WO1994029472A9

WO1994029472A9 - Mengovirus as a vector for expression of foreign polypeptides

Info

Publication number: WO1994029472A9
Application number: PCT/US1994/006177
Authority: WO
Filing date: 1994-06-03
Publication date: 1995-02-02

Abstract

This invention relates to attenuated recombinant mengoviruses expressing a heterologous amino acid sequence. The recombinant mengoviruses can express amino acid sequences comprising epitopes of various viral pathogens and are useful as live vaccines for humans and animals against these pathogens. Consequently, this invention also relates to vaccines comprising these recombinant mengoviruses or proteins thereof, cells infected with the recombinant mengoviruses, nucleic acid derived from the recombinant mengoviruses, and methods of inducing an immune response. The invention is exemplified by a recombinant mengovirus comprising amino acids 299-466 of gp120 of the MN isolate of HIV-I.

Description

MENGOVIRUS AS A VECTOR FOR EXPRESSION OF FOREIGN POLYPEPTIDES

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of United States application Serial No. 08/090,531, filed June 3, 1993. The entire disclosure of this application is relied upon and expressly incorporated by reference herein.

BACKGROUND OF THE INVENTION

This invention relates to mengoviruses modified to contain a nucleic acid encoding one or more foreign polypeptides for immunological and non-immunological purposes . Modified mengoviruses of this invention can be recombinant viruses and/or chimaeric viruses .

Mengovirus is a picornavirus belonging to the genus cardiovirus . While the natural host for mengovirus is the mouse, with infection resulting in acute murine menin- goencephalitis, mengovirus has a wide host range. In addition to the mouse, mengovirus is also able to infect various animal species including pigs, elephants, and pri¬ mates including humans .

The picornaviruses are a family of pathogenic viruses . Examples of picornaviruses include rhinovirus responsible for the common cold, poliovirus, foot and mouth disease virus (FMDV), Coxsac ie viruses, hepatitis A virus, and murine cardioviruses including mengovirus and encephalomyocarditis virus .

Picornaviruses have a non-enveloped capsid containing a small positive sense RNA genome. The capsids of all picornaviruses are composed of a 60 subunit protein shell having 5:3:2 icosahedral symmetry with each subunit containing four nonidentical polypeptide chains (VP1, VP2, VP3, and VP4 ) . The shell encapsulates a single copy of the positive sense RNA genome. The three dimensional structure of mengovirus has been determined to atomic resolution by X-ray crystallography. Luo et al . , Science 235:182-191 (1987) .

The viral genome of mengovirus is a positive stranded RNA molecule of about 7,800 nucleotides in length. The genome is polyadenylated at its 3' end and covalently linked to a small viral polypeptide VPg at its 5' end. The mengoviral genome has been cloned in the form of a complementary DNA (cDNA) molecule. The genome includes a single open reading frame encoding a viral polyprotein.

The viral proteins are located within the polyprotein in the order L-P1-P2-P3 from the N to the C terminal end of the polyprotein. The polyprotein is processed by a series of cleavage events to give rise to all structural and non- structural proteins. The details of this processing are reviewed by Ann C. Palmenberg, Proteolvtic Processing of Picornaviral Polyprotein 44 Ann. Rev. Microbiol. 603 (1990), which is incorporated herein by reference. L designates a leader polypeptide that is present in cardio and aphthoviruses. PI is a precursor to the structural proteins VP1, VP2, VP3, and VP4, which are also identified as ID, IB, 1C, and IA, respectively. P2 and P3 are precursors to the non-structural viral proteins required for the replication of the viral RNA and the processing of the polyprotein.

The viral RNA is infectious. That is, upon its introduction into permissive cells it is able to initiate a complete viral multiplication cycle regenerating infectious virus . RNA transcripts synthesized _in_ vitro by an RNA polymerase from the full-length viral cDNA were also shown to be infectious. See Duke et al., J. Virol. 63:1822 (1989).

The murine cardioviruses, such as mengovirus and encephalomyocarditis virus, and aphthoviruses can be distinguished from other positive strand RNA viruses by the presence of long homopolymeric poly(C) tracts within their 5' noncoding sequences. Although the length, generally 60-350 bases, and sequence discontinuities, e.g. uridine residues, that sometimes disrupt the homopolymeric sequence have served to characterize natural viral isolates, the exact biological function of the poly(C) region is not clear. cDNA- ediated truncation of the mengovirus poly(C) tract attenuates the pathogenicity of this virus in mice. See Duke et al., Nature 343:474 (1990).

An attenuated strain of mengovirus, vM16, has been described by Duke et al. , Attenuation of Mengovirus through Genetic Engineering of the 5' Non-coding Poly(C) Tract, Nature 343:474 (1990), which is incorporated herein by reference, and by Duke and Palmenberg, Cloning and Synthesis of Infectious Cardiovirus RNAs Containing Short, Discrete Poly(C) Tracts, J. Virol. 63:1822 (1989), which is also incorporated herein by reference. This attenuated strain contains a deletion in the poly(C) tract of the 5' non-coding region of its genome. This attenuated strain protects mice from a challenge with virulent mengovirus and encephalomyocarditis virus (EMCV).

The advent of recombinant DNA technology has permitted the development of live recombinant vaccines .

There exists a need in the art for suitable vectors by which polypeptides and/or epitopes or antigens of human or animal pathogens can be incorporated resulting in modified live viruses that can be used in vaccines .

SUMMARY OF THE INVENTION

This invention helps satisfy the needs in the art by providing, inter alia, a viable modified attenuated mengovirus where a structural or non-structural protein of the mengovirus comprises a heterologous amino acid sequence, a fusion protein of the viable modified mengovirus, a permissive cell infected with the viable modified mengovirus, a recombinant nucleic acid (RNA or DNA) comprising the full- length sequence of the modified mengovirus, a vaccine, and a method of inducing an immune response.

This invention relates, inter alia, to a viable modified mengovirus wherein the modified mengovirus is an attenuated strain and comprises a heterologous nucleotide sequence. An embodiment of this invention relates to a viable modified mengovirus where modified mengovirus is an attenuated strain and comprises a heterologous nucleotide sequence coding for a heterologous peptide or protein. In a further embodiment, the viable modified mengovirus is an attenuated strain having a mutation or a deletion in the poly (C) tract of the 5' non-coding region of the genome of the mengovirus.

In particular, an embodiment of this invention relates to a viable recombinant mengovirus where the recombinant mengovirus is an attenuated strain having a deletion in the poly(C) tract of the 5' non-coding region of the mengovirus genome, and where the leader polypeptide of the recombinant mengovirus is full-length and comprises a heterologous amino acid sequence. In one specific embodiment of this invention the recombinant mengovirus contains amino acids 299-466 of gpl20 of the MN isolate of HIV-I inserted after amino acid 6 of the leader polypeptide.

Another embodiment of this invention relates to a fusion protein comprising a full-length leader polypeptide of an attenuated mengovirus strain into which a heterologous amino acid sequence is inserted.

In a further embodiment, this invention relates to permissive cells infected with a recombinant mengovirus of this invention. In specific embodiments the permissive cells are HeLa, VERO, BHK21, and P815 cells.

An additional embodiment of this invention relates to a recombinant nucleic acid molecule (RNA or DNA) comprising a mengovirus nucleic acid sequence and a heterologous nucleic acid sequence. Preferably, the heterologous sequence is inserted within the mengovirus sequence encoding the full- length leader polypeptide. More preferably, the recombinant nucleic acid molecule comprises the full-length attenuated mengovirus sequence and the heterologous sequence inserted within the full-length leader polypeptide sequence.

In yet another embodiment, this invention relates to a viral genome of a recombinant mengovirus of this invention.

Another embodiment of this invention relates to vaccines comprising a recombinant mengovirus of this invention. In specific embodiments the vaccines comprise the recombinant mengovirus in admixture with a pharmaceutically acceptable carrier.

An additional embodiment of this invention relates to a method of inducing an immune response comprising administering a recombinant mengovirus of the invention via a parenteral or oral route to an organism such as a human or animal, in which an immune response is to be induced. The invention also concerns immunogenic compositions. Such compositions comprise the recombinant mengovirus in admixture with a pharmaceutically acceptable carrier.

A further embodiment of this invention relates to a viable recombinant mengovirus of this invention further comprising protease cleavages sites between a heterologous amino acid sequence and the leader polypeptide. In a specific embodiment of this invention, the protease cleavage site is a protease 3C cleavage site.

In yet another embodiment, this invention relates to a permissive cell infected with a viable recombinant mengovirus of this invention, where the permissive cell expresses a heterologous amino acid sequence in native form. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic depiction of the organization of the mengovirus genome.

Figure 2 is a plasmid map of pM16 depicting several restriction sites, the location of the T7 promoter (arrow), and the sequences derived from pBluescribe M13(+) (designated as pBS, thin line) and the cDNA sequences derived from mengovirus (heavy line).

Figure 3 is a plasmid map of p05156S.

Figure 4 is a plasmid map of pMRA-1. The heavy line refers to DNA sequences from mengovirus. The thin line refers to sequences from pBluescribe M13(+). The arrow refers to the T7 promoter. Various restriction sites are identified. The nucleotide numbering system refers to the position of nucleotides in the recombinant plasmid. Figure 5 is a plasmid map of pMRA-2. The heavy line refers to DNA sequences derived from mengovirus . The thin line refers to sequences from pBluescribe M13(+). The arrow refers to the T7 promoter. The stippled line refers to the sequence of Δgpl20-VCN. Various restriction sites are identified. The nucleotide numbering system refers to the position of nucleotides in the recombinant plasmid.

Figure 6 is a plasmid map of pMRA-3. The heavy line refers to DNA sequences derived from mengovirus . The thin line refers to sequences from pBluescribe M13(+). The arrow refers to the T7 promoter. The stippled line refers to the sequence of Δgpl20-VCN. Various restriction sites are identified. The nucleotide numbering system refers to the position of nucleotides in the recombinant plasmid.

Figure 7 is a diagram of the vMLN450 gene organization and the sequence of the recombinant L protein. Amino acids (aa) derived from mengovirus are written in plain letters, while amino acids derived from HIV gpl20 are written in bold letters. Amino acids derived from linkers are underlined.

Figure 8 depicts the results of a plaque assay. Fig. 8a is the plaque phenotype of vM16 and Fig. 8b is the plaque phenotype of VMLN450 stained after 72 hours.

