U.S. patent application number 09/337946 was filed with the patent office on 2002-11-07 for ebola virion proteins expressed from venezuelan equine encephalitis (vee) virus replicons.
Invention is credited to HART, MARY K., PUSHKO, PETER, SCHMALJOHN, ALAN L., SMITH, JONATHAN F., WILSON, JULIE A..
Application Number | 20020164582 09/337946 |
Document ID | / |
Family ID | 22227592 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164582 |
Kind Code |
A1 |
HART, MARY K. ; et
al. |
November 7, 2002 |
EBOLA VIRION PROTEINS EXPRESSED FROM VENEZUELAN EQUINE ENCEPHALITIS
(VEE) VIRUS REPLICONS
Abstract
Using 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.
Inventors: |
HART, MARY K.; (FREDERICK,
MD) ; WILSON, JULIE A.; (FREDERICK, MD) ;
PUSHKO, PETER; (FREDERICK, MD) ; SMITH, JONATHAN
F.; (SABILLASVILLE, MD) ; SCHMALJOHN, ALAN L.;
(FREDERICK, MD) |
Correspondence
Address: |
U S ARMY MEDICAL RESEARCH AND
MATERIAL COMMAND
ATTN MCMR JA CHARLES H HARRIS
504 SCOTT STREET
FORT DETRICK
MD
217025012
|
Family ID: |
22227592 |
Appl. No.: |
09/337946 |
Filed: |
June 22, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60091403 |
Jun 29, 1998 |
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Current U.S.
Class: |
435/5 |
Current CPC
Class: |
C12N 2770/36143
20130101; C07K 14/005 20130101; C12N 15/86 20130101; C12N 7/00
20130101; C12N 2770/36021 20130101; C12N 2770/36123 20130101; C07K
16/10 20130101; C12N 2770/36162 20130101; C12N 2760/14222 20130101;
A61K 2039/53 20130101; C12N 2760/14022 20130101; A61K 38/00
20130101; A61K 2039/5256 20130101; A61K 39/00 20130101; C12N
2770/36122 20130101 |
Class at
Publication: |
435/5 |
International
Class: |
C12Q 001/70 |
Claims
What is claimed is:
1. A DNA fragment which encodes a GP Ebola protein, said DNA
fragment comprising the sequence specified in SEQ ID NO:1, or a
polynucleotide fragment comprising at least 15 nucleotides.
2. A DNA fragment which encodes a NP Ebola protein, said DNA
fragment comprising the sequence specified in SEQ ID NO:2, or a
polynucleotide fragment comprising at least 15 nucleotides.
3. A DNA fragment which encodes a VP24 Ebola protein, said DNA
fragment comprising the sequence specified in SEQ ID NO:3, or a
polynucleotide fragment comprising at least 15 nucleotides.
4. A DNA fragment which encodes a VP30 Ebola protein, said DNA
fragment comprising the sequence specified in any of SEQ ID NO:4
and SEQ ID NO:7, or a polynucleotide fragment comprising at least
15 nucleotides.
5. A DNA fragment which encodes a VP35 Ebola protein, said DNA
fragment comprising the sequence specified in SEQ ID NO:5, or a
polynucleotide fragment comprising at least 15 nucleotides.
6. A DNA fragment which encodes a VP40 Ebola protein, said DNA
fragment comprising the sequence specified in SEQ ID NO:6, or a
polynucleotide fragment comprising at least 15 nucleotides.
7. A DNA fragment which encodes a GP Ebola protein said DNA
fragment comprising a DNA sequence encoding at least 5 amino acids
specified in SEQ ID NO:17 or a conservative substitution
thereof.
8. A DNA fragment which encodes a NP Ebola protein said DNA
fragment comprising a DNA sequence encoding at least 5 amino acids
specified in SEQ ID NO:18 or a conservative substitution
thereof.
9. A DNA fragment which encodes a VP24 Ebola protein said DNA
fragment comprising a DNA sequence encoding at least 5 amino acids
specified in SEQ ID NO:19 or a conservative substitution
thereof.
10. A DNA fragment which encodes a VP30 Ebola protein said DNA
fragment comprising a DNA sequence encoding at least 5 amino acids
specified in any of SEQ ID NO:20 and SEQ ID NO:23 or a conservative
substitution thereof.
11. A DNA fragment which encodes a VP35 Ebola protein said DNA
fragment comprising a DNA sequence encoding at least 5 amino acids
specified in SEQ ID NO:21 or a conservative substitution
thereof.
12. A DNA fragment which encodes a VP40 Ebola protein said DNA
fragment comprising a DNA sequence encoding at least 5 amino acids
specified in SEQ ID NO:22 or a conservative substitution
thereof.
13. A recombinant DNA construct comprising: (i) a vector, and (ii)
at least one of the Ebola virus DNA fragments chosen from the group
consisting of SEQ ID NO:1, 2, 3, 4, 5, 6 and 7 or a fragment
thereof comprising at least 15 nucleotides.
14. A recombinant DNA construct comprising: (i) a vector, and (ii)
at least one of the Ebola virus DNA fragments chosen from the group
consisting of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24 and 25 or a
conservative substitution thereof.
15. The recombinant DNA construct of claim 13 wherein said DNA
fragment induces a cytotoxic T lymphocyte response or antibody
response.
16. The recombinant DNA construct of claim 14 wherein said DNA
fragment induces a cytotoxic T lymphocyte response or antibody
response.
17. A recombinant DNA construct according to claim 13 wherein said
vector is an expression vector.
18. A recombinant DNA construct according to claim 13 wherein said
vector is a prokaryotic vector.
19. A recombinant DNA construct according to claim 13 wherein said
vector is a eukaryotic vector.
20. A recombinant DNA construct according to claim 14 wherein said
vector is an expression vector.
21. A recombinant DNA construct according to claim 14 wherein said
vector is a prokaryotic vector.
22. A recombinant DNA construct according to claim 14 wherein said
vector is a eukaryotic vector.
23. The recombinant DNA construct of claim 17 wherein said vector
is a VEE virus replicon vector.
24. The recombinant DNA construct of claim 20 wherein said vector
is a VEE virus replicon vector.
25. The recombinant DNA construct according to claim 23 wherein
said Ebola virus DNA fragments are from Ebola Zaire 1976.
26. The recombinant DNA construct according to claim 25 wherein
said construct is VRepEboVP24.
27. The recombinant DNA construct according to claim 25 wherein
said construct is VRepEboVP30.
28. The recombinant DNA construct according to claim 25 wherein
said construct is VRepEboVP35.
29. The recombinant DNA construct according to claim 25 wherein
said construct is VRepEboVP40.
30. The recombinant DNA construct according to claim 25 wherein
said construct is for VRepEboNP.
