Using CTL epitopes to the Ebola GP, NP, VP24, VP30, VP35 and VP40 virion proteins, a method and composition for use in inducing an immune response which is protective against infection with Ebola virus is described.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention satisfies the need discussed above. The present invention relates to a method and composition for use in inducing an immune response which is protective against infection with Ebola virus.

The inventors have induced protection against Ebola infection in mammals using virus replicon particles (VRPs) expressing the Ebola GP, NP, VP24, VP30, VP35 or VP40 genes. These VRPs and some uses are described in U.S. utility patent application Ser. No. 09/337,946 (filed Jun. 22, 1999), the entire contents of which are hereby incorporated by reference.

One embodiment of the present invention entails a DNA fragment encoding each of the Ebola Zaire 1976 GP, NP, VP24, VP30, VP35, and VP40 virion proteins (SEQUENCE ID NOS. 1-7).

Another embodiment provides the DNA fragments of Ebola virion proteins in a recombinant vector. When the vector is an expression vector, the Ebola virion proteins GP, NP, VP24, VP30, VP35, and VP40 are produced. It is preferred that the vector is an alphavirus replicon vector, especially a replicon vector that has the ability to produce the desired protein or peptide in a manner that induces protective B and T cells in vivo in mammals. Any alphavirus vector may be effective, including but not limited to the Venezuelan Equine Encephalitis (VEE) virus, eastern equine encephalitis, western equine encephalitis, Semliki forest and Sindbis. For instance, in a preferred embodiment the VEE replicon vector comprises a VEE virus replicon and a DNA fragment encoding any of the Ebola Zaire 1976 (Maying a isolate) GP, NP, VP24, VP30, VP35, or VP40 proteins. In another preferred embodiment, the VEE replicon vector comprises a VEE virus replicon and a DNA fragment encoding any of the amino acid sequences set forth in SEQ ID NOs:24-53. The construct can be used as a nucleic acid vaccine or for the production of self replicating RNA. To that end, a self replicating RNA of this invention can comprise the VEE virus replicon and any of the Ebola Zaire 1976 (Maying a isolate) RNAs encoding the GP, NP, VP24, VP30, VP35, and VP40 proteins described above, or the amino acid sequences set forth in SEQ ID NOs:24-53. The RNA can be used as a vaccine for protection from Ebola infection. When the RNA is packaged, a VEE virus replicon particle is produced.

Another embodiment entails infectious VEE virus replicon particles produced from the VEE virus replicon RNAs described above.

Another embodiment of the invention encompasses peptides that make up cytotoxic T lymphocyte (CTL) epitopes corresponding to Ebola GP, NP, VP24, VP30, VP35, or VP40 proteins. The epitopes may include the sequences identified as SEQ ID NOS:24-53, as described below. A related aspect of this embodiment provides DNA fragments that respectively encode these Ebola peptides. A further embodiment relates to recombinant DNA constructs that express these epitope peptides.

An additional embodiment includes a pharmaceutical composition that includes one or more of these CTL epitope peptides (and preferably one or more of SEQ ID NOs:24-53), in an effective immunogenic amount in a pharmaceutically acceptable carrier and/or adjuvant.

A further embodiment entails an immunological composition for the protection of mammals including humans against Ebola virus infection, comprising at least one (but preferably at least two, and more preferably at least three, and most preferably all) of the Ebola virus GP, NP, VP24, VP30, VP35, or VP40 proteins. In a related embodiment, the composition may include one or more of the CTL epitopes set forth in SEQ ID NOs: 24-53 (described below).

In a related preferred embodiment, the immunological compositions comprise alphavirus replicon particles (such as, for instance, VEE virus replicon particles) expressing the Ebola virus GP, NP, VP24, VP30, VP35, or VP40 proteins, or any combination of different VEE virus replicons each expressing one or more different Ebola proteins selected from GP, NP, VP24, VP30, VP35 and VP40--and preferably the composition includes replicons expressing each of the six proteins, in the form of a "cocktail". The six-protein cocktail, administered in the dosage, regimens and routes described below, has shown itself to be particularly successful as a protective composition. In a preferred embodiment the composition may include one or more of SEQ ID NOs: 24-53 (described below).

In a related preferred embodiment, the immunological compositions comprise alphavirus replicon particles (such as, for instance, VEE virus replicon particles) expressing only the Ebola virus GP protein. The GP protein, administered in the dosage, regimens and routes described below, has shown itself to be particularly successful as a protective composition.

An additional embodiment includes vaccines against infection by Ebola, comprising virus replicon particles (preferably VEE virus replicon particles) expressing the Ebola virus GP, NP, VP24, VP30, VP35, or VP40 proteins, or any combination of different VEE virus replicons each expressing one or more different Ebola proteins selected from GP, NP, VP24, VP30, VP35 and VP40--and preferably the vaccine includes replicons expressing each of the six proteins, in the form of a "cocktail" vaccine. For instance, in a preferred embodiment the Ebola VRPs contain one or more of the peptides specified by SEQ ID NOs: 24-53. In a related embodiment, the vaccine may include at a minimum at least one of the Ebola proteins selected from GP, NP, VP24, VP30, VP35 and VP40. In a preferred embodiment, the vaccine may include one or more of SEQ ID NOs: 24-53.

