Patent 6,958,237

Highly infectious rubella virus cDNA clones derived from infectious cDNA clone having a low specific infectivity and methods of obtaining highly infectious rubella virus cDNA clones. Togavirus expression vectors of improved stability for the expression of live, attenuated togavirus and a foreign gene, based on the nucleic acid sequence of an infectious rubella virus clone and contain a togavirus non-structural protein open reading frame; an expression element for expression of a foreign gene; a foreign gene or a multiple cloning site for insertion of a foreign gene; an expression element for the expression of the live, attenuated togavirus; and a togavirus structural protein open reading frame. The expression element is either a subgenomic promoter or an internal ribosome entry site (IRES). Administration of the vector as an immunization agents is useful for the induction of immuity against the togavirus, the foreign gene, or both.

SUMMARY OF THE INVENTION

There has been a long-standing problem of an inability to produce highly infectious rubella virus clones. Applicants discovered that by replacing a portion of a low infectivity clone with a corresponding fragment that was synthesized by a method known to produce sequences with few mutations, Applicants obtained a chimera exhibiting high infectivity. Therefore, the present invention includes methods of producing highly infectious rubella virus clones by replacing segments of a low infectivity clone with corresponding segments produced by a protocol known to generate sequences having a minimal number of mutations.

Additionally, highly infectious cDNA clones of the rubella virus are provided herein. The clones are chimeric DNA molecule constructs containing portions of a rubella virus cDNA clone having a low specific infectivity and one or more portions of at least one rubella virus genome synthesized by a method known to produce sequences having a minimal number of mutations. The highly infectious rubella virus clones of the invention are useful as molecular biology tools for studying rubella virus and can be useful for developing recombinant vaccines against rubella.

The highly infectious cDNA clones have a specific infectivity greater than 0.5 plaques/μg of transcript. In several preferred embodiments of the invention, the specific infectivities of viral transcripts are approximately 10⁴plaques/μg of transcript.

In preferred embodiments the cDNA clones are prepared by replacing one or more fragments of a known w-Therien-derived infectious cDNA clone having low specific infectivity with corresponding fragments from an f-Therien rubella virus strain synthesized by a method known to produce sequences having a minimal number of mutations.

Togavirus expression vectors for the expression of live, attenuated togavirus and a foreign gene are also dherein. The expression vector constructs contain a togavirus non-structural protein open reading frame; a first expression element for expression of a heterologous virus; a gene encoding the foreign gene or a multiple cloning site into which the foreign gene may be inserted; a second expression element for the expression of the live, attenuated togavirus; and a togavirus structural protein open reading frame. The togavirus non-structural protein open reading frame and togavirus structural protein open reading frame are preferably from an infectious rubella virus clone. The preferred foreign gene is a heterologous virus. The expression element is either a subgenomic (SG) promoter or an internal ribosome entry site (IRES). Administration of the vector as an immunization agents is useful for the induction of immuity against the togavirus, the heterologous virus, or both. The incorporation of at least one IRES in the vector results in a recombinant virus of improved stability.

In a preferred embodiment, the expression elements for expression of both the foreign gene and the togavirus are SG promoters. A multiple cloning site (MCS) is located between the two SG promoters. The MCS is useful for the insertion of the foreign genes under the control of the upstream SG promoter, including but not limited to reporter genes or heterologous virus genes. Exemplary reporter genes include green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT) genes. Exemplary heterologous virus genes include Japanese encephalitis virus genes.

In another preferred embodiment, the second expression element, which controls expression of the togavirus structural protein, is replaced by an internal ribosome entry site (IRES). The IRES is a sequence capable of promoting the entry of a ribosome into an RNA molecule at an internal site, independently of the polyadenylated cap.

This construct is prepared by replacing an indigenous SG promoter of an infectious rubella cDNA clone with the IRES, thus placing the expression of rubella virus structural genes under the control of IRES. Surprisingly, this construct gives rise to viable rubella virus. This recombinant construct is yet another embodiment of the present invention. A duplicate copy of the SG promoter region is then placed into the intermediate construct directly upstream of IRES. A MCS is placed downstream of the SG promoter to allow for the insertion of the foreign genes. Introduction of the IRES element results in improved stability of the recombinant virus, including improved expression of the foreign gene protein.

