus RNA is able to self-replicate by means of its own P1065 replicase and thus amplify the copy number of the transcribed recombinant RNA molecules.

SUMMARY OF THE INVENTION The present invention relates to modifications in the alphavirus cDNA vectors described in the aforementioned document WO 95/27044, to allow a reinforced replication of the alphavirus. In this invention, the heterologous binding site is introduced into the alphavirus replicon sequence, particularly that of Semliki Forest virus (SFV). Accordingly, in one aspect of the present invention, there is provided an expression vector comprising a DNA molecule complementary to at least part of an alphavirus RNA genome, the DNA molecule comprising the complement of the entire genome regions of alphavirus RNAs that are essential for the replication of alphavirus RNA and further comprises a heterologous DNA sequence capable of being expressed in a suitable host, for example a human or animal host, the heterologous DNA sequence is inserted within a region of the DNA molecule that is not essential to the replication thereof and the DNA molecule is placed under the transcriptional control of a functional promoter sequence for that animal or human host, wherein at least one heterologous binding site is provided in the molecule of DNA to prevent binding of aberrant alphavirus RNA. The alphavirus molecule is a large molecule and, consequently, there is a high probability of cryptic binding sites, thus damaging the replication of the alphavirus and therefore its ability to express the heterologous DNA is damaged. By introducing at least one optimal heterologous binding site according to the present invention, in the alphavirus replicon sequence, any binding is likely to be directed to the heterologous binding site and not to other cryptic binding sites, restoring the function of the replicon SFV when it is removed and can improve RNA transport from the nucleus (reference 6). In the constructs provided herein, the promoter is positioned towards the 5 'end of the alphavirus sequence, such that the resulting transcript has a true 5' end, which is required for efficient replication of the alphavirus RNA replicon. In addition, a ribozyme sequence of hepatitis delta virus can be provided at the 3 'end of the Semliki Forest virus segment to ensure adequate cleavage in vivo at the 3' end of the sequence. Any other convenient sequence can be used to achieve this effect. The sequence of the heterologous binding site can be provided by the nucleotide sequence of rabbit ß-globin intron II, as described in reference 5. This sequence of the heterologous binding site can be inserted into the complementary sequence at any convenient location that generate perfect union joints. This prevents the replication of the alphavirus, unless it is authentically removed by the union. I have identified five suitable sites in the SFV replicon that are contained within an EcoRV-Spel fragment of the replicon that is 8010 base pairs in length (Figure 3). The first of these sites is a Ppu-MI site at position 2719 within the EcoRV-Spel fragment. In constructing the modified vectors provided herein, the EcoRV-Spel fragment is cut with Ppu-MI at position 2719 and a blunt end is produced with mung bean nuclease, which removes three bases from the SFV sequence. A blunt-end ß-globin intron II of 536 base pairs is ligated to the site and replaces the three missing bases with the sequence added to the 3 'end of the ß-globin intron sequence (Figure 1). The other four sites suitable for insertion of the Intron are the PvuII sites in base pairs 2518, 3113, 6498 and 6872 of the EcoRV-Spel fragment. Intron insertion is achieved by cutting with PvuII (a blunt-ended cutter) and the blunt-ended ß-globin intron II sequence (Figure 2) is ligated into one or more of these sites. In a further aspect of the present invention there is provided a suitable cloning vector for expressing in a host cell a heterologous DNA sequence, comprising a DNA molecule that is complemented by at least part of an alphavirus RNA genome., the DNA molecule comprises the complement of the entire alphavirus RNA genome regions and has a cloning site for inserting into it a heterologous DNA sequence capable of being expressed in a host cell, the cloning site is located in a region of the DNA molecule that is not essential for the replication of it; a functional promoter sequence in the host cell and which transcriptionally controls the DNA molecule, the promoter sequence is placed towards the 5 'end of the DNA molecule, so that the resulting transcript has a genuine 5' end; at least one heterologous binding group provided in the complement of the DNA molecule to generate perfect splice junctions in the alphavirus in order to avoid aberrant binding and an additional DNA sequence at the 3 'end of the DNA molecule for direct appropriate excision in vivo at the 3 'end of the reactive mRNA transcript.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the DNA sequence of ß-globin intron II that includes three additional nucleotides at the 3 'end thereof (SEQ ID NO: 1); Figure 2 shows the DNA sequence for ß-globin intron II (SEQ ID No: 2); Figures 3A to 3C show the DNA sequence of the EcoRV-Spel fragment of the Semliki virus replicon Forest (SEQ ID No: 3); Figures 4A to 4D show the DNA sequence of the pSFV linkage (SEQ ID No: 4) prepared as illustrated in Figure 5; Figure 5 shows the construction of a pSFV link (11060 base pairs) from pSFV1, using a binding sequence (SEQ ID Nos: 5, 6); Figures 6A to 6D show the nucleotide sequence of plasmid pMP76 (SEQ ID no: 11), prepared as illustrated in Figures 8A to 8D; Figure 7 illustrates subsections of the pSFV plasmid link (see Figure 5); Figures 8A to 8D show the construction of the P1065 plasmid pMP76 from the plasmids pMP53, pMP70, pMP47, pMP55 and pMP71; Figures 9A to 9B show the construction of plasmids pMP53, pMP54 and pMP55 from plasmid pMP52; Figure 10 shows the construction of plasmid MP52 from pUC19 using a binding sequence (SEQ ID no: 7, 8); Figures HA to 11B show the construction of plasmid pMP46, pMP47 and pMP70 from pUC19 and the fragment from the pSFV link, prepared as shown in Figure 7; and Figures 12A to 12B show the construction of plasmid pMP71 from plasmid pCMV3.