31. The recombinant DNA construct according to claim 25 wherein
said construct is for VRepEboGP.
32. The recombinant DNA construct according to claim 25 wherein
said construct is for VRepEboVP30(#2).
33. Self replicating RNA produced from a construct chosen from the
group consisting of EboVP24ReP, EboVP30ReP, EboVP35ReP, EboVP40ReP,
EboVPNPReP, EboVPGPReP, and EboVP30ReP(#2).
34. Infectious alphavirus particles produced from packaging the
self replicating RNA of claim 33.
35. A pharmaceutical composition comprising infectious alphavirus
particles according to claim 34 in an effective immunogenic amount
in a pharmaceutically acceptable carrier and/or adjuvant.
36. A host cell transformed with a recombinant DNA construct
according to claim 13.
37. A host cell transformed with a recombinant DNA construct
according to claim 14.
38. A host cell according to claim 36 wherein said host cell is
prokaryotic.
39. A host cell according to claim 36 wherein said host cell is
eukaryotic.
40. A host cell according to claim 37 wherein said host cell is
prokaryotic.
41. A host cell according to claim 37 wherein said host cell is
eukaryotic.
42. A method for producing Ebola virus proteins comprising
culturing the cells according to claim 36 under conditions such
that said DNA fragment is expressed and said Ebola protein is
produced.
43. A method for producing Ebola virus proteins comprising
culturing the cells according to claim 37 under conditions such
that said DNA fragment is expressed and said Ebola protein is
produced.
44. A method for producing Ebola virus proteins comprising
culturing the cells according to claim 38 under conditions such
that said DNA fragment is expressed and said Ebola protein is
produced.
45. A method for producing Ebola virus proteins comprising
culturing the cells according to claim 39 under conditions such
that said DNA fragment is expressed and said Ebola protein is
produced.
46. An isolated and purified Ebola GP protein specified in SEQ ID
NO:17 and conservative substitutions thereof, or an immunologically
identifiable portion thereof.
47. An isolated and purified Ebola NP protein specified in SEQ ID
NO:18 and conservative substitutions thereof or an immunologically
identifiable portion thereof.
48. An isolated and purified Ebola VP24 protein specified in SEQ ID
NO:19 and conservative substitutions thereof or an immunologically
identifiable portion thereof.
49. An isolated and purified Ebola VP30 protein specified in any of
SEQ ID NO:20 and SEQ ID NO:23 and conservative substitutions
thereof or an immunologically identifiable portion thereof.
50. An isolated and purified Ebola VP35 protein specified in SEQ ID
NO:21 and conservative substitutions thereof or an immunologically
identifiable portion thereof.
51. An isolated and purified Ebola VP40 protein specified in SEQ ID
NO:22 and conservative substitutions thereof or an immunologically
identifiable portion thereof.
52. An antibody to a peptide encoded by the sequence specified in
SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 24, and 25.
53. A method for detecting Ebola virus infection comprising
contacting a sample from a subject suspected of having Ebola virus
infection with a antibody according to claim 52 and detecting the
presence or absence by detecting the presence or absence of a
complex formed between the Ebola protein and antibodies specific
therefor.
54. A method for detecting the presence or absence of Ebola virus
GP RNA in a sample using the polymerase chain reaction using
primers for Ebola GP nucleic acid sequence specified in SEQ ID NO:1
for GP.
55. An Ebola infection diagnostic kit comprising at least 12
consecutive nucleotides of SEQ ID NO:1 specific for the
amplification of DNA or RNA of Ebola virus in a sample using the
polymerase chain reaction and ancillary reagents suitable for use
in such a reaction for detecting the presence or absence of Ebola
virus DNA or RNA in a sample.
56. A vaccine for Ebola comprising alphavirus particles of claim
34.
57. A method for the diagnosis of Ebola virus infection comprising
the steps of: (i) contacting a sample from an individual suspected
of having Ebola virus infection with an antibody to Ebola proteins
according to claim 52; and (ii) detecting the presence or absence
of Ebola virus infection by detecting the presence or absence of a
complex formed between Ebola proteins and antibodies specific
therefor.
58. A pharmaceutical composition comprising the self replicating
RNA of claim 33 in an effective immunogenic amount in a
pharmaceutically acceptable carrier and/or adjuvant.
59. A pharmaceutical composition comprising one or more recombinant
DNA constructs chosen from the group consisting of VRepEboVP24,
VRepEboVP30, VRepEboVP35, VRepEboVP40, VRepEboNP, VRepEboGP, and
VRepEboVP30(#2), in a pharmaceutically acceptable amount, in a
pharmaceutically accpetable carrier and/or adjuvant.
60. A pharmaceutical composition comprising comprising a peptide
encoded by any of SEQ ID NO:24 and SEQ ID NO:25, in a
pharmaceutically acceptable amount, in a pharmaceutically
acceptable carrier and/or adjuvant.
Description
INTRODUCTION
[0001] Ebola viruses, members of the family Filoviridae, are
associated with outbreaks of highly lethal hemorrhagic fever in
humans and nonhuman primates. The natural reservoir of the virus is
unknown and there currently are no available vaccines or effective
therapeutic treatments for filovirus infections. The genome of
Ebola virus consists of a single strand of negative sense RNA that
is approximately 19 kb in length. This RNA contains seven
sequentially arranged genes that produce 8 mRNAs upon infection
(FIG. 1). Ebola virions, like virions of other filoviruses, contain
seven proteins: a surface glycoprotein (GP), a nucleoprotein (NP),
four virion structural proteins (VP40, VP35, VP30, and VP24), and
an RNA-dependent RNA polymerase (L) (Feldmann et al. (1992) Virus
Res. 24, 1-19; Sanchez et al., (1993) Virus Res. 29, 215-240;
reviewed in Peters et al. (1996) In Fields Virology, Third ed. pp.
1161-1176. Fields, B. N., Knipe, D. M., Howley, P. M., et al. eds.
Lippincott-Raven Publishers, Philadelphia). The glycoprotein of
Ebola virus is unusual in that it is encoded in two open reading
frames. Transcriptional editing is needed to express the
transmembrane form that is incorporated into the virion (Sanchez et
al. (1996) Proc. Natl. Acad. Sci. USA 93, 3602-3607; Volchkov et
al, (1995) Virology 214, 421-430. The unedited form produces a
nonstructural secreted glycoprotein (sGP) that is synthesized in
large amounts early during the course of infection. 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 to induce an immune response.