In the present embodiments, the vectors are prepared using a backbone of an infectious rubella cDNA clone containing portions of both a cDNA clone having a low specific infectivity and a second rubella virus genome Robo302, described herein, and Robo402 described in Pugachev, K. V., et al., (2000) Virology, 273, 189-197, incorporated herein by reference in its entirety.

The vectors are useful for the induction of immunity or to develop recombinant vaccines against rubella and/or a heterologous virus whose genes may be inserted into the expression vector. The vectors can also be used to study rubella, particularly rubella virus replication. The method of introduction of an IRES element into an expression vector based on rubella virus, which belongs to togavirus family (Togaviridae), can be used to develop other togavirus expression vectors of improved stability.

It is therefore an object of the present invention to provide a highly infectious cDNA clone of the rubella virus genomic RNA.

It is a further object of the present invention to provide a molecular biology tool for studying rubella, particularly rubella virus replication.

It is a further object of the present invention to provide cDNA clones for the development of a recombinant rubella virus vaccine.

It is another object of the present invention to provide an expression vector based on rubella virus.

It is a further object of the present invention to provide an expression vector based on rubella virus for the expression of protein or proteins whose genes are inserted into the vector.

It is a further object of the present invention to provide an expression vector based on rubella virus for the expression of protein or proteins in eukaryotic cells, including animal cells.

It is a further object of the present invention to provide an expression vector based on rubella virus for the induction of immunity against rubella and/or a different virus or viruses whose genes are inserted into the vector.

It is a further object of the present invention to provide an expression vector based on rubella virus for the development of recombinant vaccines against rubella virus and/or a different virus or viruses whose genes are inserted into the vector.

It is a further object of the present invention to provide an expression vector based on highly infectious cDNA clones of the rubella virus.

It is yet another object of the present invention to provide a viable cDNA rubella clone that contains IRES as one of its promoters.

It is a further object of the present invention to provide an expression vector based on rubella virus having enhanced stability.

It is yet another object of the present invention to provide a molecular biology tool to study rubella, including but not limited to rubella virus replication and protein expression.

It is yet another object of the present invention to provide a molecular biology tool to study the function of IRES elements in the context of a togavirus genome.

It is yet another object of the present invention to provide a molecular biology tool to study togaviruses other than rubella, in particular their replication and protein expression, by means of introducing IRES elements into their genome.

DETAILED DESCRIPTION OF THE INVENTION

There has been a long-standing problem of an inability to produce highly infectious rubella virus clones. Applicants discovered that, by replacing a portion of a low infectivity clone with a corresponding fragment that was synthesized by a method known to produce fragments with few mutations, Applicants obtained a chimera exhibiting high infectivity. Therefore, the present invention includes methods of producing highly infectious rubella virus clones by replacing segments of a low infectivity clone with corresponding segments produced by a protocol known to generate sequences having a minimal number of mutations.

Also disclosed are highly infectious, isolated cDNA clones of rubella virus. The infectious rubella virus clones are useful as molecular biology tools for studying rubella virus and for developing recombinant vaccines against rubella.

The term "highly infectious cDNA clone" is defined herein as a cDNA clone having a high specific infectivity, which is defined as a specific infectivity of greater than 0.5 plaques/μg of transcript. The term "low infectivity" or "low specific infectivity" is defined herein as a specific infectivity of less than or equal to 0.5 plaques/μg of transcript.

The highly infectious, isolated cDNA molecules are inserted into a vector that enables replication of the nucleotide sequence of the molecules. A preferred vector is a bacterial plasmid such as pUC 19, pGEM, or PBR-322 (all available from Promega Biotec, Madison, Wis.) or pC11921 adjacent to a bacteriophage RNA polymerase promoter sequence such as the SP6 RNA polymerase (Promega Biotec) such that RNA copies of the rubella virus DNA can be synthesized in vitro. The vector is chemically introduced into susceptible culture cells, for example, E. coli, for amplification and production of large amounts of the cDNA clone. For use, the purified infectious clone is restricted with a restriction endonuclease such as Nsi 1 (New England Biolabs, Beverly, Mass.) for linearization at the termination of the rubella virus cDNA sequences. The linearized plasmid is then transcribed with an RNA polymerase such as SP6 RNA polymerase, which results in production of RNA transcripts templated from the rubella virus cDNA sequence in the non-pathogenic infectious clone.