Figure 9 depicts Reverse Transcription PCR (RT-PCR) results . RT-PCR was performed with oligonucleotide pairs M- VDW-l/3'VCN for lanes a-e and with oligonucleotide pairs M- VDW-l/M-1094 for lanes g-k. The templates used for the reactions were a) VMLN450 RNA, b) VM16 RNA, c) negative control, d) pM16, e) pMRA-3, g) VMLN450 RNA, h) vM16 RNA,* i) negative control, j) pM16, and k) pMRA-3. Lane f) contains bacteriophage lambda DNA cut with Hindlll .

Figure 10 depicts the sequence of the Δgpl20-VCN region at the DNA level (plus strand) and the oligonucleotides used to sequence vMLN450 RNA derived PCR products. The boxed sequence is restriction site Ncol.

Figure 11 is a 12% SDS-PAGE gel of cytoplasmic extracts of a) mock infected HeLa cells, c) vM16 infected HeLa cells, e) VMLN450 infected HeLa cells. Immunoprecipitations of cytoplasmic extracts using MAb50.1 are shown in lane b) for mock infected cells, lane d) for vM16 infected cells, and lane f) VMLN450 infected cells.

Figure 12A depicts the results of an ELISA assay for sera obtained from mice infected with VMLN450, vM16, and a virus free control using gpl60 MN-LAI as an antigen.

Figure 12B depicts the results of an ELISA assay for sera obtained from Balb/c mice infected with vMLN450, vM16, and a virus free control using gpl60 LAI as an antigen. The reactivity of Balb/c sera 2 weeks after a first immunization (filled bars) and 2 weeks after a second immunization (stippled bars) with gpl60 LAI are shown. Titers are given as reciprocal values of serum dilution giving an O.D. at 490 n of 1.

Figure 12C depicts the results of an ELISA assay for sera obtained from CBA mice infected with vMLN450, vM16, and a virus free control using gpl60 LAI as an antigen. The reactivity of CBA sera 2 weeks after a first immunization (filled bars) and 2 weeks after a second immunization (stippled bars) with gpl60 LAI are shown. Titers are given as reciprocal values of serum dilution giving an O.D. at 490 nm of 1.

Figure 12D depicts the results of an ELISA assay for sera obtained from Cynomolgus monkeys infected with vMLN450, vM16, and a virus free control using gpl60 LAI as an antigen. The reactivity of Cynomolgus monkey preimmune sera (filled bars) and sera 4 weeks after immunization (stippled bars) with gpl60 LAI is shown. Titers are given as reciprocal* values of serum dilution giving an O.D. at 490 nm of 1.

Figure 13 is a diagram of the mengovirus polyprotein showing a protease 3C cleavage site at the L-VP4 junction.

Figure 14 is a diagram of the polyprotein of the recombinant mengovirus vMQG-1 showing the amino acid sequence of the Δgpl20-QG-L junction for vMQG-1 and vM16.

Figure 15 is a flow diagram of the procedure used to construct pMRA-5. Figure 16 depicts the nucleic acid sequence of pM16. The viral sequences are indicated with Us, i.e. as an RNA sequence, and the plasmid sequences are indicated with Tε, i.e. as a DNA sequence. pM16 is a DNA plasmid.

Figure 17 depicts the nucleic acid sequence of pM16-l. The first base of this sequence is the first viral base. The viral sequences are indicated by Us, i.e. as an RNA sequence, and the plasmid sequences are indicated by Ts, i.e. as a DNA sequence. pM16-l is a DNA plasmid.

Figure 18 is a comparison of the sequence of pM16 and pM16-l.

Figure 19 depicts the construction of pMLN450. The cDNA sequence encoding amino acids 299 to 445 of HIV-IMN gp!20 was inserted between amino acids 5 and 6 of the L polypeptide in pM16 cDNA at the beginning of the viral polyprotein open reading frame (A) . The sequence of the resulting fusion protein, Δgpl20-L is shown in (B). Leader amino acids are represented in normal type and gpl20 amino acids in bold characters . Additional residues encoded by the DNA linkers are underlined.

Figure 20 depicts the plaque phenotype of vM16 (A) and VMLN450 (B) viruses. Parental and recombinant viral plaques formed on HeLa cell monolayers were stained after 72 h incubation at 37°C. Each well has a diameter of 3.5 cm.

Figure 21 depicts the expression of Δgpl20-L in VMLN450 infected cells. Mock (lanes A, B), vM16 (lanes C, D) and

VMLN450 (lanes E, F) infected HeLa cells were labelled with

35 S-methionine. Cytoplasmic extracts were prepared at 7 h postinfection and analyzed by 12% SDS-PAGE as described previously (12). Some samples (lanes B, D, F) were immunoprecipitated (13) with MAb 50.1 at 2xg/ml before loading on the gel . The migration of Mengovirus marker proteins is indicated.

Figure 22 depicts the Construction of pLCMG4. Figure

22a is a portion of the protein sequence and corresponding

DNA sequence or pMCS. Figure 22b is a portion of the protein sequence and corresponding DNA sequence of pLCMG4. The sequence of the Leader peptide region at the beginning of the Open Reading Frame is displayed. Restriction sites are indicated. The cDNA sequence coding for the LCMN NP sequence (boxed sequence) was inserted between the sites SnaBI and Nhel of the plasmid pMCS. Underlined sequences result from DNA linkers.

Figure 23 depicts the plaque phenotype of vM16 and vLCMG4 virus resulting from transfection of HeLa cells. Cells were grown in 3.5cm wells and coloured after 48 hours.

Figure 24 depicts a double-stranded oligonucleotide containing restriction sites Xhol, SnaBI, and Nhel.

Figure 25 depicts the protein sequence and cDNA sequence of the L-coding region of pM16. The position of the Xhol site is indicated.

Figure 26 depicts the L-coding region of PMCS. The new restriction sites are indicated. Non-mengovirus amino acids resulting from DNA linkers are boxed.

Figure 27 depicts cytoplasmic extracts and radioimmunoprecipitations . Lane 1 is a radioi munoprecipitation of vMG-24 infected cytoplasmic extracts with monoclonal antibody RV2-22C5. Lane 2 is a cytoplasmic extract of vMG-5-24 infected cells. Lane 3 is a radioimmunoprecipitation (mAb RV2-22C5) of vM16 infected cells. Lane 4 is a cytoplasmic extract of vM16 infected cells. Lane 5 is a radioimmunoprecipitation (mAb RV2-22C5) of mock infected cells. Lane 6 is a cytoplasmic extract of mock infected cells.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention described and claimed herein may be more fully understood, the following detailed description of embodiments of this invention is provided.

Within this description various terms of art are employed. These terms are generally used in their ordinary and well recognized sense. Various terms that are employed throughout this description are defined infra.

As used herein, the term "recombinant virus" refers to a genetically modified virus. A recombinant virus can comprise protein or nucleic acid from at least one other organism. Thus, a recombinant virus can refer to a virus expressing a non-structural heterologous polypeptide as well as viruses comprising a heterologous polypeptide as a structural element. Moreover, a recombinant virus can be a chimaeric virus .

As used herein, the term "attenuated strain" refers to a strain with reduced disease-producing ability and/or pathogenicity.

As used herein, the term "genome" refers to the nucleic acid comprising all the genes of a species. The nucleic acid making up the genome may be RNA or DNA depending on the nature of the species. For example, the genome of picornaviruses or other RNA viruses is made up of RNA, while the human genome is made up of DNA.

As used herein, the term "heterologous" refers to a substance not naturally found in a given species . For example, the term "heterologous amino acid sequence" when used with reference to a specific virus refers to an amino acid sequence not found in that virus, e.g., the proteins of that virus .

As used herein, the term "nucleotide or nucleic acid sequence" refers to a linear series of nucleotides connected by covalent bonds between the 3' and 5' carbons of adjacent nucleotides . A nucleotide or nucleic acid sequence may be an RNA sequence or a DNA sequence.

As used herein, the term "amino acid sequence" refers to a linear series of amino acids connected by covalent bonds'.

As used herein, the term "fusion protein" refers to a protein comprising at least two amino acid sequences, where one of the amino acid sequences is not normally found together in nature with the other amino acid sequencer ). For example, a mengovirus fusion protein can comprise a mengovirus amino acid sequence covalently linked to a heterologous amino acid sequence.

As used herein, the term "epitope" refers to a configuration of amino acids in a protein, where the configuration of amino acids is associated with an immune response. For example, an epitope can be defined by an antigenic motif that is recognized by an antibody and that can induce an immune response. An epitope may be, but is not limited to, a linear sequence of amino acids.

As used herein, the term "permissive cell" refers to n cell that can be productively infected with a virus. Thu.-., a permissive cell to mengovirus, is a cell that can be infected by mengovirus .

As used herein, the term "recombinant nucleic acid molecule" refers to a hybrid nucleotide sequence (RNA or DNA) comprising at least two nucleotide sequences placed together by in vitro manipulation or a clone thereof .

As used herein, the term "cDNA" refers to complementary DNA. In the case of organisms whose genome is comprised of DNA, the cDNA is complementary to mRNA or a fragment thereof. In the case of organisms whose genome is comprised of RNA, the cDNA is complementary to the genome of the organism or a fragment thereof.

As used herein, the term "polypeptide" refers to a linear series of amino acids connected one to the other by peptide bonds. The term "polypeptide" includes but is not limited to proteins .

As used herein, the term "expression" refers to the process of producing a polypeptide from a structural gene.

As used herein, the term "polyprotein" refers to a covalently linked linear series of amino acids comprising more than one protein. In some cases, proteins constituting a polyprotein can be released by endoproteolytic cleavage by a specific protease.

The genomic organization of mengovirus is shown in Figure 1. This figure depicts mapping of the viral polypeptides to the genome as well as various intermediates in the processing of the polyprotein to mature components of the virus.

The poly(C) region is also identified in Figure 1. As described by Duke et al. , supra, and Duke and Palmenberg, supra, deletions in this region are associated with an attenuated phenotype. Consequently, plasmids containing cDNA of the genome of mengovirus with mutations, e.g., substitutions and deletions, in the poly(C) region can be used as the source of mengovirus DNA for the construction of various embodiments of this invention relating to recombinant mengoviruses exhibiting an attenuated phenotype.