The invention also contemplates methods for inducing in a mammal a cytotoxic T lymphocyte response to the Ebola virus GP, NP, VP24, VP30, VP35, or VP40 proteins, or to a peptide comprising at least 6 amino acids thereof. In one version of the method, a recombinant DNA construct is administered to a mammal, such as, for example, a mouse, a guinea pig, a monkey or a human, which a recombinant DNA construct expresses the amino acid sequence of at least one of the Ebola virus GP, NP, VP24, VP30, VP35, or VP40 proteins (or a peptide comprising at least 6 amino acids thereof), under such conditions that a protective CTL response is induced in that mammal. In particular, the administered peptides may include one or more of SEQ ID NOs: 24-53. In another version of the method, one of the above-described immunogenic compositions is administered to the mammal, and preferably one that comprises virus replicon particles containing one of the Ebola virus GP, NP, VP24, VP30, VP35, or VP40 proteins (or a peptide comprising at least 6 amino acids thereof), or including one of the CTL epitopes set forth in SEQ ID NOs:24-53. In another version of the method, the amino acid sequence of at least one of the Ebola virus GP, NP, VP24, VP30, VP35, or VP40 proteins (or a peptide comprising at least 6 amino acids thereof) is administered to a mammal, such as, for example, a mouse, a guinea pig, a monkey or a human, under such conditions that a protective CTL response is induced in that mammal. In particular, the administered peptides may include one or more of SEQ ID NOs: 24-53.

In a preferred method, a recombinant DNA construct is administered to a mammal, which expresses the amino acid sequence of the Ebola virus GP protein (or a peptide comprising at least 6 amino acids thereof), under such conditions that a protective CTL response is induced in that mammal. In another version of the method, the immunogenic compositions comprises virus replicon particles containing GP protein (or a peptide comprising at least 6 amino acids thereof).

In another preferred method, a recombinant DNA construct is administered to a mammal, which expresses the amino acid sequences of the six Ebola virus GP, NP, VP24, VP30, VP35 and VP40 proteins (or a peptide comprising at least 6 amino acids of each of the proteins), under such conditions that a protective CTL response is induced in that mammal. In another version of the method, the immunogenic composition comprises virus replicon particles containing the six GP, NP, VP24, VP30, VP35 and VP40 proteins (or a peptide comprising at least 6 amino acids of each of the proteins).

Another embodiment of this invention entails methods for administering a vaccine of virus replicon particles (preferably VEE virus replicon particles) expressing the Ebola virus GP protein, or VEE virus replicons each expressing one of Ebola proteins GP, NP, VP24, VP30, VP35 and VP40, to form a six-protein vaccine. It is important that the vaccines should be administered at a dosage of not greater than 1.times.10.sup.8 focus forming units (ffu) per inoculation per protein. A higher dosage, such as of 1.times.10.sup.7 ffu, is not useful. The vaccines may be administered via intramuscularly injection, which is preferred and considered a novel feature of our invention. However, subcutaneous injection is also effective as long as the dosage is 1.times.10.sup.8 ffu or lower, per inoculation. Therefore, for the GP-only vaccine, the dosage is 1.times.10.sup.8 focus forming units (ffu) or lower. For the six-peptide cocktail, the dosage is 1.times.10.sup.8 ffu or lower, per inoculation, per peptide--with a total dosage of 6.times.10.sup.8 ffu or lower. Dosage regimen can also be important, and the preferred inoculation schedule is: Day 0, one month, four months, and twelve months.

DETAILED DESCRIPTION

In the description that follows, a number of terms used in recombinant DNA, virology and immunology are extensively utilized. In order to provide a clearer and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

Filoviruses. The filoviruses (e.g. Ebola Zaire 1976) cause acute hemorrhagic fever characterized by high mortality. Humans can contract filoviruses by infection in endemic regions, by contact with imported primates, and by performing scientific research with the virus. However, there currently are no available vaccines or effective therapeutic treatments for filovirus infection in humans. The virions of filoviruses contain seven proteins: a membrane-anchored glycoprotein (GP), a nucleoprotein (NP), an RNA-dependent RNA polymerase (L), and four virion structural proteins (VP24, VP30, VP35, and VP40). Little is known about the biological functions of these proteins and it is not known which antigens significantly contribute to protection and should therefore be used in an eventual vaccine candidate.

Replicon. A replicon is equivalent to a full-length virus from which all of the viral structural proteins have been deleted. A multiple cloning site can be inserted downstream of the 26S promoter into the site previously occupied by the structural protein genes. Virtually any heterologous-gene may be inserted into this cloning site. The RNA that is transcribed from the replicon is capable of replicating and expressing viral proteins in a manner that is similar to that seen with the full-length infectious virus clone. However, in lieu of the viral structural proteins, the heterologous antigen is expressed from the 26S promoter in the replicon. This system does not yield any progeny virus particles because there are no viral structural proteins available to package the RNA into particles.

Particles which appear structurally identical to virus particles can be produced by supplying structural protein RNAs in trans for packaging of the replicon RNA. This is typically done with two defective helper RNAs which encode the structural proteins. One helper consists of a full length infectious clone from which the nonstructural protein genes and the glycoprotein genes are deleted. This helper retains only the terminal nucleotide sequences, the promoter for subgenomic mRNA transcription and the sequences for the viral nucleocapsid protein. The second helper is identical to the first except that the nucleocapsid gene is deleted and only the glycoprotein genes are retained. The helper RNAs are transcribed in vitro and are co-transfected with replicon RNA. Because the replicon RNA retains the sequences for packaging by the nucleocapsid protein, and because the helpers lack these sequences, only the replicon RNA is packaged by the viral structural proteins. The packaged replicon particles are released from the host cell and can then be purified and inoculated into animals. The packaged replicon particles will have a tropism similar to the parent virus. The packaged replicon particles will infect cells and initiate a single round of replication, resulting in the expression of only the virus nonstructural proteins and the product of the heterologous gene that was cloned in the place of the virus structural proteins. In the absence of RNA encoding the virus structural proteins, no progeny virus particles can be produced from the cells infected by packaged replicon particles.

Any alphavirus replicon may be effective in this invention, as long as it has the ability to produce the desired protein or peptide in a manner that induces protective B and T cells in vivo in mammals to which it is administered to (such as, for instance, eastern equine encephalitis, western equine encephalitis, Semlike forest, Sindbis and Venezualen Equine Encephalitis).