In preferred embodiments of the present invention, the rubella virus clones have specific infectivities of approximately 10⁴plaques/μg of transcript. In these preferred embodiments, the rubella virus cDNA clones contain portions of a cDNA clone having a low specific infectivity of approximately 0.5 plaques/ug of transcript or less. In the preferred embodiment, the cDNA clone having a low specific infectivity is the clone described by Wang, et al., J. Virol. 68:3550-3557 (1994), having the sequence shown in SEQ ID NO:1.

The chimeric constructs also contain portions, or fragments, of cDNA from a rubella virus genome in which the cDNA fragments have been produced in a manner known to generate sequences having a minimal number of mutations. The highly infectious constructs are prepared by replacing one or more portions of the cDNA clone having low infectivity with corresponding DNA fragments having fewer mutations. The corresponding DNA fragments may be derived from any rubella virus strain. The specific infectivities of the cDNA clones of the present invention exhibit an increase of at least 10⁴fold over infectivity of a cDNA clone derived solely from a strain known to have a low specific infectivity.

Rubella Genome Fragments Conferring High Infectivity

Rubella genome fragments that confer the highly infectious property upon the chimera are those produced in a manner known to generate sequences having a minimal number of mutations. The fragments that confer the highly infectious property are obtained as follows. w-Therien, f-Therien and other rubella virus genomes are available from laboratories specializing in rubella virus research. Rubella virus genomes may also be obtained by drawing blood from a person or animal infected with the rubella virus and isolating the genomes by methods that are standard in the art. Such methods can be found in standard lab manuals.

Any rubella virus strains that are or may become available can be used to produce a fragment using a protocol known to generate sequences having a minimal number of mutations. No specific rubella virus genome need be used as a template for the DNA fragments because any rubella virus genome will achieve the desired result. Possible DNA fragments include those derived from the original genome from which the low infectivity clone was produced, as long as the DNA fragments have been produced by a protocol known to generate sequences having a minimal number of mutations.

The fragments can then used for replacing a corresponding fragment of rubella virus clones having a specific infectivity of less than or equal to 0.5 plaques/μg of transcript. Materials and protocols for replacing regions of a cDNA clone with a replacement region are standard in the art. No special materials or protocols are required. Protocols can be found in standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, New York (1989). Materials can be purchased from widely used and well-known companies, such as, Sigma Chemical, Inc., Promega, and New England Biolabs.

In the most preferred embodiments of the present invention, the cDNA clone having a low specific infectivity is derived from the w-Therien rubella virus strain and the cDNA fragments used to replace portions of the cDNA clone are derived from the f-Therien rubella virus strain. Most preferably, the chimeric constructs contain one or more portions of the infectious cDNA clone Robo102, derived from the w-Therien rubella virus strain, as described in Wang, et al., J. Virol. 68:3550-3557 (1994), and in U.S. patent application Ser. No. 08/459,041, now U.S. Pat. No. 5,663,065, which is incorporated by reference herein (and shown in SEQ ID NO:1), and one or more fragments of synthesized cDNA having few mutations and derived from the f-Therien rubella virus strains.

Any method for producing corresponding DNA fragments having a minimal number of mutations may be used to make the clones of the present invention. Three preferred fragments derived from f-Therien are fragment III (SEQ ID NO:10), fragment I (SEQ ID NO:2), and fragment II (SEQ ID NO:3).

Preferably, the cDNA fragments are created using the technique known by those skilled in the art as reverse transcriptase-long polymerase chain reaction (RT-long PCR) or high-fidelity long PCR, which allows for the amplification of long nucleic acid sequences. This use of this technique results in a reduction of the number of mutations in the genomic cDNA. High-fidelity long PCR amplification of rubella virus cDNA fragments is achieved with first strand cDNA synthesis, using currently available nucleic acid synthesis kits such as the RiboClone cDNA Synthesis System kit (Promega Corporation, Madison, Wis.) according to the protocol of the manufacturer, followed by PCR amplification. In a preferred embodiment, a high-fidelity DNA polymerase, such as ExTaq polymeraserom PanVera Corp., which has a proofreading capacity, is employed for PCR amplification. Exemplary oligonucleotide primers for the generation of nucleic acid fragments, with which to replace the portions of the cDNA clone having low infectivity, are set forth in the Examples below.