In preferred embodiments of this invention, a suitable source of mengovirus nucleic acid is plasmid pM16. pM16 has been deposited at the Collection Nationale de Cultures de Micro-organismes (C.N.C.M.) in Paris, France on June 2, 1993 under accession number 1-1313. A partial plasmid map of pM16 is shown in Figure 2 and the sequence of pM16 is shown in Figure 16. This plasmid encodes a mutated poly(C) tract of C- UC-_η, but otherwise comprises a DNA sequence corresponding to the full-length genome of mengovirus inserted between the £^*c*oRI and BaιriH.1 restriction sites of the double-stranded replicative form vector pBluescribe M13(+). Consequently, this plasmid contains the mengovirus cDNA downstream from the T7 promoter.

In other embodiments of this invention, a suitable source of mengovirus nucleic acid is pM16-l. pM16-l has also been deposited at the C.N.C.M. on June 2, 1993 under accession number 1-1312. The sequence of pM16-l is shown in Figure 17.

In other embodiments of this invention other plasmids may be used as a source of mengovirus nucleic acid. For example, pM18, encoding a C_R poly(C) tract, or pMl9, encoding a C, poly(C) tract or a plasmid containing a complete deletion of the poly(C) tract can be used. If an attenuated phenotype is not required pM . , encoding the wild type poly(C) tract C_t-_nUC,_n, can be used. As each of these plasmids contains DNA complementary to the mengovirus genome outside the poly(C) tract, one plasmid can be constructed from another mengovirus plasmid (or wild type mengovirus DNA) by various in vi tro manipulations. One possibility is the replacement of the EcdRV - Avrll restriction fragment containing the poly(C) tract from one plasmid with the ap¬ propriate EcόRV - Avrl l fragment of the plasmid to be constructed.

Once a vector encoding an attenuated mengovirus genome in DNA form has been obtained, a heterologous nucleotide sequence encoding an amino acid sequence to be expressed by the recombinant mengovirus can be inserted within the coding region of the mengovirus genome. In specific embodiments of this invention the nucleic acid sequence codes for a heterologous antigen or epitope.

The site at which the heterologous nucleotide sequence is inserted can be at a restriction site. In specific embodiments of this invention, the site is at the Ncol restriction site encompassing nucleotide 729 of the mengovirus genome.

When the heterologous nucleotide sequence is inserted at a restriction site, the mengovirus DNA vector is restricted with the appropriate enzyme to cleave the DNA vector. The heterologous DNA sequence is then ligated to the restricted mengovirus vector to produce a recombinant DNA molecule comprising the mengovirus genome -- now including the heterologous nucleotide sequence.

When the heterologous nucleotide sequence is not inserted at a restriction site, one of ordinary skill in the art can select a restriction fragment of the DNA vector comprising the insertion site. A synthetic DNA fragment can then be synthesized that corresponds to the selected restriction fragment but additionally includes the heterologous nucleotide sequence inserted at the desired site. The synthetic DNA fragment can then be inserted in place of the selected restriction fragment in the DNA vector to generate a recombinant DNA molecule comprising a recombinant mengovirus genome cDNA.

The heterologous nucleotide sequence can be prepared in a variety of ways. For example, the sequence may be obtained by specifically cleaving cDNA encoding the heterologous polypeptide to be expressed by the recombinant mengovirus . For example, this may be accomplished using appropriate restriction enzymes. Alternatively, the heterologous nucleotide sequence can be chemically synthesized using methods well known in the art.

Recombinant mengoviruses and proteins or expression products thereof that comprise a desired heterologous polypeptide, e.g., an antigen or epitope, can be obtained by generating an RNA transcript from the recombinant DNA molecule comprising a heterologous nucleotide sequence inserted into the recombinant mengovirus genome. For example, in the case of pM16 and pMl6-l an RNA transcript can be produced j-n. vitro using T7 RNA polymerase. Alternative promoters and corresponding polymerases can be substituted. Aliquots of the transcription mixture can be used to transfect permissive cells. For example, the DEAE Dextran, calcium phosphate, poly-ornithin, electroporation, and synthetic transfection agents can be used to transfect mammalian cells such as HeLa, VERO, BHK21, and P815. The production of progeny viruses can be monitored by microscopy, and the viruses can be released by well known methods of cellular disruption, for example, freezing and thawing.

In specific embodiments of this invention, the heterologous polypeptide is inserted within the leader polypeptide (L) . It was determined that insertion of the foreign epitope at 6 amino acids from the N terminus of the mengovirus polyprotein does not render L non-functional and thus does not interfere with the multiplication of the virus either jLn vitro or j-n_. vivo.

Other insertion sites may be chosen within the viral genome for insertion at positions where the insert does not interfere with functions that are important for the viral life cycle. If there are no restriction sites at a suitable insertion site they can be introduced, e.g. by site directed mutagenesis. Thus in principle, all restriction sites and other locations within the genome can be envisaged for insertion with the exception of well defined functional areas, e.g. the catalytic triad of 3C. Preferred sites include non-structural regions of the genome, e.g., P2, P3 and/or regions corresponding to the N- or the C- terminus of viral proteins.

In order to produce a fusion protein comprising L and a foreign polypeptide, such as an antigen or epitope, the foreign nucleotide sequence is inserted into the mengovirus cDNA within the L polypeptide coding sequence in such a way as to conserve reading frame. The recombinant virus can then express the foreign sequences as part of the viral polyprotein. The polyprotein is processed, inter alia, to the form of a fusion protein comprising the L polypeptide with the heterologous polypeptide inserted therein. The fusion protein can then be obtained from the cytoplasm of infected cells.

In terms of this invention the heterologous DNA sequence inserted into a mengovirus vector can encode any amino acid sequence. In certain embodiments of this invention the amino acid sequence comprises a heterologous epitope. In these embodiments, the heterologous amino acid sequence can comprise several foreign epitopes or a single foreign epitope, or it can define an epitope together with other amino acids, e.g. mengovirus amino acids. In other embodi¬ ments, the amino acid sequence can consist essentially of a heterologous epitope. By way of example, amino acid se¬ quences of various embodiments of this invention are listed in Table I .

Table I

In embodiments of this invention the heterologous DNA sequence to be inserted is generally in the range of about 700-1,100 bases. Sequences given in Table I are examples of heterologous sequences for the construction of recombinant mengovirus of the invention. Recombinant mengoviruses containing heterologous sequences of species not given in the Table can be constructed as indicated herein. In cases where the heterologous sequence is too large to be expressed in mengovirus a set of recombinant mengoviruses each expressing a part of the given protein can be constructed.

The heterologous DNA sequence of this invention can encode more than one polypeptide. The DNA sequences encoding the polypeptides can be directly linked to each other or they can be separated by a joining sequence. In specific embodiments, these joining sequences can encode a cleavage site.

In embodiments of this invention the L polypeptide of the recombinant virus comprises a segment of gpl20 of HIV-I. In specific embodiments, the L polypeptide comprises amino acids 299-446 of gpl20 of the MN strain of HIV-I. In more specific embodiments, amino acids 299-446 of gpl20 of the MN strain of HIV-I are inserted after amino acid 6 of the L polypeptide. This HIV sequence comprises sequences coding for the V3 loop, which constitutes the principal neutral¬ ization determinant (PND) and sequences downstream involved in binding of the gpl20 molecule to the CD4 receptor. The resulting recombinant mengovirus expresses an HIV-I gρl20 - mengovirus L fusion protein that was recognized by HIV-I specific antibodies and induced anti HIV-I antibodies in animals .

The HIV-I gpl20 - mengovirus L fusion protein also induced a gρl20 - specific cytotoxic immune response in animals .

In terms of antigenicity, an L fusion protein comprising a foreign epitope can retain the ability to induce and bind antibodies directed to the native protein sequence. Hence, the strategy could be applied to any foreign protein that exhibits antigenic properties of interest. Consequently, in cases of single well-defined and short epitopes, the construction of recombinant mengoviruses, containing these epitopes in a larger protein, is the strategy of choice.

Recombinant viruses of this invention have been shown to induce antibodies in mice and cynomolgus monkeys that bind to the protein and/or neutralize the pathogen from which the foreign sequences are derived. Therefore, the induction of an immune response in other animal species susceptible to mengovirus, such as humans, is predicted based on the in vitro and , vivo results obtained. Thus, a protective immune response may be elicited by recombinant viruses of this invention. For example, a recombinant virus expressing sequences of the G protein of rabies can be engineered in accordance with this invention for use as a vaccine in animals, including mice. Similarly, a recombinant virus ex¬ pressing sequences from the glycoprotein of HTLV-1 could be obtained for use in macaques, other primates, or humans.

The antigenicity and immunogenicity of proteins comprising foreign epitopes expressed by the recombinant mengoviruses can be improved in various ways: the size of the heterologous nucleotide sequence encoding the foreign epitope can be increased to express larger segments of the foreign antigen (or the whole antigen) up to a maximum size of about 350 amino acids, the foreign antigen can be expressed in native form rather than as a fusion protein, and selective targeting of the foreign antigen to appropriate cell compartments can be achieved to allow post-translational modifications, such as glycosylation, that can be important for the antigenicity and/or immunogenicity of the fusion protein.

In embodiments of this invention, the recombinant mengovirus can express multiple sequences of one protein. In addition, the recombinant mengovirus can comprise multiple sequences from different proteins. Thus, this invention makes it possible to immunize or induce an immune response against multiple pathogens at the same time.

In certain embodiments of this invention a heterologous polypeptide can be expressed in native form by including protease cleavage sites between the amino acid sequence of the heterologous polypeptide and the mengovirus sequences .

In preferred embodiments of this invention mengovirus protease 3C cleavage site is used. The endogenous mengovirus protease 3C is responsible for most cleavages of the mengovirus polyprotein. For example protease 3C mediates the cleavage between the L-peptide and VP4(1A) by specifically cleaving precursor protein L-P1-2A at a Q-G amino acid linkage yielding free L-peptide and P1-2A.

Figure 13 is a depiction of the mengovirus genome with this protease 3C cleavage site indicated. The amino acid sequence in Figure 13 is a sufficient substrate for cleavage by protease 3C. See Parks et al . , J. Virol. 63: 1054 (1989), the entire disclosure of which is incorporated herein by reference. In embodiments of this invention, a DNA sequence coding for the protease 3C cleavage site can be included at each end of the heterologous DNA sequence to be inserted. In addition, the sequence Asn-Pro-Gly-Pro, which is the cleavage site between viral protein 2A and 2B can be used. There is evidence that the cleavage between 2A and 2B occurs by scission of the glycine proline bond through an autocatalytic mechanism. When such a heterologous DNA sequence is used to construct a recombinant mengovirus, the resulting heterologous polypeptide can be released from the mengovirus polypeptides by autocatalytic cleavage during infection.