[0002] Recent studies using rodent models to evaluate subunit
vaccines for Ebola virus infection using recombinant vaccinia virus
encoding Ebola virus GP (Gilligan et al., (1997) In Vaccines 97,
pp. 87-92. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.), or naked DNA constructs expressing either GP or sGP (Xu et
al. (1998) Nature Med. 4, 37-42) have demonstrated the protective
efficacy of Ebola virus GP in guinea pigs. (All documents cited
herein supra and infra are hereby incorporated in their entirety by
reference thereto.) Additionally, Ebola virus NP and GP genes
expressed from naked DNA vaccines (Vanderzanden et al., (1998)
Virology 246, 134-144) have elicited protective immunity in BALB/c
mice. However, successful vaccination of nonhuman primates with
individual Ebola virus genes has not been demonstrated. Therefore,
there exists a need for a vaccine which is efficacious for
protection from Ebola virus infection.
SUMMARY OF THE INVENTION
[0003] 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.
[0004] Because the biological functions of the individual Ebola
virus proteins are not known and the immune mechanisms necessary
for preventing and clearing Ebola virus infection are not well
understood, it was not clear which antigens significantly
contribute to protection and should therefore be included in an
eventual vaccine candidate to induce a protective immune response.
We evaluated the ability of packaged Venezuelan equine encephalitis
(VEE) virus replicons expressing GP, NP, VP40, VP35, VP30 and VP24
virion proteins of Ebola virus to elicit protective immunity in two
strains of mice which differ at the major histocompatibility locus.
There are no published reports of the VP proteins having been
assayed as antigens for the production of an immune response in a
mammal.
[0005] The VEE virus replicon (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.
[0006] For our purposes, 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 (Mayinga 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 (Mayinga 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.
[0007] 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 provide
protection in humans.
[0008] Therefore, it is one object of the present invention to
provide a DNA fragment encoding each of the Ebola Zaire 1976 GP,
NP, VP24, VP30, VP35, and VP40 virion proteins (SEQUENCE ID NOS.
1-7).
[0009] It is another object of the present invention to provide 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.
[0010] It is yet another object of the present invention to provide
a VEE virus replicon vector comprising a VEE virus replicon and a
DNA fragment encoding any of the Ebola Zaire 1976 (Mayinga isolate)
GP, NP, VP24, VP30, VP35, or VP40 proteins. The construct can be
used as a nucleic acid vaccine or for the production of self
replicating RNA.
[0011] It is another object of the present invention to provide a
self replicating RNA comprising the VEE virus replicon and any of
the Ebola Zaire 1976 (Mayinga isolate) RNAs encoding the GP, NP,
VP24, VP30, VP35, and VP40 proteins described above. 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.
[0012] It is another object of the present invention to provide
infectious VEE virus replicon particles produced from the VEE virus
replicon RNAs described above.
[0013] It is further an object of the invention to provide an
immunological composition for the protection of subjects against
Ebola virus infection, comprising VEE virus replicon particles
containing the Ebola virus GP, NP, VP24, VP30, VP35, or VP40
proteins, or any combination of different VEE virus replicons each
containing one or more different Ebola proteins selected from GP,
NP, VP24, VP30, VP35 and VP40.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] These and other features, aspects, and advantages of the
present invention will become better understood with reference to
the following description and appended claims, and accompanying
drawings where:
[0015] FIG. 1 is a schematic description of the organization of the
Ebola virus genome.
[0016] FIGS. 2A, 2B and 2C are schematic representations of the VEE
replicon constructs containing Ebola genes.
[0017] FIG. 3 shows the generation of VEE viral-like particles
containing Ebola genes.
[0018] FIG. 4 is an immunoprecipitation of Ebola proteins produced
from replicon constructs.
DETAILED DESCRIPTION
[0019] 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.
[0020] Filoviruses.
[0021] 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. 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.
[0022] Replicon.
[0023] 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.
[0024] 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.
[0025] The Venezuelan equine encephalitis (VEE) virus replicon is a
genetically reorganized version of the VEE virus genome in which
the genes encoding the VEE structural proteins are replaced with a
heterologous gene of interest. In the present invention, the
heterologous genes are the GP, NP, or VP virion proteins from the
Ebola virus. The result is 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. The
replicon and its use is further described in U.S. Pat. No 5,792,462
issued to Johnston et al. on Aug. 11, 1998.
[0026] Subject.
[0027] Includes both human, animal, e.g., horse, donkey, pig,
mouse, hamster, monkey, chicken, and insect such as mosquito.
[0028] In one embodiment, the present invention relates to DNA
fragments which encode any of the Ebola Zaire 1976 (Mayinga
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.
[0029] 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 depositied sequences.
1 VP24: (1) forward primer is 5'-GGGATCGATCTCCAGACACCAAGCA-
AGACC-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'-CCCATCCATATGTTATGCACTATCCCTGAGAAG-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)
[0030] 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).
[0031] 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).
[0032] 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).
[0033] 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 His
change at position 168, and a Pro to Gly change at position 233
(SEQ ID NO: 19).
[0034] 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 are currently examining the ability of the new VEE
replicon construct, which we believe contains the authentic
C-terminus of VP30 (VP30#2, SEQ ID NO: 23), to protect mice against
a lethal Ebola challenge.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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 the 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
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.
[0046] 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.
[0047] 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.
[0048] 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). 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). These packaged replicons will
infect host cells and initiate a single round of replication
resulting in the expression of the Ebola proteins in said 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).
[0049] 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.
[0050] 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.
[0051] 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).
[0052] 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 Abs after administration of
a vaccine.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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 could involve 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, strongly supports a
role for CTLs. These data also suggest that an eventual vaccine
candidate may include several Ebola virus proteins, or several CTL
epitopes, capable of inducing broad protection in outbred
populations (e.g. people). We have identified two sequences
recognized by CTLs. They are Ebola virus NP SEQ ID NO:24 and Ebola
virus VP24 SEQ ID NO:25. Testing is in progress to identify the
role of CTLs in protection induced by each of these Ebola virus
proteins and to define the minimal sequence requirements for the
protective response. The CTL assay is well known in the art.
[0062] An eventual vaccine candidate might comprise these CTL
sequences and others. These 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.
[0063] 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.
[0064] Vaccine formulations of the present invention 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 from about 10.sup.4-10.sup.8
focus-forming units per dose is suitable, depending upon the age
and species of the subject being treated. The subject may be
inoculated 2-3 times. Exemplary pharmaceutically acceptable
carriers include, but are not limited to, sterile pyrogen-free
water and sterile pyrogen-free physiological saline solution.
[0065] 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.
[0066] 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.
[0067] The vaccine may 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. 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, or other schedules sufficient to elicit
the desired immune responses expected to confer protective
immunity, or reduce disease symptoms, or reduce severity of
disease.
[0068] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors and
thought to function well in the practice of the invention, and thus
can be considered to constitute preferred modes for its practice.
However, those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
[0069] The following MATERIALS AND METHODS were used in the
examples that follow.