Other methods of producing fragments that generate sequences having a minimal number of mutations may become available in the future.

By employing the method of the present invention on a low infectivity rubella virus clone, Applicants discovered that some type of error or mutations in a particular region may cause low infectivity of a rubella virus clone. As discussed in more detail in Example 2, Applicants discovered the deleterious regions in Robo 102 by inserting three different fragment DNAs into three corresponding regions of Robo 102. Insertion of fragment I or fragment II, individually, did not increase the infectivity of subsequently produced viral transcripts. However, the replacement of fragment III into Robo102 did result in increased infectivity. This method may be used on other low infectivity clones to determine if specific locations are the cause of low infectivity.

The following steps may be followed to prepare a highly infectious rubella virus clone of the invention from a low infectivity clone. A low infectivity DNA molecule clone may be obtained by the method described in Wang, et al., J. Virol. 68:3550-3557 (1994). A copy of a rubella virus genome may be obtained from a laboratory specializing in this area, or from the American Type Culture Collection, or isolated from a person infected with the disease. DNA fragments of the genome may be synthesized by a method known to produce sequences having a minimal number of mutations for substitution into the DNA molecule encoding an infectious rubella virus having low infectivity. Portions of the low infectivity clone are then replaced with the newly synthesized corresponding fragments to obtain a chimeric construct exhibiting high infectivity.

As shown in FIG. 1, in a preferred embodiment of the present invention, the 5′ end portion of the cDNA clone having low specific infectivity (the w-Therien derived Robo102 construct, SEQ ID NO:1) is replaced with the corresponding cDNA fragment (fragment III) from a second rubella virus genome (the f-Therien strain of the rubella virus genome), to create a highly infectious construct (Robo202). The nucleic acid sequence of fragment III is set forth in the sequence listing as SEQ ID NO:10. Fragment III contains the entire structural protein open reading frame region (SP-ORF) of the genome. The structural protein open reading frame encodes at least three structural proteins, C, E1 and E2. Fragment III also contains a portion of the 5′-end of the non-structural protein open reading frame (NSP-ORF) and the entire structural protein open reading frame (SP-ORF). Fragment III is also described as a nucleic acid molecule between restriction endonuclease cleavage sites EcoRI and BglII. More specifically, the Robo202 chimeric construct includes nucleotides 1 to approximately 5352 of SEQ ID NO:1 and replaces nucleotides 5353 to 9734 of SEQ ID NO: 1 with the corresponding sequence from the f-Therien rubella virus genome.

In another preferred embodiment of the present invention, three fragments from a second rubella virus genome (the f-Therien rubella virus genome), are used to replace the corresponding fragments of the infectious rubella virus cDNA clone having low specific infectivity (Robo102) to create a chimeric construct having high specific infectivity (Robo302). As shown in FIG. 1, the first fragment (fragment I) contains the 3′ end of the non-structural open reading frame. Fragment I is also described as the nucleic acid molecule between restriction endonuclease cleavage sites HindIII and KpnI. The nucleic acid sequence of fragment I is set forth in the sequence listing as SEQ ID NO:2. The second fragment (fragment II) contains most of the 5′ end of the non-structural open reading frame (NSP-ORF). Fragment II is also described as the nucleic acid molecule between restriction endonuclease cleavage sites NheI and BglII. The nucleic acid sequence of fragment II is set forth in the sequence listing as SEQ ID NO:3. Fragment III (SEQ ID NO:10), also replaces the corresponding fragment in Robo102. In particular, fragment I (SEQ ID NO:2) replaces nucleotides 1 to 1723 of Robo102, fragment II (SEQ ID NO:3) replaces nucleotides 2800 to 5352 of Robo102, and fragment III (SEQ ID NO:10) replaces nucleotides 5353 to 9734 of Robo102. The resulting construct, Robo302, contains roughly 90% of the f-Therien rubella virus genome and 10% of the w-Therien strain of the rubella virus genome.