The VEE virus replicon (Vrep) is a preferred vector system. The Vrep is a genetically reorganized version of the VEE virus genome in which the structural protein genes are replaced with a gene from an immunogen of interest, such as the Ebola virus virion proteins. This replicon can be transcribed to produce a self-replicating RNA that can be packaged into infectious particles using defective helper RNAs that encode the glycoprotein and capsid proteins of the VEE virus. Since the packaged replicons do not encode the structural proteins, they are incapable of spreading to new cells and therefore undergo a single abortive round of replication in which large amounts of the inserted immunogen are made in the infected cells. The VEE virus replicon system is described in U.S. Patent to Johnston et al., U.S. Pat. No. 5,792,462 issued on Aug. 11, 1998.

Subject. Includes human, animal, e.g., horse, donkey, pig, mouse, hamster, monkey, chicken, and insect such as mosquito.

In one embodiment, the present invention relates to DNA fragments which encode any of the Ebola Zaire 1976 (Maying a isolate) GP, NP, VP24, VP30, VP35, and VP40 proteins. The GP and NP genes of Ebola Zaire were previously sequenced by Sanchez et al. (1993, supra) and have been deposited in GenBank (accession number L11365). A plasmid encoding the VEE replicon vector containing a unique ClaI site downstream from the 26S promoter was described previously (Davis, N. L. et al., (1996) J. Virol. 70, 3781-3787; Pushko, P. et al. (1997) Virology 239, 389-401). The Ebola GP and NP genes from the Ebola Zaire 1976 virus were derived from PS64-- and PGEM3ZF(-)-based plasmids (Sanchez, A. et al. (1989) Virology 170, 81-91; Sanchez, A. et al. (1993) Virus Res. 29, 215-240). From these plasmids, the BamHI-EcoRI (2.3 kb) and BamHI-KpnI (2.4 kb) fragments containing the NP and GP genes, respectively, were subcloned into a shuttle vector that had been digested with BamHI and EcoRI (Davis et al. (1996) supra; Grieder, F. B. et al. (1995) Virology 206, 994-1006). For cloning of the GP gene, overhanging ends produced by KpnI (in the GP fragment) and EcoRI (in the shuttle vector) were made blunt by incubation with T4 DNA polymerase according to methods known in the art. From the shuttle vector, GP or NP genes were subcloned as ClaI-fragments into the ClaI site of the replicon clone, resulting in plasmids encoding the GP or NP genes in place of the VEE structural protein genes downstream from the VEE 26S promoter.

The VP genes of Ebola Zaire were previously sequenced by Sanchez et al. (1993, supra) and have been deposited in GenBank (accession number L11365). The VP genes of Ebola used in the present invention were cloned by reverse transcription of RNA from Ebola-infected Vero E6 cells and subsequent amplification of viral cDNAs using the polymerase chain reaction. First strand synthesis was primed with oligo dT (Life Technologies). Second strand synthesis and subsequent amplification of viral cDNAs were performed with gene-specific primers (SEQ ID NOS:8-16). The primer sequences were derived from the GenBank deposited sequences and were designed to contain a ClaI restriction site for cloning the amplified VP genes into the ClaI site of the replicon vector. The letters and numbers in bold print indicate Ebola gene sequences in the primers and the corresponding location numbers based on the GenBank deposited sequences.

VP24: (1) forward primer is 5'-GGGATCGATCTCCAGACACCAAGCAAGACC-3' (SEQ ID NO: 8)

(10,311-10,331) (2) reverse primer is 5'-GGGATCGATGAGTCAGCATATATGAGTTAGCTC-3' (SEQ ID NO:9) (11,122-11,145) VP30: (1) forward primer is 5'-CCCATCGATCAGATCTGCGAACCGGTAGAG-3' SEQ ID NO:10) (8408-8430) (2) reverse primer is 5'-CCCATCGATGTACCCTCATCAGACCATGAGC-3' (SEQ ID NO:11) (9347-9368) VP35: (1) forward primer is 5'-GGGATCGATAGAAAAGCTGGTCTAACAAGATGA-3' (SEQ ID NO:12) (3110-3133) (2) reverse primer is 5'-CCCATCGATCTCACAAGTGTATCATTAATGTAACGT-3' (SEQ ID NO:13) (4218-4244) VP40: (1) forward primer is 5'-CCCATCGATCCTACCTCGGCTGAGAGAGTG-3' (SEQ ID NO: 14) (4408-4428) (2) reverse primer is 5'-CCCATCGATATGTTATGCACTATCCCTGAGAAG-3' (SEQ ID NO:15) (5495-5518) VP30 #2: (1) forward primer as for VP30 above (2) reverse primer is 5'-CCCATCGATCTGTTAGGGTTGTATCATACC-3' (SEQ ID NO: 16)

The Ebola virus genes cloned into the VEE replicon were sequenced. Changes in the DNA sequence relative to the sequence published by Sanchez et al. (1993) are described relative to the nucleotide (nt) sequence number from GenBank (accession number L11365).

The nucleotide sequence we obtained for Ebola virus GP (SEQ ID NO:1) differed from the GenBank sequence by a transition from A to G at nt 8023. This resulted in a change in the amino acid sequence from Ile to Val at position 662 (SEQ ID NO: 17).

The nucleotide sequence we obtained for Ebola virus NP (SEQ ID NO:2) differed from the GenBank sequence at the following 4 positions: insertion of a C residue between nt 973 and 974, deletion of a G residue at nt 979, transition from C to T at nt 1307, and a transversion from A to C at nt 2745. These changes resulted in a change in the protein sequence from Arg to Glu at position 170 and a change from Leu to Phe at position 280 (SEQ ID NO: 18).