[0070] Cells Lines and Viruses
[0071] BHK (ATCC CCL 10), Vero 76 (ATCC CRL 1587), and Vero E6
(ATCC CRL 1586) cell lines were maintained in minimal essential
medium with Earle's salts, 5-10% fetal bovine serum, and 50
.mu.g/mL gentamicin sulfate. For CTL assays, EL4 (ATCC TIB39),
L5178Y (ATCC CRL 1723) and P815 (ATCC TIB64) were maintained in
Dulbecco's minimal essential medium supplemented with 5-10% fetal
bovine serum and antibiotics.
[0072] A stock of the Zaire strain of Ebola virus originally
isolated from a patient in the 1976 outbreak (Mayinga) and passaged
intracerebrally 3 times in suckling mice and 2 times in Vero cells
was adapted to adult mice through serial passage in progressively
older suckling mice (Bray et al., (1998) J. Infect. Dis. 178,
651-661). A plaque-purified ninth-mouse-passage isolate which was
uniformly lethal for adult mice ("mouse-adapted virus") was
propagated in Vero E6 cells, aliquotted, and used in all mouse
challenge experiments and neutralization assays.
[0073] A stock of the Zaire strain of Ebola 1976 virus was passaged
spleen to spleen in strain 13 guinea pigs four times. This guinea
pig-adapted strain was used to challenge guinea pigs.
[0074] Construction and Packaging of Recombinant VEE Virus
Replicons (VRPs)
[0075] Replicon RNAs were packaged into VRPs as described (Pushko
et al., 1997, supra). Briefly, capped replicon RNAs were produced
in vitro by T7 run-off transcription of NotI-digested plasmid
templates using the RiboMAX T7 RNA polymerase kit (Promega). BHK
cells were co-transfected with the replicon RNAs and the 2 helper
RNAs expressing the structural proteins of the VEE virus. The cell
culture supernatants were harvested approximately 30 hours after
transfection and the replicon particles were concentrated and
purified by centrifugation through a 20% sucrose cushion. The
pellets containing the packaged replicon particles were suspended
in PBS and the titers were determined by infecting Vero cells with
serial dilutions of the replicon particles and enumerating the
infected cells by indirect immunofluorescence with antibodies
specific for the Ebola proteins.
[0076] Immunoprecipitation of Ebola Virus Proteins Expressed from
VEE Virus Replicons
[0077] BHK cells were transfected with either the Ebola virus GP,
NP, VP24, VP30, VP35, or VP40 replicon RNAs. At 24 h
post-transfection, the culture medium was replaced with minimal
medium lacking cysteine and methionine, and proteins were labeled
for 1 h with .sup.35S-labeled methionine and cysteine. Cell lysates
or supernatants (supe) were collected and immunoprecipitated with
polyclonal rabbit anti-Ebola virus serum bound to protein A beads.
.sup.35S-labeled Ebola virus structural proteins from virions grown
in Vero E6 cells were also immunoprecipitated as a control for each
of the virion proteins. Immunoprecipitated proteins were resolved
by electrophoresis on an 11% SDS-polyacrylamide gel and were
visualized by autoradiography.
[0078] Vaccination of Mice With VEE Virus Replicons
[0079] Groups of 10 BALB/c or C57BL/6 mice per experiment were
subcutaneously injected at the base of the neck with
2.times.10.sup.6 focus-forming units of VRPs encoding the Ebola
virus genes. As controls, mice were also injected with either a
control VRP encoding the Lassa nucleoprotein (NP) or with PBS. For
booster inoculations, animals received identical injections at 1
month intervals. Data are recorded as the combined results of 2 or
3 separate experiments.
[0080] Ebola Infection of Mice One month after the final booster
inoculation, mice were transferred to a BSL-4 containment area and
challenged by intraperitoneal (ip) inoculation of 10 plaque-forming
units (pfu) of mouse-adapted Ebola virus (approximately 300 times
the dose lethal for 50% of adult mice). The mice were observed
daily, and morbidity and mortality were recorded. Animals surviving
at day 21 post-infection were injected again with the same dose of
virus and observed for another 21 days.
[0081] In some experiments, 4 or 5 mice from vaccinated and control
groups were anesthetized and exsanguinated on day 4 (BALB/c mice)
or day 5 (C57BL/6 mice) following the initial viral challenge. The
viral titers in individual sera were determined by plaque
assay.
[0082] Passive Transfer of Immune Sera to Naive Mice.
[0083] Donor sera were obtained 28 days after the third inoculation
with 2.times.10.sup.6 focus-forming units of VRPs encoding the
indicated Ebola virus gene, the control Lassa NP gene, or from
unvaccinated control mice. One mL of pooled donor sera was
administered intraperitoneally (ip) to naive, syngeneic mice 24 h
prior to intraperitoneal challenge with 10 pfu of mouse-adapted
Ebola virus.
[0084] Vaccination and Challenge of Guinea Pigs.
[0085] EboGPVRP or EboNPVRP (1.times.10.sup.7 focus-forming units
in 0.5 ml PBS) were administered subcutaneously to inbred strain 2
or strain 13 guinea pigs (300-400 g). Groups of five guinea pigs
were inoculated on days 0 and 28 at one (strain 2) or two (strain
13) dorsal sites. Strain 13 guinea pigs were also boosted on day
126. One group of Strain 13 guinea pigs was vaccinated with both
the GP and NP constructs. Blood samples were obtained after
vaccination and after viral challenge. Guinea pigs were challenged
on day 56 (strain 2) or day 160 (strain 13) by subcutaneous
administration of 1000 LD.sub.50 (1.times.10.sup.4 PFU) of guinea
pig-adapted Ebola virus. Animals were observed daily for 60 days,
and morbidity (determined as changes in behavior, appearance, and
weight) and survival were recorded. Blood samples were taken on the
days indicated after challenge and viremia levels were determined
by plaque assay.
[0086] Virus Titration and Neutralization Assay.
[0087] Viral stocks were serially diluted in growth medium,
adsorbed onto confluent Vero E6 cells in 6- or 12-well dishes,
incubated for 1 hour at 37.degree. C., and covered with an agarose
overlay (Moe, J. et al. (1981) J. Clin. Microbiol. 13:791-793). A
second overlay containing 5% neutral red solution in PBS or agarose
was added 6 days later, and plaques were counted the following day.
Pooled pre-challenge serum samples from some of the immunized
groups were tested for the presence of Ebola-neutralizing
antibodies by plaque reduction neutralization assay. Aliquots of
Ebola virus in growth medium were mixed with serial dilutions of
test serum, or with normal serum, or medium only, incubated at
37.degree. C. for 1 h, and used to infect Vero E6 cells. Plaques
were counted 1 week later.
[0088] Cytotoxic T Cell Assays.