The heterologous polypeptide can be targeted to specific cell compartments. For example, the incorporation of a nuclear localization sequence, such as that of the SV40 T antigen, should target the protein to the nucleus. By contrast, a signal sequence, such as that of /?-2-microglobulin should target the polypeptide to the endoplasmic reticulum to permit post-translational modifica¬ tions, e.g. glycosylation, and secretion into the medium. Moreover, a sequence such as the transmembrane sequence of gp41 of HIV-I can be used as an anchoring sequence to allow the polypeptide to be inserted into the cell membrane.

In further embodiments two or more different proteins may be coexpressed in the same cell by coinfecting the target cell with two or more recombinant viruses . This can permit various intermolecular interactions in the coinfected target cell .

The recombinant mengovirus of the invention can be used as a vaccine or as part of an immunogenic composition. The subjects to be vaccinated include man, primates, non-human primates, mammals, mice, or any other animal that can be infected by mengovirus . In embodiments of this invention the recombinant mengovirus is ' present in the vaccine or immunogenic composition in admixture with a pharmaceutically acceptable carrier. Examples of suitable carriers include any buffer that supports virus stability and is acceptable for use in animals or humans. For a live vaccine an adjuvant is not necessary. In other embodiments of this invention a vaccine or immunogenic composition comprises a mengovirus fusion protein of this invention in admixture with a pharma¬ ceutically acceptable carrier, wherein the fusion protein comprises a heterologous amino acid sequence comprising an antigen.

The following properties of mengovirus make it particularly attractive as a live vaccine or an immunogen with wide potential.

1. The vM16 strain and other attenuated strains described herein are not likely to easily revert to virulence as their attenuation results from a deletion. It may be possible to use a genetically engineered mengovirus strain with the entire poly(C) tract deleted in the context of this invention.

2. The wide host range of mengovirus permits its use as a vaccine or immunogen in many different animal species for a wide variety of pathogens of medical or veterinary importance, e.g. HAV, HIV, HTLV, Rabies, FMDV, coronaviruses such as bovine, herpes virus, measles, mumps, and Respiratory Syncitial Virus (RSV) .

3. The use of mengovirus in a vaccine in the form of a live virus able to replicate in an organism after either parenteral or oral administration should permit the induction of both a humoral immune response and a cellular immune response, in particular a cytotoxic T-cell response.

4. The ability of mengovirus to multiply in the intestine after oral immunization should permit the induction of both a systemic general immune response and a local mucosal response. This is particularly relevant in the case of pathogens such as HIV, HPV HTLV, TGEV or rotaviruses .

The recombinant viruses of this invention can be used in a variety of non-immunological ways. For example, the recombinant virus can be used as a cloning or expression vector in order to produce large amounts of a desired nucleic acid or protein, e.g. in tissue culture. A heterologous protein expressed in cells infected with a recombinant mengovirus can be purified in large quantity from the infected cells.

In addition, the recombinant viruses of the invention could be used to deliver specific inhibitors of various pathogens or of cellular functions responsible for disease to various cellular or subcellular locations.

The following examples are given by way of illustration to facilitate a better understanding of the invention and are not intended to limit the invention. It should be further understood that the detailed description while indicating preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.

Examples

MATERIALS AND METHODS

Construction of VMLN450.

All DNA manipulations were performed according to standard procedures (7). Subclone pMRAl was generated by deletion of the 5.8kb Sph I - Sph I fragment (plasmid base 1928-7775) from plasmid pM16, which contains the vM16 Mengovirus cDNA downstream of the T7 promoter (5). HIV-IMN gpl20 specific DNA was amplified by PCR from plasmid pTG5156 (Transgene SA, Strasbourg), kindly provided by M. P. Kieny, using oligonucleotides 5' VCN

(ATATGTTGACCATGGAACAAATTAATTGTACAAGACCC) and 3 'VCN (TAATCCATGGCGGTCAACGTGGGTGCTACTCCTAATGG) . The PCR fragment was first cloned into pMRAl at the Nco I site (viral base 729) resulting in plasmid pMRA2. Full-length cDNA was re-established by transfer of the 5.8kb fragment of pM16 into the Sph I site of pMRA2, resulting in plasmid pMRA3. Correct orientation of cloned sequences was determined by PCR and plasmids were amplified in E. coli DH5α (Promega).

Infectious RNA transcripts derived from pMRA3 were prepared using T7 RNA polymerase and transfected into HeLa cells as described (5), resulting in recombinant Mengovirus vMLN450. Stocks of vM16 and vMLN450 were produced by passage on HeLa cells, titrated as described (8), and analyzed by RT-PCR for the presence of the inserted HIV-I sequence using Mengovirus-specific oligonucleotides, 5'194- (TAGGCCGCGGAATAAGGCCGGTGTGC) and 3 '1094- (GGAGCATGTTCGAGAAAGCATTGAC) .

Immunizations and ELISA tests.

Ten week old BALB/c mice were immunized twice intraperitoneally with 10 pfu (plaque forming units) of

VMLN450 or 10 pfu of vM16 in PBS on days 0 and 56. Control animals received PBS alone. Adult cynomolgus monkeys were immunized intramuscularly with 10 pfu of vMLN450 or vM16 in

PBS. ELISA plates (Nunc Maxisorb) were coated with 50ng per well of purified recombinant gpl60LAI or gpl60MN-LAI

(Transgene), incubated with serial dilutions of sera, followed by horseradish peroxidase conjugated sheep anti mouse-IgG (H+L) antibody (Diagnostics Pasteur) or rabbit anti monkey-IgG (H+L) antibody (Nordic). ELAVIA tests (Diagnostics

Pasteur) were carried out following the manufacturer's protocol. HIV-IMN neutralization assays were performed as

5 described (9) using 3.5x10 MT4 cells. Syncytia formation was monitored between days 6 and 10.

Example 1 Construction of pMRA-3 In order to facilitate cloning in the Ncol site 729 of the Mengovirus cDNA, a deletion clone of pM16 was constructed by elimination of the 5.8 kb Sphl ( 1928 ) - Sphl ( 7775 ) fragment, which contains two Ncol sites. The resulting clone, designated pMRA-1 (Figure 4), contains a unique Ncol site at position 729. A 478 bp HIV-I-MN specific DNA fragment was generated using Polymerase Chain Reaction (PCR) from plasmid p05156S with the following oligonucleotides:

5 'VCN: ATATGTTGACCATGGAACAAATTAATTGTACAAGACCC 3'VC : TAATCCATGGCGGTCAACGTGGGTGCTACTCCTAATGG. Plasmid p05156S is depicted in Figure 3 and was obtained from Transgene S.A., Strasbourg, France. p05156S is described in patent application W092/19742. The MN sequence of the genome has been published by Gurgo et al., 1988. Virology 164: 531- 536 (1988), which is incorporated herein by reference.

The amplified sequence, Δgpl20-VCN, corresponds to amino acids 299 to 446 of the glycoprotein 120 of HIV-T-MN. Δgpl20-VCN was generated with oligonucleotides 5 'VCN and 3'VCN at a concentration of about 100 mM on 5 ng of template, p05156S with the PFU Polymerase obtained from Stratagene using a PFU amplification buffer also obtained from Stratagene and standard thermocycling conditions.

The Δgpl20-VCN fragment was then cloned into Ncol site 729 of pMRA-1 to yield pMRA-2. pMRA-2 is depicted in Figure 5. The correct orientation and length of the Δgpl20-VCN insert in pMRA-2 was confirmed by PCR, using the oligonucleotides M-1094, 5'VCN and 3'VCN. The oligonucleotide sequence of M-1094 is:

M-1094 : GGAGGCATGTTCGAGAAAGCATTGAC. Correct clones gave an 800 bp PCR fragment with M-1094 and 5'VCN and no amplified DNA with M-1094 and 3'VCN, whereas clones containing Δgpl20-VCN in the incorrect orientation gave the opposite results .

The full-length cDNA was reconstituted by cloning the 5.8 kb Sp (1928) - Sphl ( lll ) fragment from pM16 back into pMRA-2, which resulted in the plasmid pMRA-3. pMRA-3 is depicted in Figure 6. This plasmid contains 459 additional bp inserted in frame in the L-peptide coding region of the mengovirus cDNA, and codes for the 25.8 kD fusion protein LN450 (Figure 7) .

The correct orientation of the 5.8 kb Sp (1928) - Sphl ( 1115 ) fragment of pM16 in pMRA-3 was confirmed by a double restriction digest with BarriΑl (Boehringer Mannheim) and HϊΛdll l (Boehringer Mannheim) . This double restriction digest of a plasmid in the correct orientation results in two DNA fragments of 30 and 11356 bases. Whereas, the incorrect clones exhibit two DNA fragments of 5821 and 5565 bases in length.

Example 2 Growth characteristics of VMLN450

Infectious RNA transcripts of VMLN450 and vM16 cDNA were produced in. vitro by incubating T7 RNA Polymerase (Pharmacia) with Bairill digested plasmids pM16 and pMRA-3 in an appropriate buffer. Aliquots of this transcription mixture were adjusted to 1 mg/ml DEAE Dextran and subsequently transfected into HeLa cells by incubating the solution on monolayer cells for 30 minutes, as described by Sylvie van der Werf et al . , Synthesis of Infectious Poliovirus RNA by Purified T7 RNA Polymerase, Proc. Natl. Acad. Sci. (USA) 83:2330 (1986), the entire disclosure of which is incorpo¬ rated herein by reference.

Transfection of RNA derived from plasmid pMRA-3 into HeLa cells resulted in production of the virus VMLN450. The production of progeny virus was monitored by microscopy. Stock virus was grown by infection of a confluent monolayer of HeLa cells at a multiplicity of infection (MOI) of 1. Virus was harvested after complete cpe (cytopathic effect). Intracellular virus was released by freezing and thawing cells three times and pelleting cellular debris and nuclei by centrifugation. Complete cpe was observed after 72 hours, compared to 48 hours when cells were transfected with pM16 RNA. vMLN450 grows to high titers, albeit approximately 3 to 4 times lower than vM16.