The Ebola virus VP24 nucleotide sequence (SEQ ID NO:3) differed from the GenBank sequence at 6 positions, resulting in 3 nonconservative changes in the amino acid sequence. The changes in the DNA sequence of VP24 consisted of a transversion from G to C at nt 10795, a transversion from C to G at nt 10796, a transversion from T to A at nt 10846, a transversion from A to T at nt 10847, a transversion from C to G at nt 11040, and a transversion from C to G at nt 11041. The changes in the amino acid sequence of VP24 consisted of a Cys to Ser change at position 151, a Leu to H is change at position 168, and a Pro to Gly change at position 233 (SEQ ID NO: 19).

Two different sequences for the Ebola virus VP30 gene, VP30 and VP30#2 (SEQ ID NOS: 4 and 7) are included. Both of these sequences differ from the GenBank sequence by the insertion of an A residue in the upstream noncoding sequence between nt 8469 and 8470 and an insertion of a T residue between nt 9275 and 9276 that results in a change in the open reading frame of VP30 and VP30#2 after position 255 (SEQ ID NOS: 20 and 23). As a result, the C-terminus of the VP30 protein differs significantly from that previously reported. In addition to these 2 changes, the VP30#2 nucleic acid in SEQ ID NO:7 contains a conservative transition from T to C at nt 9217. Because the primers originally used to clone the VP30 gene into the replicon were designed based on the GenBank sequence, the first clone that we constructed (SEQ ID NO: 4) did not contain what we believe to be the authentic C-terminus of the protein. Therefore, in the absence of the VP30 stop codon, the C-terminal codon was replaced with 37 amino acids derived from the vector sequence. The resulting VP30 construct therefore differed from the GenBank sequence in that it contained 32 amino acids of VP30 sequence (positions 256 to 287, SEQ ID NO:20) and 37 amino acids of irrelevant sequence (positions 288 to 324, SEQ ID NO:20) in the place of the C-terminal 5 amino acids reported in GenBank. However, inclusion of 37 amino acids of vector sequence in place of the C-terminal amino acid (Pro, SEQ ID NO: 23) did not inhibit the ability of the protein to serve as a protective antigen in BALB/c mice. We have also determined that a VEE replicon construct, which contains the authentic C-terminus of VP30 (VP30#2, SEQ ID NO: 23), protects mice against a lethal Ebola challenge.

The nucleotide sequence for Ebola virus VP35 (SEQ ID NO:5) differed from the GenBank sequence by a transition from T to C at nt 4006, a transition from T to C at nt 4025, and an insertion of a T residue between nt 4102 and 4103. These sequence changes resulted in a change from a Ser to a Pro at position 293 and a change from Phe to Ser at position 299 (SEQ ID NO: 21). The insertion of the T residue resulted in a change in the open reading frame of VP35 from that previously reported by Sanchez et al. (1993) following amino acid number 324. As a result, Ebola virus VP35 encodes a protein of 340 amino acids, where amino acids 325 to 340 (SEQ ID NO: 21) differ from and replace the C-terminal 27 amino acids of the previously published sequence.

Sequencing of VP30 and VP35 was also performed on RT/PCR products from RNA derived from cells that were infected with Ebola virus 1976, Ebola virus 1995 or the mouse-adapted Ebola virus. The changes noted above for the Vrep constructs were also found in these Ebola viruses. Thus, we believe that these changes are real events and not artifacts of cloning.

The Ebola virus VP40 nucleotide sequence (SEQ ID NO:6) differed from the GenBank sequence by a transversion from a C to G at nt 4451 and a transition from a G to A at nt 5081. These sequence changes did not alter the protein sequence of VP40 (SEQ ID NO: 22) from that of the published sequence.

Each of the Ebola virus genes were individually inserted into a VEE virus replicon vector. The VP24, VP30, VP35, and VP40 genes of Ebola Zaire 1976 (Maying a isolate) were cloned by reverse transcription of RNA from Ebola-infected Vero E6 cells and viral cDNAs were amplified using the polymerase chain reaction. The Ebola Zaire 1976 (Maying a isolate) GP and NP genes were obtained from plasmids already containing these genes (Sanchez, A. et al., (1989) Virology 170, 81-91; Sanchez, A. et al., (1993) Virus Res. 29, 215-240) and were subcloned into the VEE replicon vector.

After characterization of the Ebola gene products expressed from the VEE replicon constructs in cell culture, these constructs were packaged into infectious VEE virus replicon particles (VRPs) and subcutaneously injected into BALB/c and C57BL/6 mice. As controls in these experiments, mice were also immunized with a VEE replicon expressing Lassa nucleoprotein (NP) as an irrelevant control antigen, or injected with PBS buffer alone. The results of this study demonstrate that VRPs expressing the Ebola GP, NP, VP24, VP30, VP35 or VP40 genes induced protection in mice and may reasonably to expected to provide protection in humans.

DNA or polynucleotide sequences to which the invention also relates include sequences of at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, most preferably at least about 15-20 nucleotides corresponding, i.e., homologous to or complementary to, a region of the Ebola nucleotide sequences described above. Preferably, the sequence of the region from which the polynucleotide is derived is homologous to or complementary to a sequence which is unique to the Ebola genes. Whether or not a sequence is unique to the Ebola gene can be determined by techniques known to those of skill in the art. For example, the sequence can be compared to sequences in databanks, e.g., GenBank and compared by DNA:DNA hybridization. Regions from which typical DNA sequences may be derived include but are not limited to, for example, regions encoding specific epitopes, as well as non-transcribed and/or non-translated regions.

The derived polynucleotide is not necessarily physically derived from the nucleotide sequences shown in SEQ ID NO: 1-7, but may be generated in any manner, including for example, chemical synthesis or DNA replication or reverse transcription or transcription, which are based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. In addition, combinations of regions corresponding to that of the designated sequence may be modified in ways known in the art to be consistent with an intended use. The sequences of the present invention can be used in diagnostic assays such as hybridization assays and polymerase chain reaction assays, for example, for the discovery of other Ebola sequences.