[0089] BALB/c and C57BL/6 mice were inoculated with VRPs encoding
Ebola virus NP or VP24 or the control Lassa NP protein. Mice were
euthanized at various times after the last inoculation and their
spleens removed. The spleens were gently ruptured to generate
single cell suspensions. Spleen cells (1.times.10.sup.6/ml) were
cultured in vitro for 2 days in the presence of 10-25 .mu.M of
peptides synthesized from Ebola virus NP or VP24 amino acid
sequences, and then for an additional 5 days in the presence of
peptide and 10% supernatant from concanavalin A-stimulated
syngeneic spleen cells. Synthetic peptides were made from Ebola
virus amino acid sequences predicted by a computer algorithm (HLA
Peptide Binding Predictions, Parker, K. C., et al. (1994) J.
Immunol. 152:163) to have a likelihood of meeting the MHC class I
binding requirements of the BALB/c (H-2.sup.d) and C57BL/6
(H-2.sup.b) haplotypes. Only 2 of 8 peptides predicted by the
algorithm and tested to date have been identified as containing CTL
epitopes. After in vitro restimulation, the spleen cells were
tested in a standard .sup.51chromium-release assay well known in
the art (see, for example, Hart et al. (1991) Proc. Natl. Acad.
Sci. USA 88: 9449-9452). Percent specific lysis of peptide-coated,
MHC-matched or mismatched target cells was calculated as: 1
Experimental cpm - Spontaneous cpm .times. 100 Maximum cpm -
Spontaneous cpm
[0090] Spontaneous cpm are the number of counts released from
target cells incubated in medium. Maximum cpm are obtained by
lysing target cells with 1% Triton X-100. Experimental cpm are the
counts from wells in which target cells are incubated with varying
numbers of effector (CTL) cells. Target cells tested were L5178Y
lymphoma or P815 mastocytoma cells (MHC matched to the H2.sup.d
BALB/c mice and EL4 lymphoma cells (MHC matched to the H2.sup.b
C57BL/6 mice). The effector:target (E:T) ratios tested were 25:1,
12:1, 6:1 and 3:1.
EXAMPLE 1
[0091] Survival of Mice Inoculated With VRPs Encoding Ebola
Proteins.
[0092] Mice were inoculated two or three times at 1 month intervals
with 2.times.10.sup.6 focus-forming units of VRPs encoding
individual Ebola virus genes, or Lassa virus NP as a control, or
with phosphate buffered saline (PBS). Mice were challenged with 10
pfu of mouse-adapted Ebola virus one month after the final
immunization. The mice were observed daily, and morbidity and
mortality data are shown in Table 1A for BALB/c mice and Table 1B
for C57BL/6 mice. The viral titers in individual sera of some mice
on day 4 (BALB/c mice) or day 5 (C57BL/6 mice) following the
initial viral challenge were determined by plaque assay.
2TABLE 1 Survival Of Mice Inoculated With VRPs Encoding Ebola
Proteins # VRP Injections. S/T.sup.1 (%) MDD.sup.2 V/T.sup.3
Viremia.sup.4 A. BALB/c Mice EboNP 3 30/30 (100%) 5/5 5.2 2 19/20
(95%) 7 5/5 4.6 EboGP 3 15/29 (52%) 8 1/5 6.6 2 14/20 (70%) 7 3/5
3.1 EboVP24 3 27/30 (90%) 8 5/5 5.2 2 19/20 (95%) 6 4/4 4.8 EboVP30
3 17/20 (85%) 7 5/5 6.2 2 11/20 (55%) 7 5/5 6.5 EboVP35 3 5/19
(26%) 7 5/5 6.9 2 4/20 (20%) 7 5/5 6.5 EboVP40 3 14/20 (70%) 8 5/5
4.6 2 17/20 (85%) 7 5/5 5.6 LassaNP 3 0/29 (0%) 7 5/5 8.0 2 0/20
(0%) 7 5/5 8.4 none (PBS) 3 1/30 (3%) 6 5/5 8.3 2 0/20 (0%) 6 5/5
8.7 B. C57BL/6 Mice EboNP 3 15/20 (75%) 8 5/5 4.1 2 8/10 (80%) 9
ND.sup.5 ND EboGP 3 19/20 (95%) 10 0/5 -- 2 10/10 (100%) -- ND ND
EboVP24 3 0/20 (0%) 7 5/5 8.6 EboVP30 3 2/20 (10%) 8 5/5 7.7
EboVP35 3 14/20 (70%) 8 5/5 4.5 EboVP40 3 1/20 (5%) 7 4/4 7.8
LassaNP 3 1/20 (5%) 7 4/4 8.6 2 0/10 (0%) 7 ND ND none (PBS) 3 3/20
(15%) 7 5/5 8.6 2 0/10 (0%) 7 ND ND .sup.1S/T, Survivors/total
challenged. .sup.2MDD, Mean day to death .sup.3V/T, Number of mice
with viremia/total number tested. .sup.4Geometric mean of
Log.sub.10 viremia titers in PFU/mL. Standard errors for all groups
were 1.5 or less, except for the group of BALB/c mice given 2
inoculations of EboGP, which was 2.2. .sup.5ND, not determined.
EXAMPLE 2
[0093] VP24-Immunized BALB/c Mice Survive a High-Dose Challenge
With Ebola Virus.
[0094] BALB/c mice were inoculated two times with 2.times.10.sup.6
focus-forming units of EboVP24VRP. Mice were challenged with either
1.times.10.sup.3 pfu or 1.times.10.sup.5 pfu of mouse-adapted Ebola
virus 1 month after the second inoculation. Morbidity and mortality
data for these mice are shown in Table 2.
3TABLE 2 VP24-Immunized BALB/c Mice Survive A High- Dose Challenge
With Ebola virus Replicon Challenge Dose Survivors/Total Ebo VP24 1
.times. 10.sup.3 pfu 5/5 (3 .times. 10.sup.4 LD.sub.50) Ebo VP24 1
.times. 10.sup.5 pfu 5/5 (3 .times. 10.sup.6 LD.sub.50) None 1
.times. 10.sup.3 pfu 0/4 (3 .times. 10.sup.4 LD.sub.50) None 1
.times. 10.sup.5 pfu 0/3 (3 .times. 10.sup.6 LD.sub.50)
EXAMPLE 3
[0095] Passive Transfer of Immune Sera Can Protect Naive Mice from
a Lethal Challenge of Ebola Virus.
[0096] Donor sera were obtained 28 days after the third inoculation
with 2.times.10.sup.6 focus-forming units of VRPs encoding the
indicated Ebola virus gene, the control Lassa NP gene, or from
unvaccinated control mice. One mL of pooled donor sera was
administered intraperitoneally (ip) to naive, syngeneic mice 24 h
prior to intraperitoneal challenge with 10 pfu of mouse-adapted
Ebola virus.