The plaque phenotype of the recombinant viruses was tested. Virus stock solutions were diluted in Dulbecco's Modified Eagle Medium (DMEM) without Fetal Calf Serum (FCS) and used to infect a confluent monolayer of HeLa cells. After 30 minutes incubation, cells were covered with DMEM containing 0.9% soft agar. The cells were then incubated for 72 hours at 37°C and intact cells were stained with 0.2% crystal violet solution.

The plaque phenotype of the recombinant viruses harvested from culture supernatant gives medium-size plaques with an average diameter of approximately 65% that of wild- type-virus plaques (Figure 8).

Example 3 Genetic stability of vMLN450

The transfection supernatant containing VMLN450 was passaged three times on HeLa cells, and the viral RNA was extracted. Viral RNA from passage 3 of the recombinant virus VMLN450 was analyzed by RT-PCR (Reverse Transcription PCR), using the oligonucleotides 5 'VCN, 3 'VCN, M-1094 and M-VDW-1. The sequence of oligonucleotide M-VDW-1 is: TAGGCCGCGGAATAAGGCCGGTGTGC.

Sequence analysis was performed on RT-PCR products by a PCR sequencing method derived from the Sanger dideoxy sequencing method described by Adams and Blakesley, Focus 13:56-57 (1991) and Adams and Blakesley, Focus 14:31-33 (1992). PCR fragments were extracted from low-melting point agarose gels and approximately 200 ng of DNA was added to each of four reaction mixtures, each containing different concentrations of dNTP's and ddNTP's and 4 pmol of 32P-end labelled primer. The reaction was started by adding TAQ Polymerase (Amersham) and incubating for 20 rounds of am¬ plification at standard thermocycling conditions. The sequencing reaction was analyzed by polyacrylamide gel electrophoresis (PAGE) under standard conditions.

Analyses with oligonucleotide pairs M-VDW-1 / M-1094 and 5'VCN / M-1094 demonstrated that specific DNA fragments, 1382 bp and 932 bp respectively, could be amplified from vMLN450 RNA. There was no apparent difference in size between these DNA fragments and DNA fragments generated from plasmid pMRA- 3. The results of these experiments are shown in Figure 9.

No wild-type-size fragments, indicating a reversion to vM16 like viruses, could be detected. The 1382 bp fragment was partially sequenced to further exclude non-specificity. A partial sequence obtained for vMLN450 is depicted as an underlined sequence in Figure 10 and shows no difference from that of the original insert.

Example 4 Expression of LN450 in HeLa cells

In order to analyze expression of the fusion protein

LN450, HeLa cells in monolayer were infected with VMLN450 at a MOI of 10 and labelled with 35S methionine from 5 1/2 to 7

1/2 hours post infection, essentially as described by Lee and

Wimmer, Virology 166: 405 (1988) which is incorporated herein by reference. At 7 hours post infection, cells were lysed with NP40 and cytoplasmic extracts were prepared as described by Harber et al., Journal of Virology 65: 326 (1991) which is incorporated herein by reference. The 35S methionine labelled proteins were then analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Gel electrophoresis and autoradiography were carried out according to standard conditions .

HeLa cells were infected with VMLN450 and vM16 respectively and viral proteins were labelled with 35S- methionine. Cytoplasmic extracts were prepared at 7 1/2 hours post infection and aliquots analyzed subsequently by

SDS-PAGE. VMLN450 infected cells show the presence of a single additional protein with an apparent molecular weight of 26 kDa (Figure 11, e), which is absent in vM16 or mock infected cells (Figure 11, a, c). The molecular weight of this protein corresponds to the expected size for LN450.

Example 5

Antigenicity

Antigenic properties of LN450 were assayed by im- munoprecipitation using monoclonal antibody 50.1 (obtained from Repligen).^* Aliquots of 35S methionine labelled VMLN450 infected cell lysate were incubated with lμg of MAb 50.1 for 1 hour at 4°C, then an equal volume of a protein A-Sepharose suspension was added and the mixture was incubated for 1 hour. The protein A-sepharose complexes were pelleted, washed three times and analyzed by SDS-PAGE followed by autoradiography.

Cellular extracts of vMLN450, vM16 and mock infected HeLa cells were immunoprecipitated with the monoclonal antibody 50.1 (MAb 50.1). This antibody recognizes the V3 loop of the MN strain of HIV-I, the principal neutralizing determinant of HIV-I-MN. The data shown in Figure 11, b), d) and f) demonstrate that only the 26 kDa protein that is produced in VMLN450 infected cells, can be immunoprecipitated with MAb 50.1, confirming the identity of the 26 kd protein as LN450.

Example 6 Immunogenicity

In one experiment, ten week old Balb/c or CBA mice were immunized intraperitoneally with 10 pfu of vMLN450 (4 animals for Balb/c and 5 animals for CBA) or 10 pfu of M16

(4 animals for Balb/c and 2 animals for CBA) in PBS. Three animals (Balb/c) or 2 animals (CBA) received PBS as a virus free control. Animals were boosted 8 weeks after the initial immunization with an equal dose of virus. Blood samples were taken 14 days after immunization and sera were prepared.

Cynomolgus monkeys (Macaca fasciculariε ) were immunized

5 intramuscularly with approximately 4 x 10 pfu of VMLN450 (3 animals) or vM16 (1 animal) in PBS. Blood samples were taken

28 days after immunization and sera were prepared.

ELISA plates (Nunc Maxisorb) were coated with 100 μl per well of recombinant gpl60 MN-LAI or gpl60 LAI (500 ng/ml in PBS). gpl60 MN-LAI is a fusion protein containing the gpl20 moiety from the MN viral strain, and gp41 from the IIIB

(LAI) strain. The plates were washed, and incubated with serial dilutions of mouse or monkey sera. After incubation for 2 hours at 37°C, plates were washed and a monoclonal antibody (antimouse IgG (H+L) (Diagnostics Pasteur) or antimonkey IgG (H+L) (Nordic Immunology) ) conjugated to horseradish peroxidase was added and incubated for 1 hour at

37°C. After a final wash the test was developed by adding an

OPD (670 μg/ml), H„0₂ (0.042%) solution. The reaction was stopped by addition of 4N H-SO. and the O.D. was read at 490 nm in a Dynatech ELISA plate reader.

Serial 1 in 2 dilutions starting from a 1/100 dilution of mouse sera were tested in a syncitia inhibition assay.

The diluted sera were added to a virus solution, containing

HIV-I-MN at the minimal concentration inducing syncitia formation. The mixture was incubated for one hour and a

5 suspension of 3.5x10 MT4 cells were added. Cells were grown in 48 well plates, diluted 1 in 4 at day 3, and syncitia formation was monitored between day 6 and 10.

Balb/c mice, CBA mice, or cynomolgus monkeys were immunized with either 10 pfu of vMLN450 in the case of mice or 2 x 10 pfu of VMLN450 in the case of monkeys, 10 pfu vM16 or a virus free control. The mice were bled two weeks after immunization and 10 weeks after immunization (2 weeks after boost). Sera were analyzed by ELISA, using recombinant gplδO MN-LAI or gpl60 LAI as antigen. The monkeys were bled 4 weeks after immunization and were analyzed similarly. The data shown in Figure 12 and Table 3 demonstrate that specific anti HIV antibodies were only detected in sera from mice infected with vMLN450.

Anti-HIV-I titers raised approximately 35 fold after the animals were boosted with VMLN450 and remained at a high level for at least 10 weeks after the second immunization. Similar results were obtained using CBA mice.

When analyzed in a neutralization assay using MT4 cells and HIV-I-MN (Table 2), two out of four Balb/c sera obtained 2 weeks after immunization inhibited virus induced syncitia formation completely at dilutions of 1 in 100. Table 2

Neutralization Assay (Inhibition of syncitia formation

HIV-I-specific antibody response in mice and monkeys.

Table 3

ELISA reactivity of mouse sera with HIV-I gpl60LAl

Days after immunization

Immunization 0 14 70 112

PBS(3)* <100 <100 <100 <100 vM16(4) <100 <100 <100 <100 vMLN450(4) <100 4677 169824 95499

Sera of Balb/c mice were prepared at the indicated time point, pooled and tested as described in Materials and

Methods . Titers are given as reciprocal values of the dilution giving an 0D.g_nnm of 1.

* Number of animals in the lot.

HIV-I neutralizing antibody titers were determined in a syncytia formation inhibition assay using HIV-IMN (9). Neutralization titers ranged from 1:100 to 1:400 in the day 70 sera from VMLN450 immunized BALB/c mice, whereas values for control sera were below 1:100. The V3 specific response in the animals is currently being determined.

Comparable experiments were conducted using cynomolgus monkeys. Animals were immunized with a single intramuscular injection of 10 pfu of live vM16 or VMLN450. HIV-I-specific antibodies were measured one month later by ELISA, using either purified recombinant gpl60MN-LAI (Table 4 ) or a commercial ELISA kit (ELAVIA) (data not shown). Both tests demonstrated the presence of elevated gpl60 specific antibodies in the VMLN450 immunized monkeys, whereas the vM16 immunized animal showed no reactivity.

Table 4 ELISA reactivity of monkey sera with HIV-I gpl60MN-LAl Monkey (Immunization )

Days 339B 46698 320A 5343A after immunization(vM16) (VMLN450) (VMLN450) (vMLN450)

0 <20 43 <20 45

28 23 933 141 2089

Titers are given as reciprocal values of the dilution giving an 0D._qnnm of 1.

Example 7 Gpl20-specific cytotoxic immune response.

In addition to B-cell and T-helper cell epitopes, the V3-loop sequence of gpl20 of HIV-IMN has been shown to contain a MHC class I-restricted cytotoxic T-lymphocyte (CTL) epitope that is recognized in mice in the context of the H-2 haplotype (10). We therefore searched for the presence of a cytotoxic cellular immune response towards this epitope in BALB/c mice immunized with vMLN450. Spleen cells from immunized mice were stimulated in vi tro with P18-MN peptide and assayed for cytotoxic activity towards syngeneic target cells (P815) that had been pulsed with peptide PI 8-MN, comprising the V3 CTL-epitope sequence (10). As shown in Table 5, a clear HIV-IMN-specific cytotoxic activity could be demonstrated at an effector to target ratio of 25:1 in the animals immunized with VMLN450 but not in those immunized with the parental vM16. This activity was MHC class I-restricted. See Ref . 11.