In another embodiment, the present invention relates to a recombinant DNA molecule that includes a vector and a DNA sequence as described above. The vector can take the form of a plasmid, a eukaryotic expression vector such as pcDNA3.1, pRcCMV2, pZeoSV2, or pCDM8, which are available from Invitrogen, or a virus vector such as baculovirus vectors, retrovirus vectors or adenovirus vectors, alphavirus vectors, and others known in the art.

In a further embodiment, the present invention relates to host cells stably transformed or transfected with the above-described recombinant DNA constructs. The host cell can be prokaryotic (for example, bacterial), lower eukaryotic (for example, yeast or insect) or higher eukaryotic (for example, all mammals, including but not limited to mouse and human). Both prokaryotic and eukaryotic host cells may be used for expression of the desired coding sequences when appropriate control sequences which are compatible with the designated host are used.

Among prokaryotic hosts, E. coli is the most frequently used host cell for expression. General control sequences for prokaryotes include promoters and ribosome binding sites. Transfer vectors compatible with prokaryotic hosts are commonly derived from a plasmid containing genes conferring ampicillin and tetracycline resistance (for example, pBR322) or from the various pUC vectors, which also contain sequences conferring antibiotic resistance. These antibiotic resistance genes may be used to obtain successful transformants by selection on medium containing the appropriate antibiotics. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985) for general cloning methods. The DNA sequence can be present in the vector operably linked to sequences encoding an IgG molecule, an adjuvant, a carrier, or an agent for aid in purification of Ebola proteins, such as glutathione S-transferase.

In addition, the Ebola virus gene products can also be expressed in eukaryotic host cells such as yeast cells and mammalian cells. Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Pichia pastoris are the most commonly used yeast hosts. Control sequences for yeast vectors are known in the art. Mammalian cell lines available as hosts for expression of cloned genes are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), such as CHO cells, Vero cells, baby hamster kidney (BHK) cells and COS cells, to name a few. Suitable promoters are also known in the art and include viral promoters such as that from SV40, Rous sarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus (BPV), and cytomegalovirus (CMV). Mammalian cells may also require terminator sequences, poly A addition sequences, enhancer sequences which increase expression, or sequences which cause amplification of the gene. These sequences are known in the art.

The transformed or transfected host cells can be used as a source of DNA sequences described above. When the recombinant molecule takes the form of an expression system, the transformed or transfected cells can be used as a source of the protein described below.

In another embodiment, the present invention relates to Ebola virion proteins such as

GP having an amino acid sequence corresponding to SEQ ID NO:17 encompassing 676 amino acids,

NP, having an amino acid sequence corresponding to SEQ ID NO: 18 encompassing 739 amino acids,

VP24, having an amino acid sequence corresponding to SEQ ID NO: 19 encompassing 251 amino acids,

VP30, having an amino acid sequence corresponding SEQ ID NO:20 encompassing 324 amino acids,

VP35, having an amino acid sequence corresponding to SEQ ID NO:21 encompassing 340 amino acids, and

VP40, having an amino acid sequence corresponding to SEQ ID NO:22, encompassing 326 amino acids, and

VP30#2, having an amino acid sequence corresponding to SEQ ID NO:23 encompassing 288 amino acids, or any allelic variation of these amino acid sequences. By allelic variation is meant a natural or synthetic change in one or more amino acids which occurs between different serotypes or strains of Ebola virus and does not affect the antigenic properties of the protein. There are different strains of Ebola (Zaire 1976, Zaire 1995, Reston, Sudan, and Ivory Coast). The NP and VP genes of all these different viruses have not been sequenced. It would be expected that these proteins would have homology among different strains and that vaccination against one Ebola virus strain might afford cross protection to other Ebola virus strains.

A polypeptide or amino acid sequence derived from any of the amino acid sequences in SEQ ID NO:17, 18, 19, 20, 21, 22, and 23 refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 2-5 amino acids, preferably at least 8-10 amino acids, and more preferably at least 11-15 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence.

A recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence, or the DNA sequence found in GenBank accession number L11365. It may be generated in any manner, including for example, chemical synthesis, or expression from a recombinant expression system.

When the DNA or RNA sequences described above are in a replicon expression system, such as the VEE replicon described above, the proteins can be expressed in vivo. The DNA sequence for any of the GP, NP, VP24, VP30, VP35, and VP40 virion proteins can be cloned into the multiple cloning site of a replicon such that transcription of the RNA from the replicon yields an infectious RNA encoding the Ebola protein or proteins of interest (see FIGS. 2A, 2B and 2C (see Original Patent)). The replicon constructs include Ebola virus GP (SEQ ID NO: 1) cloned into a VEE replicon (VRepEboGP), Ebola virus NP (SEQ ID NO:2) cloned into a VEE replicon (VRepEboNP), Ebola virus VP24 (SEQ ID NO:3) cloned into a VEE replicon (VRepEboVP24), Ebola virus VP30 (SEQ ID NO:4) or VP30#2 (SEQ ID NO:7) cloned into a VEE replicon (VRepEboVP30 or VRepEboVP30(#2)), Ebola virus VP35 (SEQ ID NO:5) cloned into a VEE replicon (VRepEboVP35), and Ebola virus VP40 (SEQ ID NO:6) cloned into a VEE replicon (VRepEboVP40). The replicon DNA or RNA can be used as a vaccine for inducing protection against infection with Ebola.

Use of helper RNAs containing sequences necessary for packaging of the viral replicon transcripts will result in the production of virus-like particles containing replicon RNAs (FIG. 3 (see Original Patent)). These packaged replicons will infect host cells and initiate a single round of replication resulting in the expression of the Ebola proteins in the infected cells. The packaged replicon constructs (i.e. VEE virus replicon particles, VRP) include those that express Ebola virus GP (EboGPVRP), Ebola virus NP (EboNPVRP), Ebola virus VP24 (EboVP24VRP), Ebola virus VP30 (EboVP30VRP or EboVP30VRP(#2)), Ebola virus VP35 (EboVP35VRP), and Ebola virus VP40 (EboVP40VRP).