4TABLE 3 Passive Transfer of Immune Sera Can Protect Unvaccinated
Mice from a Lethal Challenge of Ebola Virus Survivors/ Mean Day
Total of Death A. BALB/c Mice Specificity of Donor Sera- Ebola GP
15/20 8 Ebola NP 1/20 7 Ebola VP24 0/20 6 Ebola VP30 0/20 7 Ebola
VP35 ND.sup.1 ND Ebola VP40 0/20 6 Lassa NP 0/20 7 Normal mouse
sera 0/20 6 B. C57BL/6 Mice Specificity of Donor Sera- Ebola GP
17/20 7 Ebola NP 0/20 7 Ebola VP24 ND ND Ebola VP30 ND ND Ebola
VP35 0/20 7 Ebola VP40 ND ND Lassa NP 0/20 7 Normal mouse sera 0/20
7 .sup.1ND, not determined
EXAMPLE 4
[0097] Immunogenicity and Efficacy of VRepEboGP and VRepEboNP in
Guinea Pigs.
[0098] EboGPVRP or EboNPVRP (1.times.10.sup.7 IU in 0.5 ml PBS)
were administered subcutaneously to inbred strain 2 or strain 13
guinea pigs (300-400 g). Groups of five guinea pigs were inoculated
on days 0 and 28 at one (strain 2) or two (strain 13) dorsal sites.
Strain 13 guinea pigs were also boosted on day 126. One group of
Strain 13 guinea pigs was vaccinated with both the GP and NP
constructs. Blood samples were obtained after vaccination and after
viral challenge.
[0099] Sera from vaccinated animals were assayed for antibodies to
Ebola by plaque-reduction neutralization, and ELISA. Vaccination
with VRepEboGP or NP induced high titers of antibodies to the Ebola
proteins (Table 4) in both guinea pig strains. Neutralizing
antibody responses were only detected in animals vaccinated with
the GP construct (Table 4).
[0100] Guinea pigs were challenged on day 56 (strain 2) or day 160
(strain 13) by subcutaneous administration of 1000 LD.sub.50
(10.sup.4 PFU) of guinea pig-adapted Ebola virus. Animals were
observed daily for 60 days, and morbidity (determined as changes in
behavior, appearance, and weight) and survival were recorded. Blood
samples were taken on the days indicated after challenge and
viremia levels were determined by plaque assay. Strain 13 guinea
pigs vaccinated with the GP construct, alone or in combination with
NP, survived lethal Ebola challenge (Table 4). Likewise,
vaccination of strain 2 inbred guinea pigs with the GP construct
protected {fraction (3/5)} animals against death from lethal Ebola
challenge, and significantly prolonged the mean day of death (MDD)
in one of the two animals that died (Table 4). Vaccination with NP
alone did not protect either guinea pig strain.
5TABLE 4 Immunogenicity and efficacy of VRepEboGP and VRepEboNP in
guinea pigs Survivors/ Viremia.sup.c VRP ELISA.sup.a PRNT.sub.50
total (MDD.sup.b) d7 d14 A. Strain 2 guinea pigs GP 4.1 30 3/5 (13
+ 2.8) 2.3 1.8 NP 3.9 <10 0/5 (9.2 + 1.1) 3.0 -- Mock <1.5
<10 0/5 (8.8 + 0.5) 3.9 -- B. Strain 13 guinea pigs GP 4.0 140
5/5 <2.0 <2.0 GP/NP 3.8 70 5/5 <2.0 <2.0 NP 2.8 <10
1/5 (8.3 + 2.2) 4.6 -- Lassa NP <1.5 <10 2/5 (8.3 + 0.6) 4.8
-- .sup.aData are expressed as geometric mean titers, log.sub.10.
.sup.bMDD, mean day to death .sup.cGeometric mean of log.sub.10
viremia titers in PFU/mL. Standard errors for all groups were 0.9
or less.
EXAMPLE 5
[0101] Induction of Murine CTL Responses to Ebola Virus NP and
Ebola Virus VP24 Proteins.
[0102] BALB/c and C57BL/6 mice were inoculated with VRPs encoding
Ebola virus NP or VP24. Mice were euthanized at various times after
the last inoculation and their spleens removed. Spleen cells
(1.times.10.sup.6/ml) were cultured in vitro for 2 days in the
presence of 10 to 25 .mu.M of peptides, and then for an additional
5 days in the presence of peptide and 10% supernatant from
concanavalin A-stimulated syngeneic spleen cells. After in vitro
restimulation, the spleen cells were tested in a standard
.sup.51chromium-release assay. Percent specific lysis of
peptide-coated, MHC-matched or mismatched target cells was
calculated as: 2 Experimental cpm - Spontaneous cpm .times. 100
Maximum cpm - Spontaneous cpm
[0103] In the experiments shown, spontaneous release did not exceed
15%.
6TABLE 5 Induction of murine CTL responses to Ebola virus NP and
Ebola virus VP24 proteins. % Specific Lysis E:T ratio Mice,
VRP.sup.1 Peptide.sup.2 Cell.sup.3 25 BALB/c, VP24 None P815 55
BALB/c, VP24 SEQ ID NO:25 P815 93 C57BL/6, Ebo NP None EL4 2
C57BL/6, Ebo NP.sup.4 SEQ ID NO:24 EL4 70 C57BL/6, Ebo NP Lassa NP
EL4 2 C57BL/6, Lassa NP None L5178Y 1 C57BL/6, Lassa NP SEQ ID
NO:24 L5178Y 0 C57BL/6, Lassa NP None EL4 2 C57BL/6, Lassa NP SEQ
ID NO:24 EL4 6 .sup.1Indicates the mouse strain used and the VRP
used as the in vivo immunogen. In vitro restimulation was performed
using SEQ ID NO:24 peptide for BALB/c mice and SEQ ID NO:23 for all
C57BL/6 mice shown. .sup.2Indicates the peptide used to coat the
target cells for the chromium release assay. .sup.3Target cells are
MHC-matched to the effector cells, except for the L5178Y cells that
are C57BL/6 mismatched. .sup.4High levels of specific lysis
(>40%) were also observed using E:T ratios of 12, 6, 3, or
1:1.