Table 5 HIV-IMN V3 specific cytolysis Percent specific lysis

Immunization Control P18-MN pulsed

P815 cells P815 cells

vM16

VMLN450 3 36

Spleen cells from vM16 or VMLN450 immunized BALB/c mice were pooled and subsequently restimulated in vi tro as described in Materials and Methods . Cytolytic activity was measured against P815 cells pulsed or not with P18-MN peptide at an effector:target ratio of 25:1.

The cytotoxicity assay was performed as follows: BALB/c mice were immunized intraperitoneally on days 0 and 21 with 10 pfu of VM16 or VMLN450. Spleen cells from three mice per lot were recovered 10 days later, pooled and restimulated in vi tro for 7 days with P18-MN peptide (10), then for 5 days with P18-MN peptide in the presence of 5% concanavalin A supernatant-containing medium as a source of growth factor.

Cytolytic activity of stimulated splenocytes was determined by a 5h 51Cr-release assay. Target cells were peptide-pulsed

51 Cr-labelled P815 tumor cells. Percentage of specific 51Cr release was calculated as: [(experimental release - spontaneous release)/(maximal release - spontaneous release)] x 100. Spontaneous release was less than 20% of maximal release obtained by incubation with 1% triton X-100.

Example 8

Construction of vMOG-1

The HIV-I-MN gpl20 sequences used in this construction were generated as described supra in the description of the vMLN450 construction. However, the amino acid sequence was altered as indicated in Figure 14 at the C-terminal end of the HIV insert and the N-terminal end of the L-peptide.

The generation of recombinant mengovirus vMQG-1, containing a heterologous truncated gpl20, or fragments thereof, at the N-terminus of the L-peptide, and separated from the latter by protease cleavage site(s) e.g., 3C, bears the potential to augment expression and immunogenicity of the gpl20 sequences, as compared to VMLN450. The fragments of gpl20 are chosen to elicit an immune response in an animal. In specific embodiments these fragments are composed of peptides between 20 and 100 amino acids.

A flow diagram of the construction of pMRA-5, the cDNA of vMQG-1, is depicted in Figure 15. The sequence of the synthetic double-stranded oligonucleotide labelled LQGd/s in Figure 15 is:

TGA AAC TCA GGG TAA CTC TAC TAC

ACT TTG AGT CCC ATT GAG ATG ATG GTA C

Briefly, the PCR amplified sequence Δgpl20-VCN was re¬ stricted with Hindi and Ncol and a 451 bp fragment was isolated. This fragment was ligated to a synthetic double- stranded oligonucleotide LQGd/s coding for the 3C cleavage site. The resulting ligated fragment was restricted with Ncol and inserted at the Ncol restriction site of pMRA-1, The resulting plasmid was named pMRA-4. The 5.8 kb SphI fragment of pM16 described supra is inserted in pMRA-4 at SphI (1926) to restore the full-length mengovirus sequence, and produce pMRA-5. pMRA-5 is transcribed m_. vitro and the resulting RΝA is used to infect HeLa cells to generate virus .

The insertion of 486 additional bases into the mengovirus genome, in the case of vMQG-1, is unlikely to interfere with the viability of the virus since the insertion of 459 bases yielded a perfectly viable recombinant mengovirus in the case of VMLΝ450. Along with the introduction of the 3C cleavage site between the heterologous amino acid sequence and the L-peptide a myristylation signal has been retained at the N-terminus of L*, a mutant of mengovirus L shown in Figure 14. Moreover, the change of the N-terminal amino acid sequences in L is not expected to interfere with the function of the L -peptide since the al¬ teration only comprises the replacement of the first two amino acids (MA) by the amino acid sequence GNS . A similar deletion of the N-terminal amino acid and fusion with het¬ erologous amino acids has been shown to have no dramatic effect on virus viability in the case of VMLN450.

Expression of vMQG-1 proteins should yield the proteins Δgpl20-Q and L after proteolytic cleavage by protease 3C. The difference between gpl20-Q and gpl20-VCN is at the C- terminus of the protein: vMQG-1 . . . RWFETQ

VMLN450 . . . RWLTAMEQ.

The entire sequence of Δgpl20-Q is:

MATTMEQINC TRPNYNKRKR IHIGPGRAFY TTKNIIGTTR QAHCNISRAK

WNDTLRQIVS KLKEQFKNKT IVFNQSSGGD PEIVMHSFNC GGEFFYCNTS

PLFNSTWNGN NTWNNTTGSN NNITLQCKIK QIINMWQEVG KAMYAPPIEG

QIRWFETQ- (COOH).

The cellular localization of Δgpl20-Q is expected to be

* cytoplasmic and independent of the L -peptide localization.

In addition, this strategy provides information that is applicable to the cloning of a signal sequence at the N-terminus of the Δgpl20 sequence, or any other heterologous insert, leading to the secretion of the foreign insert thus likely to result in increased immunogenicity.

Example 9 PM16-1 as Source of Mengovirus DNA

The previous Examples may also be carried out using' plasmid pM16-l as a source of mengovirus nucleic acid. pM16-l contains a deletion of nucleotides 10,931 to 10,950 in the plasmid region resulting in an increase in the infectivity of the transcript. The sequence of pM16-l is depicted in Figure 17 and a comparison of the sequence of pM16 and pM16-l is provided in Figure 18. pM16-l or any other version of mengovirus cDNA could be used for construction of recombinant viruses like VMLN450. Example 10

Protection of Mice from Lymphocytic Choriomeningitis Infection Using Recombinant Mengovirus

The well-characterized immunodominant cytotoxic

T-lymphocyte (CTL) epitope from the lymphocytic choriomeningitis virus (LCMV) nucleoprotein (NP) was used aε a model. It has previously been shown that the introduction of this epitope into Vaccinia virus allows the induction of protective immunity against lethal LCMV infection in BALB/ c (H-2^d) mice. J.C. Whitton et al., J. Virology 67:348-356

(1993). A Mengovirus chimaera, vLCMG4, was constructed that expresses 14 amino acids (aa) of the LCMV NP including the 9 aa CTL epitope; ProGlnAlaSerGlyValTyrMetGly.

Materials and Methods

Construction and production of vLCMG4

DNA manipulations, transcription, transfection, tissue culture of HeLa cells, and production of Mengovirus were carried out as described above. Cytotoxicity assays

Primary cytotoxic activity against LCMV was checked in livers from immunized animals essentially as described by P. L. Gossens, H. JOUIN, and G. Milon, Dynamics of Lymphocytes and Inflammatory Cells Recruited in Liver during Murine Listenosis, J. Immunol . , 147, 3514-3520.

Each liver was homogenized in a potter glass grinder containing 10ml HBSS + antibiotics, passed through a nylon sieve and centrifuged for 10 min. at 4°C at 150g. Each pellet was resuspended in 45ml HBSS adjusted to 0.03% Trypsin and 33μg/ml DNAse-1. Cells were incubated for 45 minutes at

37°C under gentle shaking, before adjusting the mixture to

10% fetal calf serum (FCS). Cells were washed twice with

HBSS and the final pellet was resuspended in 1ml of complete

RPMI 1640 culture medium. (10% FCS, 1% L-Glutamine, 4xl0^~ M

/?-mercaptoethanol) . Cells were counted and adjusted to the appropriate concentration for the CTL assay. Non-infected or

LCMV infected J774 target cells were radiolabelled with

200μCi of ⁵¹ Cr for lh at 37°C, washed and distributed into

5 96 well plates at 10 cells/well and 100μl, containing diluted effector cells. Plates were centrifuged at lOOg for 2 min. before a 4 h incubation at 37°C. After another centrifugation supernatants were collected and counted in a gamma counter. Specific cytotoxicity was calculated according to the following formula: experimental release - spontaneous release/maximum release - spontaneous release, where spontaneous release was determined in the absence of effector cells and 100% release in the presence of 1% triton. Immunizations

8 week old female BALB/c mice obtained from Iffa-Credo were immunized intraperitoneally with 0.2ml of either PBS or different doses of either vM16, vLCMG4 or the Armstrong strain of LCMV (LCMV-ARM, was obtained from Dr. Oldstone, Scripps Research Institute, LaJolla, USA). LCMV-ARM has been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 under accession number

ATCC VR-134. Mice were challenged intracranially ten days after immunization (p.i.) with 10 2' 8 pfu of LCMV-ARM in 30μl to test for the primary protective response and 43 or 45 days p.i. to test for the memory response.

Results

Construction of vLCMG4 and growth characteristics

A double stranded synthetic oligonucleotide, coding for the LCMV NP as 117 to 130, was inserted in frame between the restriction sites SnaBI (747) and Nhel (754) of the Mengovirus cDNA pMCS, yielding plasmid pLCMG4 (Fig. 1). Plasmid DNA was sequenced through the mutated region. RNA transcripts of pLCMG4 were transfected into HeLa cells giving rise to the recombinant virus vLCMG4. Virus from transfection and infection supernatants showed the same plaque phenotype as the parental vM16 or vMCS (Fig. 2). After passage in HeLa cells VLCMG4 grows to high titers comparable to vM16 or vMCS. LCMV specific cytotoxicity

Whereas PBS and vM16 immunized animals show no lytic activity towards syngeneic LCMV-infected J775 target cells, LCMV and vLCMG4 immunized animals exhibit specific cytotoxicity even at low effector/target ratios (Table 6).

Table 6

PBS LCMV vM16 VLCMG

Targets 2X10⁵ pfu ip 10⁶ pfu ip 10⁶ pfu ip

26* 13 6.5 50 25 12.5 16 8 36 18 9

J774 ** 6 5 3 3 4 3 4 0 0 0

J774 + LCMV 17 16 7 84 81 75 0 1 74 78 77

* Effector/target ratio

** Percentage specific cytotoxicity

Vaccination of BALB/c mice against LCMV challenge

Single dose immunization

Mice were immunized intraperitoneally with 10 pfu of either vM16 or vLCMG4, 2 x 10⁵ pfu of LCMV-ARM or tissue culture medium as virus-free control. Animals were challenged by intracranial injection of LCMV on day 10 or 43 as described in Materials and Methods and monitored daily. All deaths occurred before day 10. No protection was obtained for animals that had received vM16 or medium, whereas complete protection was observed for LCMV and vLCMG4 immunized animals (Table 7). Table 7

Immunization

vM16 vLCMGA LCMV Medium

Day of 10 43 10 43 10 43 10 challenge p.i.