GENERAL DESCRIPTION OF THE INVENTION As discussed in the foregoing, the present invention provides a modified alphavirus DNA. The alphavirus of preference is the Semliki Forest virus. In particular, the present invention provides a cloning vector for the expression of a heterologous gene in a host, for example an animal or a human. The promoter sequence may comprise a promoter of eukaryotic or prokaryotic origin. Suitable promoters are the cytomegalovirus immediate early promoter (pCMV), although other promoters such as the long terminal repeat promoter of Rous sarcoma virus (pRSV) can be used, since in the case of these and other similar promoters the transcription it is carried out by the DNA-dependent RNA polymerase of the host cell. Additionally SP6, T3 or T7 promoters can be used as long as the cell has been first transformed with genes encoding SP6, T3 or T7 RNA polymerase molecules, which are either inserted into the chromosome or remain in episomal form. The expression of these genes coding for RNA polymerase (SP6, T3, T7) depends on the RNA polymerase dependent on DNA of the host cell. The heterologous DNA insert may comprise the coding sequence for a desired product, which may be a biologically active polypeptide or protein, for example, the heterologous DNA insert may encode the HIV sequences, eg, a polypeptide or immunogenic protein or antigenic or a therapeutically active polypeptide or protein. The heterologous DNA may also comprise other additional sequences, for example a sequence complementary to an RNA sequence which is a self-cleaving ribozyme sequence. The DNA vectors provided herein may be administered to a host, including a host P1065 human, for the in vivo expression of a heterologous DNA sequence, according to a further aspect of the invention, in order to generate an immune response in the host, which could be a protective immune response. DNA vectors can also be formulated into immunogenic compositions to perform this administration.

BIOLOGICAL DEPOSITS Some vectors that contain the Semliki Forest virus replicon and referred to here have been deposited in the American Type Culture Collection (ATCC) located at 10801 University Boulevard, Manassas, VA 20110-2209, E.U.A., according to the Budapest Treaty and before the submission of this application. Samples of the deposited plasmids will be available to the public when a patent is granted based on this United States patent application and any restriction on access to the deposits will be withdrawn at that time. Non-viable deposits will be replaced. The invention described and claimed herein is not limited by the scope of the deposited plasmids, since the deposited mode is intended only as an illustration of the invention.

Deposit Summary PLASM Designation Deposit Date pMP76 EXAMPLES The foregoing discussion generally describes the present invention. A broader understanding may be obtained by reference to the following specific Examples. These Examples are described solely for illustrative purposes and are not intended to limit the scope of the invention. Changes in the form and substitution of equivalents are contemplated as circumstances that may arise or will be more expeditious. Although specific terms have been used herein, these terms are intended to be descriptive and in no way intended to be limiting. The methods of molecular genetics, protein biochemistry and immunology that are used and not explicitly described in this exposition and in the Examples are widely reported in the scientific literature and are within the normal skill and expertise of those engaged in this area.