RESULTS AND DISCUSSION
[0104] Ebola Zaire 1976 (Mayinga) virus causes acute hemorrhagic
fever characterized by high mortality. There are no current
vaccines or effective therapeutic measures to protect individuals
who are exposed to this virus. In addition, it is not known which
genes are essential for evoking protective immunity and should
therefore be included in a vaccine designed for human use. In this
study, the GP, NP, VP24, VP30, VP35, and VP40 virion protein genes
of the Ebola Zaire 1976 (Mayinga) virus were cloned and inserted
into a Venezuelan equine encephalitis (VEE) virus replicon vector
(VRep) as shown in FIGS. 2A and 2B. These VReps were packaged as
VEE replicon particles (VRPs) using the VEE virus structural
proteins provided as helper RNAs, as shown in FIG. 3. This enables
expression of the Ebola virus proteins in host cells. The Ebola
virus proteins produced from these constructs were characterized in
vitro and were shown to react with polyclonal rabbit anti-Ebola
virus antibodies bound to Protein A beads following SDS gel
electrophoresis of immunoprecipitated proteins (FIG. 4).
[0105] The Ebola virus genes were sequenced from the VEE replicon
clones and are listed here as SEQ ID NO:1 (GP), 2 (NP), 3 (VP24), 4
(VP30), 5 (VP35), 6 (VP40), and 7 (VP30#2) as described below. The
corresponding amino acid sequences of the Ebola proteins expressed
from these replicons are listed as SEQ ID NO: 17, 18, 19, 20, 21,
22, and 23, respectively. 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).
[0106] The 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).
[0107] The DNA 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).
[0108] The Ebola virus VP24 (SEQ ID NO:3) gene 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 His change at
position 168, and a Pro to Gly change at position 233 (SEQ ID NO:
19).
[0109] We have included 2 different sequences for the Ebola virus
VP30 gene (SEQ ID NOS:4 and SEQ ID NO:7). 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 SEQ ID NO: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 gene in SEQ ID
NO:23 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 are currently examining the ability of the new VEE
replicon construct (SEQ ID NO:7), which we believe contains the
authentic C-terminus of VP30 (VP30#2, SEQ ID NO:23), to protect
mice against a lethal Ebola challenge.
[0110] The DNA 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 for 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.
[0111] 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.
[0112] The Ebola virus VP40 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.
[0113] To evaluate the protective efficacy of individual Ebola
virus proteins and to determine whether the major
histocompatibility (MHC) genes influence the immune response to
Ebola virus antigens, two MHC-incompatible strains of mice were
vaccinated with VRPs expressing an Ebola protein. As controls for
these experiments, some mice were injected with VRPs expressing the
nucleoprotein of Lassa virus or were injected with
phosphate-buffered saline (PBS). Following Ebola virus challenge,
the mice were monitored for morbidity and mortality, and the
results are shown in Table 1.
[0114] The GP, NP, VP24, VP30, and VP40 proteins of Ebola virus
generated either full or partial protection in BALB/c mice, and may
therefore be beneficial components of a vaccine designed for human
use. Vaccination with VRPs encoding the NP protein afforded the
best protection. In this case, 100% of the mice were protected
after three inoculations and 95% of the mice were protected after
two inoculations. The VRP encoding VP24 also protected 90% to 95%
of BALB/c mice against Ebola virus challenge. In separate
experiments (Table 2), two or three inoculations with VRPs encoding
the VP24 protein protected BALB/c mice from a high dose
(1.times.10.sup.5 plaque-forming units (3.times.10.sup.6
LD.sub.50)) of mouse-adapted Ebola virus.
[0115] Vaccination with VRPs encoding GP protected 52-70% of BALB/c
mice. The lack of protection was not due to a failure to respond to
the VRP encoding GP, as all mice had detectable Ebola
virus-specific serum antibodies after vaccination.
[0116] Some protective efficacy was also observed in BALB/c mice
vaccinated two or three times with VRPs expressing the VP30 protein
(55% and 85%, respectively),or the VP40 protein (70% and 80%,
respectively). The VP35 protein was not efficacious in the BALB/c
mouse model, as only 20% and 26% of the mice were protected after
either two or three doses, respectively.
[0117] Geometric mean titers of viremia were markedly reduced in
BALB/c mice vaccinated with VRPs encoding Ebola virus proteins
after challenge with Ebola virus, indicating an ability of the
induced immune responses to reduce virus replication (Table 1A). In
this study, immune responses to the GP protein were able to clear
the virus to undetectable levels within 4 days after challenge in
some mice.
[0118] When the same replicons were examined for their ability to
protect C57BL/6 mice from a lethal challenge of Ebola virus, only
the GP, NP, and VP35 proteins were efficacious (Table 1B). The best
protection, 95% to 100%, was observed in C57BL/6 mice inoculated
with VRPs encoding the GP protein. Vaccination with VRPs expressing
NP protected 75% to 80% of the mice from lethal disease. In
contrast to what was observed in the BALB/c mice, the VP35 protein
was the only VP protein able to significantly protect the C57BL/6
mice. In this case, 3 inoculations with VRPs encoding VP35
protected 70% of the mice from Ebola virus challenge. The reason
behind the differences in protection in the two mouse strains is
not known but is believed to be due to the ability of the
immunogens to sufficiently stimulate the cellular immune system. As
with the BALB/c mice, the effects of the induced immune responses
were also observed in reduced viremias and, occasionally, in a
prolonged time to death of C57BL/6 mice.
[0119] VRPs expressing Ebola virus GP or NP were also evaluated for
protective efficacy in a guinea pig model. Sera from vaccinated
animals were assayed for antibodies to Ebola by western blotting,
IFA, plaque-reduction neutralization, and ELISA. Vaccination with
either VRP (GP or NP) induced high titers of antibodies to the
Ebola proteins (Table 4) in both guinea pig strains. Neutralizing
antibody responses were only detected in animals vaccinated with
the VRP expressing GP (Table 4).
[0120] Vaccination of strain 2 inbred guinea pigs with the GP
construct protected {fraction (3/5)} animals against death from
lethal Ebola challenge, and significantly prolonged the mean day of
death in one of the two animals that died (Table 4). All of the
strain 13 guinea pigs vaccinated with the GP construct, alone or in
combination with NP, survived lethal Ebola challenge (Table 4).
Vaccination with NP alone did not protect either guinea pig strain
from challenge with the guinea pig-adapted Ebola virus.
[0121] To identify the immune mechanisms that mediate protection
against Ebola virus and to determine whether antibodies are
sufficient to protect against lethal disease, passive transfer
studies were performed. One mL of immune sera, obtained from mice
previously vaccinated with one of the Ebola virus VRPs, was
passively administered to unvaccinated mice 24 hours before
challenge with a lethal dose of mouse-adapted Ebola virus.
Antibodies to GP, but not to NP or the VP proteins, protected mice
from an Ebola virus challenge (Table 3). Antibodies to GP protected
75% of the BALB/c mice and 85% of the C57BL/6 mice from death. When
the donor sera were examined for their ability to neutralize Ebola
virus in a plaque-reduction neutralization assay, a 1:20 to 1:40
dilution of the GP-specific antisera reduced the number of viral
plaque-forming units by at least 50% (data not shown). In contrast,
antisera to the NP and VP proteins did not neutralize Ebola virus
at a 1:20 or 1:40 dilution. These results are consistent with the
finding that GP is the only viral protein found on the surface of
Ebola virus, and is likely to induce virus-neutralizing
antibodies.