Percent Protection 02 02 1002 1002 1002 1002 02 (Number of (5) (4) (10) (11) (5) (5) (5) animals)

Dose dependent protection

In order to evaluate the minimal dose requirement for effective vaccination with vLCMG4, different doses of vLCMG4 were used to immunize BALB/c mice. 100% protection could be observed 10 days p.i. at a dose as low as 100 pfu, the protection being still at 60% (2 dead out of 5) when animals were immunized with 10 pfu. At 45 days p.i. protection levels remained very high with single cases of death with 10, 10³ and 10⁴ pfu (Table 8).

Table 8

* Each lot contained 5 animals Discussion and Conclusion

The results obtained for the Mengo-LCMV recombinant vLCMG4 confirm and extend the results obtained for the HIV recombinant vMLN450:

1/ The 14 amino acid foreign sequence was inserted into the Mengovirus genome without loss of viability. vLCMG4 grows to high titers, comparable to vM16 and vMCS, and the plaque phenotype is the same as for the parental virus . 2/ A specific immune response can be induced in vLCMG4 immunized animals against LCMV. Strong primary CTL activity can be detected against LCMV infected target cells in livers of immunized animals without secondary antigen stimulation of effector cells in vitro. In addition to the capacity of Mengovirus to induce a humoral response against foreign antigens, this result confirms the possibility to use Mengovirus as a vector to induce cellular immune responses against heterologous sequences .

3/ After immunization with vLCMG4 animals were protected against a lethal dose challenge of LCMV.

DISCUSSION Picornaviruses such as poliovirus, rhinovirus or Mengovirus are attractive models for the development of recombinant vaccines because their RNA genomes are expressed exclusively in the cytoplasm of infected cells. We have developed Mengovirus as a new viral vector. Mengovirus is a cardiovirus and shares the same serotype as encephalomyocarditis virus (EMCV), Columbia SK, and Maus-Elberfeld viruses (1,2). Mengovirus is able to replicate in a wide range of animal species including primates (3,4). Genetic engineering has shown that the pathogenic potential of Mengovirus is controlled by a homopolymeric poly(C) tract (C-._QUC-_Π) within the 5' non-coding region of the genome (5,6). Truncation or deletion of the poly(C) tract leads to stably attenuated Mengovirus strains that can be propagated with ease in cell culture and are highly resistant to reversion.

Like all picornaviruses, the Mengovirus genome encodes a large polyprotein that is cleaved proteolytically into a series of mature structural and non-structural proteins (2) . Unlike entero- and rhinoviruses, the PI capsid region of cardio- and aphthoviruses is preceded by a leader (L) polypeptide. The Mengovirus L polypeptide is 67 amino acids in length. Its functional relevance to the virus is unknown, as it is not a protease like the L protein in aphthoviruses . Release of L from the polyprotein requires proteolysis by viral protease 3C, an enzyme encoded downstream in the viral genome. To determine whether attenuated Mengovirus could be used as a viral vector and have potential to serve as live recombinant vaccine, we engineered a segment encoding the V3-C4 domains of HIV-IMN gpl20 into the region of the vM16 genome that encodes the L polypeptide. The resulting recombinant virus expressed the gpl20-L fusion protein along with the normal Mengovirus proteins and elicited a strong humoral as well as cellular immune response to HIV-I in immunized animals. We have demonstrated here that Mengovirus can be used as a vector for the expression of immunogenic foreign protein sequences. In this case, 147 amino acids from the HIV-IMN gpl20 were fused in frame into the N-terminus of the L polypeptide of the vM16 strain of Mengovirus. The recombinant was viable, although showing somewhat smaller plaque size and reduced virus yields as compared to vM16. The vMLN450 virus could be stably passaged for at least four cycles in cell culture with complete retention of the HIV-I sequence.

It has been reported that the size of the poliovirus genome can be increased by up to 17% in recombinant bicistronic constructions and still be encapsidated, albeit with reduced viability. Poliovirus genomes 31% longer than wild-type are not encapsidated (13). The virions of VMLN450 carry an HIV-I/Mengo recombinant RNA genome, but the viral capsids are identical to those of parental Mengovirus . The HIV-I sequence is replicated and expressed only during infection, as is typical for other Mengo non-structural proteins. The viral 3C cleavage site between the fusion protein and PI region is recognized and processed in a normal manner. Polyprotein translation directed by the 5' non-coding IRES (internal ribosome entry site) (14-16) is also normal, and initiated at the appropriate AUG of the fusion-protein sequence. The ability of this IRES to direct efficient translation of a wide variety of heterologous protein sequences is already well established (13,17). We have, for example, recently constructed a new Mengovirus recombinant encoding part of the rabies virus G protein which expresses a ΔG-L fusion protein that can be immunopreci¬ pitated by a rabies-specific antibody. We have also engineered a vector cassette that will allow easy insertion of almost any antigen-encoding cDNA segment into the L protein region of an attenuated Mengovirus genome.

The cytoplasmic location of the infectious cycle of picornaviruses and the fact that picornavirus genomic RNA does not undergo reverse transcription are desirable features for any viral expression vector to be used as a live recombinant vaccine. The vM16 system could potentially show broad applicability and safety in a wide variety of mammalian hosts .

The gratifying and unexpected aspect reported in this current study is that the immunogenic response to attenuated Mengovirus clearly extends to heterologous antigens that are carried and expressed by the virus during its limited replication. The Δgpl20-L protein synthesized within cells, retained natural antigenic properties and could be recognized by a gpl20 V3-loop specific monoclonal antibody. Infection of mice or monkeys with VMLN450, produced high titer polyclonal sera that reacted with HIV-I gpl60. The efficacy of the response means that the fusion protein was efficiently expressed in an immunologically relevant configuration. Different modes of expression, such as those of non-fused and glycosylated gpl20 segments, need now to be investigated in order to determine the optimal antigen presentation to achieve high HIV-specific neutralization titers. Given the wide host range of Mengovirus and the strong humoral response typically elicited by its nonstructural proteins, it is likely that vM16-based recombinants expressing appropriate heterologous antigens will induce high titer protective responses against various pathogens in many different animal hosts.

Example 11

Construction of A Mengovirus cDNA, pMCS, Allowing Facilitated Cloning at The N-Terminus of The Leader Peptide

In embodiments of this invention, cloning of foreign sequences into the Mengovirus genome at the Ncol 729 site within the L coding region of the Mengovirus genome, involves a two-step cloning procedure, as it was realized for the construction of pMRA3/vMLN450. First a sequence is cloned into the subclone pMRAl, then the orientation of the insert is verified, since cloning into a single site allows insertion in the proper as well as the inverse orientation. In order to accelerate cloning of foreign sequences into the leader peptide, we engineered a synthetic oligonucleotide at the site of the Ncol site 729, which contains the restriction sites for the enzymes Xhol , Sna BI and Nhe I. These restriction sites do not occur in the Mengovirus cDNA, and allow easy one step cloning into the Mengovirus cDNA. Furthermore if different enzymes are chosen for each end of the insert sequence, e.g., the 5' end of the insert carries the Sna BI site and the 3' end the Nhe I site, the ligation/ insertion of the insert sequence into the Mengovirus genome will be directed/forced and does not require screening for orientation.

Generation of the cassette containing cDNA pMCS:

A double stranded oligonucleotide containing the restriction sites Xhol , Sna BI and Nhe I (Figure 24) was inserted at the Ncol site at position 729 of pMRAl, giving pMΔLFUSL. The 5.8 kb Sph I-Sph I fragment of pM16 was subsequently transferred into the Sph I site 1928 of pMΔLFUSL, resulting in plasmid pMCS (Figure 26). All DΝA manipulations have been carried out according to standard procedures (ref 11, page 43).

The oligonucleotide linker sequence codes for non-Mengo amino acids (Figure 26). R A derived from pMCS and transfected into permissive HeLa cells gives large size plaques like the parental vM16 RΝA.

The utility of pMCS has been demonstrated by the insertion of an LCMV epitope, allowing the generation of a recombinant Mengovirus, vLCMG4, that is able to induce a protective immune response in mice against lethal LCMV infection. Conclusion

The Mengovirus cDNA pMCS allows easy and orientation directed/forced cloning of foreign sequences at the N- ter inus of the Mengovirus leader peptide. Viable recombinant Mengoviruses can be generated from pMCS as demonstrated by the construction of the Mengo LCMV recombinant, vLCMG4.

Example 12 Construction of A Recombinant Mengovirus Encoding For A Segment of the Rabies Virus Glycoprotein.

A PCR fragment of 350 base pairs was generated from the plasmid pRb56, See W. Tordo et al, Proc. Natl. Acad. Sci. (USA) 83, 3914-3918 (1986), which includes the sequence for the linear neutralizing epitope G5-24, B. Dietzschold et al., J. Virol. 64: 3804-3809 (1990), of the glycoprotein of the PV isolate of Rabies virus. The amplified DNA carries at the 5' end a Xhol site and at the 3' end a Sna BI site. The PCR fragment and the pMCS plasmid were digested with restriction enzymes Xhol and Sna BI, purified and ligated according to standard procedures (ref 11, page 43). The resulting plasmid, pMG5-24 was used to prepare RNA for transfection, as described above. The recombinant genome codes for a L-ΔG fusion protein of an expected size of about 20 kDa . Viable recombinant virus was obtained after transfection of pMG5-24 RNA. Virus stocks were prepared and used to infect HeLa cells in the presence of 35S methionine, as described earlier.

VMG5-24 infected cells show the presence of an additional protein of an apparent molecular weight of 22—

25 kDa, which is absent from vM16 or Mock infected cells

(Figure 27). In order to identify the identity of this protein, the cytoplasmic extract was immunoprecipitated with the Rabies G5-24 specific monoclonal antibody RV2-22C5, as described earlier. See H. Burschoten et al., J. Gen. Virol.

70:291-298 (1989). The 22-25 kDa protein could be immunoprecipitated from cytoplasmic extracts from VMG5-24 infected cells. These results demonstrate that an additional foreign sequence can be expressed from a Mengovirus genome in an antigenic manner.

REFERENCES

1) Rueckert, R. (1991) In Fields (ed.), Virology 2nd ed . , pp. 507-548.

2) Palmenberg, A.C. (1990) Ann. Rev. Microbiol. 44, 603-623.

3) Hubbard, G.B., Soike, K.F., Butler, T.M., Carey, K.D., Davis H., Butcher, W.I. & Gauntt, C.J. (1992) Lab. Ani . Sci. 42, 233-239.