In another embodiment, the present invention relates to RNA molecules resulting from the transcription of the constructs described above. The RNA molecules can be prepared by in vitro transcription using methods known in the art and described in the Examples below. Alternatively, the RNA molecules can be produced by transcription of the constructs in vivo, and isolating the RNA. These and other methods for obtaining RNA transcripts of the constructs are known in the art. Please see Current Protocols in Molecular Biology. Frederick M. Ausubel et al. (eds.), John Wiley and Sons, Inc. The RNA molecules can be used, for example, as a direct RNA vaccine, or to transfect cells along with RNA from helper plasmids, one of which expresses VEE glycoproteins and the other VEE capsid proteins, as described above, in order to obtain replicon particles.

In a further embodiment, the present invention relates to a method of producing the recombinant or fusion protein which includes culturing the above-described host cells under conditions such that the DNA fragment is expressed and the recombinant or fusion protein is produced thereby. The recombinant or fusion protein can then be isolated using methodology well known in the art. The recombinant or fusion protein can be used as a vaccine for immunity against infection with Ebola or as a diagnostic tool for detection of Ebola infection.

In another embodiment, the present invention relates to antibodies specific for the above-described recombinant proteins (or polypeptides). For instance, an antibody can be raised against a peptide having the amino acid sequence of any of SEQ ID NO:17-25, or against a portion thereof of at least 10 amino acids, preferably, 11-15 amino acids. Persons with ordinary skill in the art using standard methodology can raise monoclonal and polyclonal antibodies to the protein (or polypeptide) of the present invention, or a unique portion thereof.

Materials and Methods for producing antibodies are well known in the art (see for example Goding, In Monoclonal Antibodies: Principles and Practice, Chapter 4, 1986).

In another embodiment, the present invention relates to an Ebola vaccine comprising VRPs that express one or more of the Ebola proteins described above. The vaccine is administered to a subject wherein the replicon is able to initiate one round of replication producing the Ebola proteins to which a protective immune response is initiated in said subject.

One particularly effective vaccine comprises virus replicon particles expressing the GP Ebola peptide. Preferably the GP Ebola peptide has the sequence specified in SEQ ID NO:29, or a peptide fragment comprising at least 9 consecutive amino acids. For instance, the peptide fragment may has the sequence of YFGPAAEGI (SEQ ID NO:42).

Another particularly effective vaccine comprises virus replicon particles expressing the Ebola peptides GP, NP, VP24, VP30, VP35, and VP40, in an effective immunogenic amount, in a pharmaceutically acceptable and/or adjuvant. Thus, this vaccine contains all six of the Ebola peptides. The virus replicon particles are preferably produced from a replicon vector selected from the group consisting of Venezuelan Equine Encephalitis (VEE) virus, eastern equine encephalitis, western equine encephalitis, Semliki forest and Sindbis. Preferably, the GP peptide has the sequence specified in SEQ ID NO:29, or a peptide fragment comprising at least 9 consecutive amino acids; the NP peptide has the sequence specified in SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28, or a peptide fragment comprising at least 9 consecutive amino acids; the VP24 peptide has the sequence specified in SEQ ID NO:25, SEQ ID NO:30, or SEQ ID NO:31, or a peptide fragment comprising at least 9 consecutive amino acids; the VP30 peptide has the sequence specified in SEQ ID NO:32 or SEQ:ID NO:33, or a peptide fragment comprising at least 9 consecutive amino acids; the VP35 peptide has the sequence specified in SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36, or a peptide fragment comprising at least 9 consecutive amino acids; and the VP40 peptide has the sequence specified in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:41, or a peptide fragment comprising at least 9 consecutive amino acids. The GP peptide fragment may the sequence of YFGPAAEGI (SEQ ID NO:42); the VP24 peptide fragment may have the sequence of KFINKLDAL, (SEQ ID NO:43), NYNGLLSSI (SEQ ID NO:44), or PGPAKFSLL (SEQ ID NO:45); the VP30 peptide fragment may have the sequence of LSLLCETHLR (SEQ ID NO:46) or MFITAFLNI (SEQ ID NO:47); and the VP40 peptide fragment may have the sequence of EFVLPPVQL (SEQ ID NO:48), FLVPPV (SEQ ID NO:49), QYFTFDLTALK (SEQ ID NO:50), TSPEKIQAI (SEQ ID NO:51), RIGNQAFL (SEQ ID NO:52) or QAFLQEFV (SEQ ID NO:53).

The method of administering the vaccine is also a novel aspect of our invention. For the GP-alone vaccine, a mammal, including a human, may be injected subcutaneously or preferably intramuscularly with a composition comprising virus replicon particles expressing the GP Ebola peptide, in a dosage not greater than 1.times.10.sup.8 focus forming units. For the six-protein vaccine, a mammal, including a human, may be injected subcutaneously or preferably intramuscularly with a composition comprising virus replicon particles expressing the GP Ebola peptide, in a dosage not greater than 1.times.10.sup.8 focus forming units per inoculation per peptide--with a total dosage of 6.times.10.sup.8 focus forming units. Injections are preferably repeated as needed or desired for maximum immunoprotection, such as at one month, four months, and twelve months.

It is likely that the protection afforded by these genes is due to both the humoral (antibodies (Abs)) and cellular (cytotoxic T cells (CTLs)) arms of the immune system. Protective immunity induced to a specific protein may comprise humoral immunity, cellular immunity, or both. The only Ebola virus protein known to be on the outside of the virion is the GP. The presence of GP on the virion surface makes it a likely target for GP-specific Abs that may bind either extracellular virions or infected cells expressing GP on their surfaces. Serum transfer studies in this invention demonstrate that Abs that recognize GP protect mice against lethal Ebola virus challenge.