In another preferred embodiment of the present invention, fragments I (SEQ ID NO:2) and III replace the corresponding portions of the infectious cDNA clone having low infectivity (Robo102) to produce a highly infectious cDNA clone (Robo202/I). As shown in FIG. 1, the resulting cDNA construct contains both the 5′ and 3′ ends of the f-Therien strain of the rubella virus genome corresponding to nucleotides 1 to 1723 and 5352 to 9734, respectively. The central portion of the Robo202/I cDNA is derived from nucleotides 1723 to 5352 of the w-Therien strain.

In another preferred embodiment of the present invention, fragments II (SEQ ID NO:3) and III as described above, replace the corresponding portions of the infectious cDNA clone having low infectivity (Robo102) to produce a highly infectious cDNA clone (Robo202/II). As shown in FIG. 1, the resulting cDNA construct contains the 5′ end of the w-Therien rubella virus genome up to nucleotide 2800 with the remaining section consisting of the f-Therien rubella virus genome.

The specific infectivity of highly infectious clones Robo 202, Robo 302, Robo 202/I, and Robo 202/II is approximately 10⁴plaques per μg. As a comparison, the specific infectivity of the rubella virus RNA is 10⁵plaques per μg.

Recombinant togavirus expression vector constructs are described herein. The vectors are useful for protein expression in vitro or in vivo, induction of immunity, or for development recombinant vaccines against rubella and/or a heterologous virus whose genes may be inserted into the expression vector. The expression vectors can also be used as molecular biology tools to study togaviruses, particulary rubella viruses, more particularly rubella virus replication and protein expression. The vectors can also be used to study the function of IRES elements in the context of a togavirus genome. The method of incorporating IRES elements into the rubella virus expression vectors can be used to study togaviruses other than rubella, particularly their replication and protein expression.

The expression vector constructs contain a togavirus non-structural protein open reading frame; a first expression element for expression of a heterologous virus; a gene encoding the foreign gene or a multiple cloning site into which the foreign gene may be inserted; a second expression element for the expression of the live, attenuated togavirus; and a togavirus structural protein open reading frame. The togavirus non-structural protein open reading frame and togavirus structural protein open reading frame are preferably from an infectious rubella virus clone. The preferred foreign gene is a heterologous virus gene. The expression element is either a subgenomic (SG) promoter or an internal ribosome entry site (IRES). The incorporation into the vector of at least one IRES results in a recombinant virus of improved stability. Administration of the vector as an immunization agent is useful for the induction of immunity against the togavirus, the heterologous virus, or both.

The term "improved stability" is defined herein as the ability to maintain the expression of foreign genes by the recombinant virus for longer than three passages through the cell culture, wherein the recombinant virus results from the infection of cells by the virus expression vector.

The term "foreign gene" as used herein means a heterologous gene whose expression by the expression vector described herein is desirable.

In a preferred embodiment, the expression elements for expression of both the foreign gene and the togavirus are SG promoters. A multiple cloning site (MCS) is located between the two SG promoters. The MCS is useful for the insertion of the foreign genes under the control of the upstream SG promoter, including but not limited to reporter genes or heterologous virus genes. Exemplary reporter genes include green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT) genes. Exemplary heterologous virus genes include encephalitis virus, hepatitis and Dengue virus genes.

In another preferred embodiment, the second expression element, which controls expression of the togavirus structural protein, is replaced by an internal ribosome entry site (IRES). The IRES is a sequence capable of promoting the entry of a ribosome into an RNA molecule at an internal site, independently of the polyadenylated cap

This construct is prepared by replacing an indigenous SG promoter of an infectious rubella cDNA clone with the IRES, thus placing the expression of rubella virus structural genes under the control of IRES. Surprisingly, this construct gives rise to viable rubella virus. This recombinant construct is yet another embodiment of the present invention. A duplicate copy of the SG promoter region is then placed into the intermediate construct directly upstream of IRES. A MCS is placed downstream of the SG promoter to allow for the insertion of the foreign genes. Introduction of the IRES element results in improved stability of the recombinant virus, including improved expression of the foreign gene protein.