4) Helwig, F.C. & Schmidt, E.C.H. (1945) Science 102, 31-33.

5) Duke, G.M. & Palmenberg, A.C. (1989) J. Virol. 63, 1822-1826.

6) Duke, G.M., Osorio, J.E. & Palmenberg, A.C. (1990) Nature (London) 343, 474-476.

7) Sambrook, J., Fritch, E.F., & Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press.

8) Emini, E.A., Jameson, B.A., Lewis, A.J., Larsen, G.R. & Wimmer, E. (1982) J. Virol. 43, 997-1002.

9) Rey, F., Barre-Sanoussi, F., Schmidtmayerova, H., & Chermann, J.C. (1987) J. of Virol. Meth. 16, 239-249.

10) Takahashi, H. , Merli, S., Putney, S.D., Houghton. R., Moss, B., Germain, R.N.,& Berzofsky, J.A. (1989) Science 246, 118-120.

11) Harber, J.J., Bradley, J., Anderson, C.W., & Wimmer, E. (1991) J. Virol. 65, 326-334.

12) Harlow, E. & Lane, D. (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press.

13) Alexander, L., Lu, H.H. & Wimmer, E. (1994) Proc Natl Acad Sci USA 91, 1406-1410.

14) Parks, G.D., Duke, G.M., & Palmenberg, A.C. (1986) J. Virol. 60, 376-384.

15) Jang, S.K., Krausslich, H.G., Nicklin, M.J., Duke, G.M,, Palmenberg, A.C, & Wimmer, E. (1988) J. Virol. 62, 2636-2643.

16) Pelletier, J. & Sonnenberg, N. , (1988) Nature (London) 334, 320-325. 17) Kaufman, R.J., Davies, M.V., Wasley, L.C., & Michnick, D. (1991) Nuc. Acids Res. 19, 4485-4490.

Any publications, patents, and patent applications mentioned, referred to, or cited in this specification are expressly incorporated herein by reference to the same extent as if such publication, patent, and patent application was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A viable modified mengovirus, wherein said modified mengovirus is an attenuated strain having a deletion in th<^ poly(C) tract of the 5' non-coding region of the genome of said mengovirus, and comprises a heterologous nucleotide sequence.

2. A viable recombinant mengovirus, wherein:

- said recombinant mengovirus is an attenuated strain having a deletion in the poly(C) tract of the 5' non-coding region of the genome of said mengovirus;

- the leader polypeptide L of said recombinant mengovirus comprises a heterologous amino acid sequence; and

- said leader polypeptide is full length.

3. The recombinant mengovirus as claimed in claim 2, wherein said heterologous amino acid sequence is inserted after amino acid 6 of the leader polypeptide L.

4. The recombinant mengovirus as claimed in claim 3, wherein cells infected with said mengovirus express a fusion protein comprising the heterologous amino acid sequence covalently linked to the mengovirus leader polypeptide.

5. The recombinant mengovirus as claimed in claim 4, wherein said cells are cells of human origin.

6. The recombinant mengovirus as claimed in claim 4, wherein said heterologous amino acid sequence comprises aI: least one epitope of HAV, HIV, Rabies virus, FMDV, coronaviruε, herpes virus, measles, mumps, or RSV.

7. The recombinant mengovirus as claimed in claim 6, wherein said heterologous amino acid sequence comprises at least one epitope of gpl20 of HIV.

8. The recombinant mengovirus as claimed in claim 7, wherein said heterologous amino acid sequence comprises amino acids 299-466 of gpl20 of the MN isolate of HIV-I.

9. A fusion protein comprising a full-length leadeu: polypeptide of an attenuated mengovirus strain into which a heterologous amino acid sequence is inserted. - so -

io. The fusion protein as claimed in claim 9, wherein said heterologous amino acid sequence is inserted after amino acid 6 of the leader polypeptide of mengovirus .

11. The fusion protein as claimed in claim 10, wherein said heterologous amino acid sequence comprises at least one epitope of HAV, HIV, Rabies virus, FMDV, coronavirus, herpes virus, measles, mumps, or RSV.

12. The fusion protein as claimed in claim 11, wherein said fusion protein has the sequence:

MATTMEQINC TRPNYNKRKR IHIGPGRAFY TTKNIIGTIR QAHCNISRAK WNDTLRQIVS KLKEQFKNKT IVFNQSSGGD PEIVMHSFNC GGEFFYCNTS PLFNSTWNGN NTWNNTTGSN NNITLQCKIK QIINMWQEVG KAMYAPPIEG QIRWLTAMEQ EICAHSMTFE ECPKCSALQY RNGFYLLKYD EEWYPEESLT DGEDDVFDPD LDMEWFETQ GNSTS

13. A permissive cell infected with a recombinant mengovirus as claimed in claim 2.

14. A permissive cell as claimed in claim 13, wherein said permissive cell is of human origin.

15. A permissive cell as claimed in claim 14, wherein said permissive cell is a HeLa cell.

16. A recombinant nucleic acid molecule comprising a nucleic acid sequence of mengovirus and a heterologous nucleotide sequence inserted within the full-length leader polypeptide encoding sequence of said nucleic acid sequence of mengovirus .

17. The recombinant nucleic acid molecule as claimed in claim 16, wherein the heterologous nucleic acid sequence is inserted at a Ncol restriction site at position 729 of said full-length leader polypeptide encoding sequence.

18. The recombinant nucleic acid molecule as claimed in claim 17, wherein the heterologous nucleic acid sequence comprises a sequence encoding at least one epitope of HAV, HIV, Rabies virus, FMDV, coronavirus, herpes virus, measles, mumps, or RSV. 19. The recombinant nucleic acid molecule as claimed in claim 18, wherein the heterologous nucleic acid sequence comprises a sequence encoding at least one epitope of gpl20 of HIV.

20. The recombinant nucleic acid molecule as claimed in claim 19, wherein said heterologous nucleic acid sequence encodes amino acids 299-446 of gpl20 of the MN isolate of HIV-I.

21. The viral genome of a recombinant mengovirus as claimed in claim 2.

22. A vaccine comprising a recombinant mengovirus as claimed in claim 2.

23. The vaccine as claimed in claim 22, wherein said recombinant mengovirus is in admixture with a pharmaceutically acceptable carrier.

24. A vaccine comprising a fusion protein as claimed in claim 9 in admixture with a pharmaceutically acceptable carrier.

25. A method of inducing an immune response, wherein said method comprises administering a recombinant mengovirus as claimed in claim 2 via a parenteral or oral route to an organism in which an immune response is to be induced.

26. The method of inducing an immune response as claimed in claim 25, wherein the recombinant mengovirus is administered orally.

27. A viable recombinant mengovirus as claimed in claim 2, wherein said mengovirus further comprises protease cleavage sites between said heterologous amino acid sequence and said leader polypeptide.

28. The viable recombinant mengovirus as claimed in claim 27, wherein said protease cleavage site is a protease 3C cleavage site.

29. A permissive cell infected with the viable recombinant mengovirus as claimed in claim 27, wherein said permissive cell expresses said heterologous amino acid sequence in native form. 30. The recombinant nucleic acid molecule as claimed in claim 16, wherein said recombinant nucleic acid molecule further comprises a nucleic acid sequence encoding protease cleavage sites between said heterologous sequence and said mengovirus sequence.

31. pMRA-5.

32. VMLN450.

33. pMRA-3.

34. The recombinant mengovirus as claimed in claim 4, wherein said heterologous amino acid sequence comprises at least one epitope of lymphocytic choriomeningitis virus .

35. The recombinant mengovirus as claimed in claim 34, wherein said epitope comprises the amino acid sequence

Pro-Gln-Ala-Ser-Gly-Val-Tyr-Met-Gly.

36. The fusion protein as claimed in claim 10, wherein said heterologous amino acid sequence comprises at least one epitope of lymphocytic choriomeningitis virus .

37. The fusion protein as claimed in claim 36, wherein said heterologous amino acid sequence comprises the amino acid sequence

Pro-Gln-Ala-Ser-Gly-Val-Tyr-Met-Gly.

38. The recombinant nucleic acid molecule as claimed in claim 17, wherein the heterologous sequence comprises a sequence encoding at least one epitope of lymphocytic choriomeningitis virus .

39. The recombinant nucleic acid molecule as claimed in claim 38, wherein said epitope comprises the amino acid sequence

Pro-Gln-Ala-Ser-Gly-Val-Tyr-Met-Gly.

40. A vaccine comprising a recombinant mengovirus as claimed in claim 35.

41. The vaccine as claimed in claim 40, wherein said recombinant mengovirus is in admixture with a pharmaceutically acceptable carrier.

42. The recombinant mengovirus as claimed in claim 4, wherein said heterologous amino acid sequence comprises at least one epitope of rabies virus. 43. The recombinant mengovirus as claimed in claim 42, wherein said epitope is the linear neutralizing epitope G5-24 and the glycoprotein of the PV isolate of rabies virus.

44. The fusion protein as claimed in claim 10, wherein said heterologous amino acid sequence comprises at least one epitope of rabies virus.

45. The fusion protein as claimed in claim 44, wherein said epitope is the linear neutralizing epitope G5-24 and the glycoprotein of the PV isolate of rabies virus .

46. The recombinant nucleic acid molecule as claimed in claim 17, wherein the heterologous sequence comprises a sequence encoding at least one epitope of rabies virus.

47. The recombinant nucleic acid molecule as in claim 46, wherein said epitope is the linear neutralizing epitope G5-24 and the glycoprotein of the PV isolate of rabies virus.

48. A vaccine comprising a recombinant mengovirus as claimed in claim 43.

49. The vaccine as claimed in claim 48, wherein said recombinant mengovirus is in admixture with a pharmaceutically acceptable carrier.

50. The recombinant nucleic acid as claimed in claim 16, wherein said recombinant nucleic acid is DNA.

51. A recombinant nucleic acid comprising a nucleic acid sequence of mengovirus and a oligonucleotide comprising multiple restriction sites, wherein said oligonucleotide is inserted in the nucleic acid encoding leader polypeptide of said mengovirus.

52. The recombinant nucleic acid as claimed in claim 51, wherein said restriction sites are selected from the group consisting of Xhol, SnaBI, Nhel and said oligonucleotide is inserted at the NcoJ site at position 729,

54. The recombinant nucleic acid as claimed in claim 52, wherein said recombinant nucleic acid is pMCS .