In contrast, transfer of Abs specific for NP, VP24, VP30, VP35, or VP40 did not protect mice against lethal Ebola challenge. This data, together with the fact that these are internal virion proteins that are not readily accessible to Abs on either extracellular virions or the surface of infected cells, suggest that the protection induced in mice by these proteins is mediated by CTLs.

CTLs can bind to and lyse virally infected cells. This process begins when the proteins produced by cells are routinely digested into peptides. Some of these peptides are bound by the class I or class II molecules of the major histocompatability complex (MHC), which are then transported to the cell surface. During virus infections, viral proteins produced within infected cells also undergo this process. CTLs that have receptors that bind to both a specific peptide and the MHC molecule holding the peptide lyse the peptide-bearing cell, thereby limiting virus replication. Thus, CTLs are characterized as being specific for a particular peptide and restricted to a class I or class II MHC molecule.

CTLs may be induced against any of the Ebola virus proteins, as all of the viral proteins are produced and digested within the infected cell. Thus, protection to Ebola virus involves CTLs against GP, NP, VP24, VP30, VP35, and/or VP40. It is especially noteworthy that the VP proteins varied in their protective efficacy when tested in genetically inbred mice that differ at the MHC locus. This, together with the inability to demonstrate a role for Abs in protection induced by the VP proteins and the data in Table A below (see Original Patent), demonstrates a role for CTLs. Thus, in this invention a vaccine may include several Ebola virus proteins (e.g., at least two), or several CTL epitopes (e.g., at least two), capable of inducing broad protection to different Ebola viruses in outbred populations (e.g. people). To that end, the inventors have identified 18 sequences recognized by CTLs, as determined initially by measuring gamma interferon production by intracellular cytokine staining and gamma interferon secretion by the ELISpot assay. The ability to lyse cells was measured in chromium release assays and protection was evaluated by adoptive transfer of cells into Ebola-naive mice. Where sequence information is available, the conservation of these CTL epitopes in other Ebola viruses is noted in Table A. Conserved sequences should be capable of inducing protective CTLs to each of the viruses in which the sequence is present.

The identified CTL epitopes are:

Ebola virus NP SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28;

Ebola virus GP SEQ ID NO:29 (encompassing YFGPAAEGI, SEQ ID NO:42);

Ebola virus VP24 SEQ ID NO:460 (encompassing KFINKLDAL, SEQ ID NO:43), SEQ ID NO:30 (encompassing NYNGLLSSI, SEQ ID NO:44), and SEQ ID NO:31 (encompassing PGPAKFSLL, SEQ ID NO:45);

Ebola virus VP30 SEQ ID NO:32 (encompassing LSLLCETHLR, SEQ ID NO:46), and SEQ ID NO:33 (encompassing MFITAFLNI, SEQ ID NO:47);

Ebola virus VP35 SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36; and

Ebola virus VP40 SEQ ID NO:37 (encompassing EFVLPPVQL, SEQ ID NO:48), SEQ ID NO:38 (encompassing FLVPPV, SEQ ID NO:49 and QYFTFDLTALK, SEQ ID NO:50), SEQ ID NO:39 (encompassing TSPEKIQAI, SEQ ID NO:51); SEQ ID NO:40 (encompassing RIGNQAFL, SEQ ID NO:52), and SEQ ID NO:41 (encompassing QAFLQEFV, SEQ ID NO:53).

See Table A below -- see Original Patent.

Testing to identify the role of CTLs in protection was performed by obtaining CTLs from mice, expanding them in vitro, and transferring the cells into an unvaccinated mouse of the same genetic background. Several hours later, the recipient mice were challenged with 10-1000 pfu of mouse-adapted Ebola virus. They were observed for signs of illness for 28 days. Control animals received no cells or cells that were not specific for Ebola virus. Testing of the cells in vitro indicated that they were CD8+, a marker indicating class I-restriction. The inventors were able to demonstrate that CTLs to these sequences protected at least 80% of recipient mice from challenge. In all of our examples, the ability to lyse peptide-pulsed target cells has predicted protection in mice receiving those cells.

In another vaccine embodiment, the invention relates to a vaccine against Ebola infection including at least one of these CTL epitope sequences, and preferably at least one CTL epitope having the amino acid sequence of SEQ ID NOs:24-53. Preferably, the vaccine includes at least two of the CTL epitope sequences, more preferably at least three, more preferably at least four, more preferably at least five, and more preferably all of the sequences. As shown in the examples below, protection is increased as the number of CTL epitopes in the immunogenic composition or vaccine is increased, and also as the number of epitopes from different Ebola proteins is increased.

In another vaccine embodiment, the vaccine includes a CTL epitope sequence from at least two different proteins selected from the group consisting of GP, NP, VP24, VP30, VP35 and VP40. More preferably, the vaccine includes a CTL epitope sequence from at least three different proteins from that group, more preferably at least four, more preferably at least five, and most preferably includes at least one CTL epitope sequence from each of the six proteins. The CTL epitopes may have the amino acid sequences as set forth in SEQ ID NOs:24-53. It is noted that administering the GP peptide alone may prevent the induction of protective antibodies, which may be undesirable.

In another vaccine embodiment, the vaccine may include at a minimum at least one of the Ebola proteins selected from GP, NP, VP24, VP30, VP35 and VP40, but preferably contains at least two, more preferably at least three, more preferably at least four, more preferably at least five, and most preferably all of them. In another preferred embodiment, the vaccine may include one or more of SEQ ID NOs: 24-53.

When considering which type of vaccine may be most effective for an individual, it is noted that the same protective response could be induced by the peptide or the full protein produced from the VRPs. Production of the peptide intracellularly is generally preferred because it is usually (but not always) more effective than providing it extracellularly. Thus, vaccines containing VRPs may be preferred because the VRPs infect cells and therefore achieve intracellular production.

Such vaccines might be delivered as synthetic peptides, or as fusion proteins, alone or co-administered with cytokines and/or adjuvants or carriers safe for human use, e.g. aluminum hydroxide, to increase immunogenicity. In addition, sequences such as ubiquitin can be added to increase antigen processing for more effective CTL responses.