In the present embodiments, the vectors are prepared using a backbone of an infectious rubella cDNA clone containing portions of both a cDNA clone having a low specific infectivity and a second rubella virus genome, such as Robo302, described herein, or Robo402 described in Pugachev, K. V., et al., (2000) Virology, 273, 189-197, incorporated herein by reference in its entirety, or a combination of the two clones.

The expression vector is constructed using an infectious rubella cDNA clone and modifying its subgenomic promoter-containing site. The molecular biology techniques employed to perform such modifications are well-known to the one skilled in the art and are detailed in such common in the art manuals as Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^nded. Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989). A preferred starting cDNA clone is a chimeric DNA construct based on a bacterial plasmid such as pUC 19, pGEM, or PBR-322 (all available from Promega Biotec, Madison, Wis.) containing a cDNA copy of a viral genome positioned adjacent to an RNA polymerase promoter, such as an the SP6 RNA Polymerase (Promega Biotec) such that infectious in vitro transcripts can be synthesized. The most preferred cDNA clone is a highly infectious cDNA clone such as wild-type Therien strain rubella infectious clone Robo302 described herein, or Robo402 described in Pugachev, K. V., et al., (2000) Virology, 273, 189-197, incorporated herein by reference in its entirety.

In the preferred embodiments of the present invention, the subgenomic (SG) promoter containing site of a cDNA rubella virus clone is modified to contain, between a non-structural-protein open reading frame (ORF) and structural protein ORF, a promoter followed by restriction nuclease recognition (cloning) site or sites that may be used to introduce a foreign gene, including but not limited to reporter genes, such as green fluorescent protein or chloramphenicol acetyltransferase, and heterologous virus, such as Japanese encephalitis virus, genes. The subgenomic structural protein genes of rubella virus either remain under the control of another promoter, such as the indigenous subgenomic promoter, or an internal ribosome entry site. For use, the vector is chemically introduced into susceptible culture cells, for example, E. coli, for amplification and production of large amounts of the cDNA clone. For use, the purified infectious clone is restricted with a restriction endonuclease such as EcoRI (New England Biolabs, Beverly, Mass.) for linearization at the termination of the rubella virus cDNA sequences. The linearized plasmid is then transcribed in vitro with an RNA polymerase such as SP6 RNA polymerase, which results in production of RNA transcripts. The resulting RNA transcripts are used to transfect the cells by transfection procedures known to those skilled in the art. The cells, in turn, will produce both the native structural proteins of the rubella virus and the protein encoded by the foreign gene. The replication of the RNA sequences and the expression of the encoded protein by the cells may be monitored by various means known to the ones skilled in the art. The cells will further produce recombinant virus particles which, in turn, may be used to infect cells or organisms.

When an appropriate amount of the infectious clone RNA transcript is transfected into susceptible cells by transfection procedures known to those skilled in the art, less virulent togavirus is recovered from the culture fluid within several days incubation. The identity of the virus recovered from the transfected cells can be confirmed by sequencing a specific region of the infectious clone in which a mutation exists which distinguishes it from the wild-type virus.

The less virulent togavirus is then combined with a pharmaceutically acceptable carrier to provide a safe, effective vaccine, such as a rubella virus vaccine. The carrier can be oil, water, saline, phosphate buffer, polyethylene glycol, glycerine, propylene glycol, and combinations thereof, or other vehicles routinely used by the pharmaceutical industry for these purposes. The vaccine is usually provided in lyophilized form and therefore is free of preservatives.

It will be understood by those skilled in the art that modified cDNA for other DNA or RNA viruses could be inserted into the vector in combination with the rubella virus cDNA to make a vaccine effective in immunizing a patient against more than one virus. For example, the modified cDNA of RNA viruses such as encephalitis, hepatitis or Dengue fever virus, is inserted into the vector to produce a combined recombinant vaccine, particularly Japanese encephalitis or hepatitis C virus.