EXAMPLE 1 This Example describes the construction of the P1065 plasmid pMP76 as outlined in Figures 5, 7, 8A, 8B, 8C, 8D, 9A, 9B, 10, HA, 11B, 12A and 12B. The binding of plasmid pSFV was created by restriction of plasmid pSFV1 (Gibco) with BamHI. This plasmid was then ligated with a binder (SEQ ID no: 5 and 6) to produce the binding of plasmid pSFV (Figures 4A to 4D, Figure 5). Some of the SFV replicon fragments were subcloned by restricting the pSFV binding with EcoRV and Spel and isolating the EcoRV-Spel fragment from 890 base pairs. This fragment was then restricted with EcoRI and the EcoRV-EcoRI fragments of 1906 base pairs, EcoRI-EcoRI of 1578 base pairs and 3627 base pairs and EcoRI-Spel of 899 base pairs were isolated (Figure 7). The EcoRV-EcoRI fragment of SFB of 1909 base pairs was cloned into the plasmid pMP52 restricted with EcoRV-EcoRI in order to produce the plasmid pMP53 (Figure 9A). The EcoRI-Spel fragment of 899 base pairs of SFV was cloned into pMP52 restricted by EcoRI-Spel to produce pMP54 (Figure 9A). Plasmid pMP54 was restricted with Spel and its end blunted with mung bean nuclease. Subsequently, the plasmid was restricted with BglII, dephosphorylated and ligated to the delta hepatitis virus ribozyme ligand (SEQ ID Nos. 9 and 10), which had been phosphorylated to produce pMP55 (Figure 9B).

P1065 Plasmid pMP52 was created by ligating a binder (SEQ ID Nos: 7, 8), in the EcoRI site of pUC19 (Figure 10). The 1578 base pair EcoRI-SFV fragment was cloned into the EcoRI site of pUC19 to produce pMP45 (Figure HA). This plasmid was then restricted with PpuMl and its terminus blunted with mung bean nuclease. The PCR fragment of rabbit ß-globin intron II (Figure 1) was treated with mung bean nuclease to make its blunt end, phosphorylated and ligated to pMP46 restricted by PpuMI to produce plasmid pMP70 (Figure 11B). The 3627 bp SFRI EcoRI fragment was cloned into the EcoRI site of pUC19 to produce pMP47 (Figure 11A). The plasmid pCMV3, which contains the CMV promoter, the Intron A sequence, the BGH poly A sequence and the SU40 poly A sequence was restricted with Ndel and EcoRV. The Ndel-EcoRV fragment of 3191 base pairs was isolated and dephosphorylated. The Ndel-EcoRV fragment of 1321 base pairs was isolated and restricted with Sacl. The 334 base pair Ndel-Sacl fragment was isolated (Figure 12A). The isolated Sacl-EcoRV PCR fragment containing the 5 'end of SFV was ligated with the previously isolated Ndel-Sacl fragment of 334 base pairs and the Ndel-EcoRV fragment of 3191 base pairs to produce pMP71 (Figure 12A and 12B).

P1065 Plasmid pMP53 was then restricted with EcoRI and BamHI were ligated to the EcoRI fragment of 2151 base pairs, dephosphorylated and isolated from pMP70 (Figure 8A). This ligand was then restricted with EcoRV and the EcoRV-EcoRI fragment of 4057 base pairs was purified (Figure 8A). Plasmid pMP47 was restricted with EcoRI and the EcoRI fragment of 3627 base pairs was isolated and dephosphorylated (Figure 8B). Plasmid pMP55 was then restricted with BglII, dephosphorylated and restricted with EcoRI. The 985 base pair EcoRI-BglII fragment was isolated and ligated with the previously isolated EcoRI fragment from pMP47 (Figure 8B). The binding reaction was subsequently phosphorylated and the EcoRI-BglII fragment of 4612 base pairs was isolated. Plasmid pMP71 was restricted with EcoRV and BamHI and then dephosphorylated. This fragment was used in a 3-way ligand with the EcoRI-BglII fragment of 4612 base pairs from pMP47 and pMP55, and the EcoRV-EcoRI fragment of 4057 base pairs from pMP53 and pMP70 to produce pMP76 (FIGS. 8B and 8C). The 5 'end of the SFV replicon was produced by PCR amplification of pSFV1 using SFV-5'-3 primers having the sequence 5'-ATCTATGAGCTCGTTTAGTGAACCGTATGGCGGATGTGTGACATACA-3' P1065 and EcoR-SPE having the sequence 5 '-TCCACCTCCAAGGATATCCAAGATGAGTGTG-3' (SEQ ID no: 9 and SEQ ID no: 10, respectively) between the CMV promoter and the 5 'end of the SFV replicon. The resulting PCR fragment was restricted with Sacl and EcoRV (Figure 13, SEQ ID no: 11) and the fragment was isolated.

EXHIBITION SUMMARY As a summary of this disclosure, the present invention provides an expression vector based on a modified alphavirus wherein at least one optimal binding site is introduced to the alphavirus replicon to prevent aberrant binding of the alphavirus genome, and improve RNA transport outside the nucleus. Modifications are possible within the scope of the invention.

REFERENCES Fulginiti, V.A., Eller, J.J., Sieber, O.F., Joyner, J.W., Minamitani, M. and Meiklejohn, G., (1969) Am. J. Epidemiol. 89 (4), 435-44B.