In yet another embodiment, the present invention relates to a method for providing immunity against Ebola virus, said method comprising administering one or more VRPs expressing any combination of the GP, NP, VP24, VP30 or VP30#2, VP35 and VP40 Ebola proteins to a subject such that a protective immune reaction is generated. In another related embodiment, the method may entail administering one or more VRPs expressing any combination of the peptides designated SEQ ID NOs:24-53, or simply one or more of the peptides designated SEQ ID NOs:24-53.

Vaccine formulations of the present invention may comprise an immunogenic amount of a VRP, such as for example EboVP24VRP described above, or, for a multivalent vaccine, a combination of replicons, in a pharmaceutically acceptable carrier. An "immunogenic amount" is an amount of the VRP(s) sufficient to evoke an immune response in the subject to which the vaccine is administered. An amount of about 1.times.10.sup.8 focus forming units or less, per dose, is suitable. The dosage may vary a bit depending upon the age and species of the subject being treated. For instance, in two species of monkeys the dosage of about 1.times.10.sup.8 focus forming units was quite successful. The subject may be inoculated 2-4 times. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sterile pyrogen-free water and sterile pyrogen-free physiological saline solution.

Administration of the VRPs disclosed herein may be carried out by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), in ovo injection of birds, orally, or by topical application of the virus (typically carried in a pharmaceutical formulation) to an airway surface. Topical application of the virus to an airway surface can be carried out by intranasal administration (e.g., by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally). Topical application of the virus to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the replicon as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed. Oral administration may be in the form of an ingestable liquid or solid formulation. As noted above, one preferred way is subcutaneous injection, and most preferred is intramuscular injection.

When the replicon RNA or DNA is used as a vaccine, the replicon RNA or DNA can be administered directly using techniques such as delivery on gold beads (gene gun), delivery by liposomes, or direct injection, among other methods known to people in the art. Any one or more DNA constructs or replicating RNA described above can be use in any combination effective to elicit an immunogenic response in a subject. Generally, the nucleic acid vaccine administered may be in an amount of about 1-5 ug of nucleic acid per dose and will depend on the subject to be treated, capacity of the subject's immune system to develop the desired immune response, and the degree of protection desired. Precise amounts of the vaccine to be administered may depend on the judgement of the practitioner and may be peculiar to each subject and antigen.

As noted above, the preferred vaccine schedule, especially for the GP-only vaccine and the six-protein vaccine, is Day 0, one month, four months, and twelve months. However, generally speaking, the vaccines described herein may also be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. Other examples of suitable immunization schedules include: (i) 0, 1 months and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, (v) 0, 1 and 2 months, or other schedules sufficient to elicit the desired immune responses expected to confer protective immunity, or reduce disease symptoms, or reduce severity of disease.

In a further embodiment, the present invention relates to a method of detecting the presence of antibodies against Ebola virus in a sample. Using standard methodology well known in the art, a diagnostic assay can be constructed by coating on a surface (i.e. a solid support for example, a microtitration plate, a membrane (e.g. nitrocellulose membrane) or a dipstick), all or a unique portion of any of the Ebola proteins described above or any combination thereof, and contacting it with the serum of a person or animal suspected of having Ebola. The presence of a resulting complex formed between the Ebola protein(s) and serum antibodies specific therefor can be detected by any of the known methods common in the art, such as fluorescent antibody spectroscopy or colorimetry. This method of detection can be used, for example, for the diagnosis of Ebola infection and for determining the degree to which an individual has developed virus-specific antibodies after administration of a vaccine.

In yet another embodiment, the present invention relates to a method for detecting the presence of Ebola virion proteins in a sample. Antibodies against GP, NP, and the VP proteins could be used for diagnostic assays. Using standard methodology well known in the art, a diagnostics assay can be constructed by coating on a surface (i.e. a solid support, for example, a microtitration plate or a membrane (e.g. nitrocellulose membrane)), antibodies specific for any of the Ebola proteins described above, and contacting it with serum or a tissue sample of a person suspected of having Ebola infection. The presence of a resulting complex formed between the protein or proteins in the serum and antibodies specific therefor can be detected by any of the known methods common in the art, such as fluorescent antibody spectroscopy or colorimetry. This method of detection can be used, for example, for the diagnosis of Ebola virus infection.

In another embodiment, the present invention relates to a diagnostic kit which contains any combination of the Ebola proteins described above and ancillary reagents that are well known in the art and that are suitable for use in detecting the presence of antibodies to Ebola in serum or a tissue sample. Tissue samples contemplated can be from monkeys, humans, or other mammals.

In yet another embodiment, the present invention relates to DNA or nucleotide sequences for use in detecting the presence of Ebola virus using the reverse transcription-polymerase chain reaction (RT-PCR). The DNA sequence of the present invention can be used to design primers which specifically bind to the viral RNA for the purpose of detecting the presence of Ebola virus or for measuring the amount of Ebola virus in a sample. The primers can be any length ranging from 7 to 400 nucleotides, preferably at least 10 to 15 nucleotides, or more preferably 18 to 40 nucleotides. Reagents and controls necessary for PCR reactions are well known in the art. The amplified products can then be analyzed for the presence of viral sequences, for example by gel fractionation, with or without hybridization, by radiochemistry, and immunochemistry techniques.

In yet another embodiment, the present invention relates to a diagnostic kit which contains PCR primers specific for Ebola virus and ancillary reagents for use in detecting the presence or absence of Ebola in a sample using PCR. Samples contemplated can be obtained from human, animal, e.g., horse, donkey, pig, mouse, hamster, monkey, or other mammals, birds, and insects, such as mosquitoes.

Claim 1 of 3 Claims

1. An Ebola virus vaccine comprising virus replicon particles expressing GP Ebola protein comprising the sequence specified in SEQ ID NO: 29.

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