The vaccine can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, intramuscularly, subcutaneously, or topically, in liquid or solid form, in a single dose or a dose repeated after a certain time interval. Preferably, the administration of the vaccine will result in in vivo protein expression of the proteins encoded by the open reading frames contained in the expression vector construct. Most preferably, the administration of the vaccine will result in the induction of immunity against the viruses whose proteins are encoded by the open reading frames. The vaccine is preferably administered subcutaneously at a concentration range from 10²to 10⁴TCID₅₀/person. (TCID is an abbreviation for tissue culture infectious doses). Preferably, the vaccine is provided to the physician in a lyophilized form, reconstituted in an appropriate solvent such as deionized water or saline, and administered as a single injection.

Expression Vector Construction

In a preferred embodiment of the present invention, a rubella expression vector is constructed using the wild-type Therien strain rubella infections clone Robo302 described herein. As shown in FIG. 4, an additional SG promoter is located between the non-structural protein and structural protein ORFs. The production of alphavirus (other members of togavirus family) expression vector constructs from infectious clones of alphaviruses by duplicating the subgenomic promoter is described by Bredenbeek P., et al., J. Virol. (1993); Liljestrom P., et al. Bio/Technology (1991), Smerdou C., et al., J. Virol. (1999); et. al. Virology (1997); and Schlesinger S. et al. Curr. Opin. Biotechnol. (1999).

In the alphavirus-based vectors, the second SG promoter is placed both between the ORFs and downstream of the SP-ORF within the 3′ untranslated region, which is 400 to 500 nucleotides long in these viruses. In the vectors described herein, the region between the structural and non-structural protein ORFs, rather than the region downstream of the structural protein ORF (3′ untranslated region), was chosen for the location of the additional SG promoter because the rubella virus 3′ untranslated region is relatively short (60 nucleotides) and the 3′ 300 nucleotides (including the 3′ end of the structural protein ORF) appear to be necessary for efficient virus replication, as reported by Chen, et al., J. Virol. 73:3386-3403 (1999).

As the rubella SG promoter has not been mapped, a region consisting of the 3′-terminal 126 nucleotides of the nonstructural protein ORF (NSP-ORF) and the entire 120-nt noncoding region between the NSP-ORF and the SP-ORF is duplicated. A multiple cloning site (MCS) containing convenient restriction sites (including unique sites for restriction endonucleases XbaI, BstBI, HpaI, and NsiI, all available from New England Biolabs, Beverly, Mass.) is located between the SG promoters for insertion of foreign genes. Thus, in this construct the SG RNA transcribed from the upstream SG promoter is translated to produce the foreign gene that may be placed in MCS, while the SG RNA transcribed from the downstream SG promoter is equivalent to the standard SG RNA and is translated to produce the virus structural proteins. The plasmid is termed dsRobo302.

In another preferred embodiment, an IRES element is incorporated into the rubella expression vector in place of the second SG promoter. Construction of this vector is initiated by replacing the SG promoter with the IRES in Robo402. Surprisingly, transcripts from this construct, Robo402/IRES, shown in FIG. 4, give rise to viable virus which formed plaques on Vero cells but do not produce subgenomic RNA. This shows that an IRES element can drive expression of a togavirus structural protein even in the absence of SG promoter or the corresponding subgenomic RNA.

In another preferred embodiment, the non-structural protein ORF is followed by a SG promoter followed, in turn, by the MCS for the introduction of foreign genes, such as the gene for the green fluorescent protein gene (GFP), followed by an IRES element, followed by the structural protein ORF. In this particular embodiment, the construct is developed from the intermediate construct Robo402/IRES. This expression vector results in a virus of improved stability when passaged multiple times through the Vero cells compared to dsRobo302.

Modifications and variations of the DNA encoding an infectious rubella virus and rubella virus expression vectors, methods of making and use thereof, methods of making a less virulent rubella virus and use thereof, an improved rubella virus vaccine and methods making and use thereof are intended to come within the scope of the present invention.

Claim 1 of 5 Claims

1. A rubella virus expression vector, comprising a rubella virus non-structural protein open reading frame, a subgenomic promoter for expression of a foreign gene, operably linked to the foreign gene or a multiple cloning site for insertion of the foreign gene, and an internal ribosome entry site, operably linked to a rubella virus structural protein open reading frame.

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