Chin, J., Magoffin, R.L., Shearer, L.A., Schieble, J.H. and Lennette, E.H. (1969) Am. J. Epidemiol. 89 (4), 449-463.

P1065 Jensan, K.E., Peeler, B.E. and Dulworth, W.G. (1962) J. Immunol. 89, 216-226.

Murphy, B.R., Prince, G.A. , Collins, P.L., Van Wyke-Coelingh, K., Olmstead, R.A. , Spriggs, M.K., Parrott, R.H., Kim, H.-Y., Brandt, C.D. and Chanock, R.N. (1988) Vir. Res. 11, 1-15. Chapman, B.S .; Thayer, R.M .; Vincent, K.A. Y Haigwood, N.L., Nucí. Acids Res. 1991, 19: 3979-3986.

Huang, Zhi-ming and Yen, T. S. Benediot, Molecular and Cell Biology, July 1995, p. 3864-3869.

Claims (13)

1. CLAIMS; 1. An expression vector characterized by a DNA molecule complementary to at least part of an alphavirus RNA genome, the DNA molecule comprises the complement of the entire genome regions of alphavirus RNA which are essential for the replication of the Alphavirus RNA and a heterologous DNA sequence capable of being expressed in a host, the heterologous DNA sequence is inserted into a region of the DNA molecule that is not essential for replication thereof and the DNA molecule is placed under transcriptional control of a functional promoter sequence in the host, wherein at least part of the heterologous binding site is provided in the DNA molecule to prevent aberrant binding of alphavirus RNA.
2. 2. The vector according to claim 1, characterized in that the promoter is positioned towards the 5 'end of the DNA molecule, so that the resulting transcript has an authentic 5' end.
3. 3. The vector according to claim 1 or 2, characterized in that the promoter is the cytomegalovirus immediate early promoter.
4. 4. The vector according to any of claims 1 to 3, characterized by an additional DNA sequence at the 3 'end of the DNA molecule for P1065 direct adequate cleavage in vivo at the 3 'end of the DNA molecule.
5. 5. The vector according to claim 4, characterized in that the additional DNA sequence comprises a hepta delta ribozyme sequence.
6. 6. The vector according to any of claims 1 to 5, characterized in that the sequence of the heterologous binding site is provided by the DNA sequence of rabbit ß-globin intron II.
7. The vector according to any of claims 1 to 6, characterized in that the sequence of the heterologous binding site is inserted into the DNA molecule in a location that generates perfect splicing junctions and restores the function of the SFV replicon upon removal.
8. 8. The vector according to any of claims 1 to 7, characterized in that the alpha virus is a Semliki Forest virus.
9. The vector according to claim 8, characterized in that at least one heterologous binding site is located at a Ppu-MI site at position 2719 and / or at a Pvull site at positions 2518, 3113, 6498 and / or 6872 within an EcoRV-Spel fragment of the SFV replicon.
10. 10. A suitable cloning vector for pioes expression in a host cell of a heterologous DNA sequence, which is characterized by: a DNA molecule complementary to at least part of an alphavirus RNA genome, the DNA molecule comprises the complement of the genome replication regions complete of alphavirus RNA and has a cloning site for inserting therein a heterologous DNA sequence capable of expression in a host cell, the cloning site is located in a region of the DNA molecule that is not essential for replication Of the same; a promoter sequence functional in the host cell and transcriptionally controlling the DNA molecule, the promoter sequence is placed towards the 5 'end of the DNA molecule, so that the resulting transcript has a genuine 5' end; at least one heterologous binding site provided in the complement of the DNA molecule to prevent aberrant binding of RNA and to generate perfect splicing linkages in the alphavirus; and an additional DNA sequence at the 3 'end of the DNA molecule to direct appropriate cleavage in vivo at the 3' end of the reactive RNA molecule.
11. 11. The cloning vector according to claim 10, characterized in that the binding site P1065 heterologous is provided by the DNA sequence of the Intron II of rabbit ß-globin.
12. 12. The cloning vector according to claims 10 or 11, characterized in that the additional sequence comprises a hepatitis delta ribozyme sequence.
13. 13. The cloning vector according to any of claims 10 to 12, characterized in that the alpha virus is a Semliki Forest virus. The cloning vector according to claim 13, characterized in that at least one heterologous binding site is located at a Ppu-MI site at position 2719 and / or at a Pvull site at the positions 2518, 3113, 6498 and / or 6872 within an EcoRV-Spel fragment of the SFV replicon. 15. The cloning vector according to any of claims 10 to 14, having the identification characteristics of the plasmid pMP76 shown in Figure 8D. 16. The cloning vector according to any of claims 10 to 15, having SEQ ID no: 11. P1065