(19) (CA) APPLICATION FOR CANADIAN PATENT (12)

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1 MUM - ram., OPIC mem CIPO 71 OFFICE DE LA PROPRIETP - 1 CANADIAN INTELLECTUAL INTELLECTUELLE DU CANADA PROPERTY OFFICE Ottawa Hull K1A 0C9 (21) (Al) 2,164,297 (86) 1994/06/03 (43) 1994/12/22 6 (51) Int.C1. C12N 15/86; C12N 15/62; C12N 5/10; CO7K 19/00; CO7K 14/08; A61K 39/12; A61K 39/295 (19) (CA) APPLICATION FOR CANADIAN PATENT (12) (54) Mengovirus as a Vector for Expression of Foreign Polypeptides (72) Altmeyer, Ralf - France ; Van Der Werf, Sylvie - France ; Girard, Marc - France ; Palmenberg, Ann C. - U.S.A. (71) Institut Pasteur - France ; (30) (US) 08/090, /06/03 (57) 54 Claims Notice: This application is as filed and may therefore contain an incomplete specification. I*. Industrie Canada Industry Canada OPIC - CIPO 191 Canada.

2 [ CORRECTED VERSION* PCT CORRECTED VERSION** (51) International Patent Classification 5 : C12N 15/86, CO7K 14/085, 14/16, 14/145, A61K 39/12, 39/125, 39/21, 39/205, 39/295 (21) International Application Number: PCT/US94/06177 (22) International Filing Date: 3 June 1994 ( ) WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) A3 (11) International Publication Number: WO 94/29472 (43) International Publication Date: 22 December 1994 ( ) (74) Agents: TURNER, John, B. et al.; Finnegan, Henderson, Farabow, Garrett & Dunner, 1300 L Street, N.W., Washington, DC (US). (30) Priority Data: 08/090,531 3 June 1993 ( ) US (81) Designated States: CA, JP, US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, 1E, IT, LU, MC, NL, FT, SE). (60) Parent Application or Grant (63) Related by Continuation US 08/090,531 (CIP) Filed on 3 June 1993 ( ) (71) Applicant (for all designated States except US): INSTITUT PASTEUR [FR/FR]; Rue du Docteur-Roux, F Paris Cedex 15 (FR). Published With international search report. With an indication in relation to a deposited microorganism furnished under Rule 13bis separately from the description. Date of receipt by the International Bureau: 12 December 1994 ( ) (88) Date of publication of the international search report: 16 February 1995 ( ) (72) Inventors; and (75) Inventors/Applicants (for US only): ALTMEYER, Ralf [DE/FR]; 12, part de Beam, F Saint-Cloud (FR). VAN DER WERF, Sylvie [FR/FR]; 112, all& de la Pointe Genete, F Gif-Sur-Yvette (FR). GIRARD, Marc [FR/FR]; 6, rue Cesar-Franck, F Paris (FR). PALMENBERG, Ann, C. [US/US]; 5010 Manor Cross, Madison, WI (US). (54) Title: MENGOVIRUS AS A VECTOR FOR EXPRESSION OF FOREIGN POLYPEPTIDES (57) 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 of gp120 of the MN isolate of HIV-I. * (Referred to in PICT Givette No. 06/1995, Section II) * *(Referred to in PCI. Gazette No. II/1995, Section II)

3 WO 94/ PCT/US94/ 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, 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 meningoencephalitis, mengovirus has a wide host range. In addition to the mouse, mengovirus is also able to infect various animal species including pigs, elephants, and primates 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), Coxsackie 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

4 WO 94/ PCT/US94/ X-ray crystallography. Luo et al., Science 235: (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-Pl-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 nonstructural proteins. The details of this processing are reviewed by Ann C. Palmenberg, Proteolytic 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. P1 is a precursor to the structural proteins VP1, VP2, VP3, and VP4, which are also identified as 1D, 1B, 1C, and 1A, 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 bases, and sequence discontinuities, e.g. uridine residues, that sometimes disrupt the homopolymeric sequence have served to characterize natural viral isolates, the exact biological

5 WO 94/ PCT/US94/06177 function of the poly(c) region is not clear. cdna-mediated 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 fulllength 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

6 WO 94/ PCT/US94/ 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 of gp120 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

7 WO 94/ PCT/US94/06177 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.

8 WO 94/ PCT/US94/ 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 ilgp120-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 Agp120-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 gp120 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-1/3'VCN for lanes a-e and with oligonucleotide pairs M- VDW-1/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 RIndIII. Figure 10 depicts the sequence of the bogp120 -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

9 WO 94/ '64297 PCT/US94/ 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 gp160 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 gp160 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 gp160 LAI are shown. Titers are given as reciprocal values of serum dilution giving an O.D. at 490 nm 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 gp160 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 gp160 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 gp160 LAI as an antigen. The reactivity of Cynomolgus monkey preimmune sera (filled bars) and sera 4 weeks after immunization (stippled bars) with gp160 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,&gp120-qg-l junction for vmqg-1 and vm16. Figure 15 is a flow diagram of the procedure used to construct pmra-5.

10 WO 94/ PCT/US94/06177 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 Ts, i.e. as a DNA sequence. pm16 is a DNA plasmid. Figure 17 depicts the nucleic acid sequence of pm16-1. 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. pm16-1 is a DNA plasmid. pm16-1. Figure 18 is a comparison of the sequence of pm16 and Figure 19 depicts the construction of pmln450. The cdna sequence encoding amino acids 299 to 445 of HIV-IMN gpi20 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, Agp120-L is shown in (B). Leader amino acids are represented in normal type and gp120 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 Lgp120-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 2/.1g/m1 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

11 WO 94/ PCT/US94/ 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 SnaBl 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 XhoI, SnaBI, and NheI. Figure 25 depicts the protein sequence and cdna sequence of the L-coding region of pm16. The position of the XhoI 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 radioimmunoprecipitation 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

12 WO 94/ PCT/US94/ 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 sequence(s). 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

13 WO 94/ PCT/US94/ 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 c.1 cell that can be productively infected with a virus. Thus, 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,

14 WO 94/29472 PCT/US94/ 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 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 13 UC 10, but otherwise comprises a DNA sequence corresponding to the full-length genome of mengovirus inserted between the EcoRI and BamHI 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 pm16-1. pm16-1 has also been deposited at the C.N.C.M. on June 2, 1993 under accession number The sequence of pm16-1 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 8 poly(c) tract, or pm19, encoding a C 12 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 pmwt, encoding the wild type poly(c) tract C 5 ec 10, 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 vitro manipulations. One possibility is the replacement of the EcoRV - AvrII restriction fragment

15 WO 94/ PCT/US94/ containing the poly(c) tract from one plasmid with the appropriate EcoRV - AvrII 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.

16 WO 94/ PCT/US94/06177 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 pml6-1 an RNA transcript can be produced in 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 in vitro or in 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

17 WO 94/ PCT/US94/ 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 embodiments, the amino acid sequence can consist essentially of a heterologous epitope. By way of example, amino acid sequences of various embodiments of this invention are listed in Table I.

18 WO 94/29472 Cd.* A t PCT/US94/06177 Table I PATHOGEN PROTEIN SUBDOMAIN AMINO ACIDS 1/Hepatitis B Virus S / Streptococcus type 24 M 12 N terminal spec. type 5 M type 6 M as type 19 M 3/ Influenza virus NS1 HA2 4/ Plasmodium 93kd blood falciparium stage protein 5/ Schistosoma Immunodominant mansoni Calcium binding 6/ Hepatitis C Virus proteins (CaBP's) C El , , / Borrelia OSP A burgdoferi 8/ HTLV-1 Env / Chlamydia spec. MOMP variable domain (Outer membrane IV protein) 10/ HIV-I, HIV-2, glycoprotein, e.g. V3 loop SIV gp120 (PND) 11/TGEV S S 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

19 WO 94/ PCT/US94/06177 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 gp120 of HIV-I. In specific embodiments, the L polypeptide comprises amino acids of gp120 of the MN strain of HIV-I. In more specific embodiments, amino acids of gp120 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 neutralization determinant (PND) and sequences downstream involved in binding of the gp120 molecule to the CD4 receptor. The resulting recombinant mengovirus expresses an HIV-I gp120 - mengovirus L fusion protein that was recognized by HIV-I specific antibodies and induced anti HIV-I antibodies in animals. The HIV-I gp120 - mengovirus L fusion protein also induced a gp120 - 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 in 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

20 WO 94/ PCT/US94/ accordance with this invention for use as a vaccine in animals, including mice. Similarly, a recombinant virus expressing 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

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