[0122] Since the NP and VP proteins of Ebola virus are internal
virion proteins to which antibodies are not sufficient for
protection, it is likely that cytotoxic T lymphocytes (CTLs) are
also important for protection against Ebola virus. Initial studies
aimed at identifying cellular immune responses to individual Ebola
virus proteins expressed from VRPs identified CTL responses to the
VP24 and NP proteins (Table 5). One CTL epitope that we identified
for the Ebola virus NP is recognized by C57BL/6 (H-2 b) mice, and
has an amino acid sequence of, or contained within, the following
11 amino acids: VYQVNNLEEIC (SEQ ID NO:24). Vaccination with
EboNPVRP and in vitro restimulation of spleen cells with this
peptide consistently induces strong CTL responses in C57BL/6
(H-2.sup.b) mice. In vivo vaccination to Ebola virus NP is required
to detect the CTL activity, as evidenced by the failure of cells
from C57BL/6 mice vaccinated with Lassa NP to develop lytic
activity to peptide (SEQ ID NO:24) after in vitro restimulation
with it. Specific lysis has been observed using very low
effector:target ratios (<2:1). This CTL epitope is H-2.sup.b
restricted in that it is not recognized by BALB/c (H-2.sup.d) cells
treated the same way (data not shown), and H-2.sup.b effector cells
will not lyse MHC-mismatched target cells coated with this
peptide.
[0123] A CTL epitope in the VP24 protein was also identified. It is
recognized by BALB/c (H-2.sup.d) mice, and has an amino acid
sequence of, or contained within, the following 23 amino acids:
LKFINKLDALLVVNYNGLLSSIF (SEQ ID NO:25). In the data shown in Table
5, high (>90%) specific lysis of P815 target cells coated with
this peptide was observed. The background lysis of cells that were
not peptide-coated was also high (>50%), which is probably due
to the activity of natural killer cells. We are planning to repeat
this experiment using the L5178Y target cells, which are not
susceptible to natural killer cells.
[0124] Future studies will focus on determining the fine
specificities of these CTL responses and the essential amino acids
that constitute these CTL epitopes. Additional studies to identify
other CTL epitopes on Ebola virus GP, NP, VP24, VP30, VP35, and
VP40 will be performed. To evaluate the role of these CTLs in
protection against Ebola virus, lymphocytes will be restimulated in
vitro with peptides containing the CTL epitopes, and adoptively
transferred into unvaccinated mice prior to Ebola virus challenge.
In addition, future studies will examine the CTL responses to the
other Ebola virus proteins to better define the roles of the cell
mediated immune responses involved in protection against Ebola
virus infection.
Sequence CWU 1
1
25 1 2298 DNA Ebola Zaire 1 atcgataagc tcggaattcg agctcgcccg
gggatcctct 40 agagtcgaca acaacacaat gggcgttaca ggaatattgc 80
agttacctcg tgatcgattc aagaggacat cattctttct 120 ttgggtaatt
atccttttcc aaagaacatt ttccatccca 160 cttggagtca tccacaatag
cacattacag gttagtgatg 200 tcgacaaact agtttgtcgt gacaaactgt
catccacaaa 240 tcaattgaga tcagttggac tgaatctcga agggaatgga 280
gtggcaactg acgtgccatc tgcaactaaa agatggggct 320 tcaggtccgg
tgtcccacca aaggtggtca attatgaagc 360 tggtgaatgg gctgaaaact
gctacaatct tgaaatcaaa 400 aaacctgacg ggagtgagtg tctaccagca
gcgccagacg 440 ggattcgggg cttcccccgg tgccggtatg tgcacaaagt 480
atcaggaacg ggaccgtgtg ccggagactt tgccttccat 520 aaagagggtg
ctttcttcct gtatgatcga cttgcttcca 560 cagttatcta ccgaggaacg
actttcgctg aaggtgtcgt 600 tgcatttctg atactgcccc aagctaagaa
ggacttcttc 640 agctcacacc ccttgagaga gccggtcaat gcaacggagg 680
acccgtctag tggctactat tctaccacaa ttagatatca 720 ggctaccggt
tttggaacca atgagacaga gtacttgttc 760 gaggttgaca atttgaccta
cgtccaactt gaatcaagat 800 tcacaccaca gtttctgctc cagctgaatg
agacaatata 840 tacaagtggg aaaaggagca ataccacggg aaaactaatt 880
tggaaggtca accccgaaat tgatacaaca atcggggagt 920 gggccttctg
ggaaactaaa aaaaacctca ctagaaaaat 960 tcgcagtgaa gagttgtctt
tcacagttgt atcaaacgga 1000 gccaaaaaca tcagtggtca gagtccggcg
cgaacttctt 1040 ccgacccagg gaccaacaca acaactgaag accacaaaat 1080
catggcttca gaaaattcct ctgcaatggt tcaagtgcac 1120 agtcaaggaa
gggaagctgc agtgtcgcat ctaacaaccc 1160 ttgccacaat ctccacgagt
ccccaatccc tcacaaccaa 1200 accaggtccg gacaacagca cccataatac
acccgtgtat 1240 aaacttgaca tctctgaggc aactcaagtt gaacaagatc 1280
accgcagaac agacaacgac agcacagcct ccgacactcc 1320 ctctgccacg
accgcagccg gacccccaaa agcagagaac 1360 accaacacga gcaagagcac
tgacttcctg gaccccgcca 1400 ccacaacaag tccccaaaac cacagcgaga
ccgctggcaa 1440 caacaacact catcaccaag ataccggaga agagagtgcc 1480
agcagcggga agctaggctt aattaccaat actattgctg 1520 gagtcgcagg
actgatcaca ggcgggagaa gaactcgaag 1560 agaagcaatt gtcaatgctc
aacccaaatg caaccctaat 1600 ttacattact ggactactca ggatgaaggt
gctgcaatcg 1640 gactggcctg gataccatat ttcgggccag cagccgaggg 1680
aatttacata gaggggctaa tgcacaatca agatggttta 1720 atctgtgggt
tgagacagct ggccaacgag acgactcaag 1760 ctcttcaact gttcctgaga
gccacaactg agctacgcac 1800 cttttcaatc ctcaaccgta aggcaattga
tttcttgctg 1840 cagcgatggg gcggcacatg ccacattctg ggaccggact 1880
gctgtatcga accacatgat tggaccaaga acataacaga 1920 caaaattgat
cagattattc atgattttgt tgataaaacc 1960 cttccggacc agggggacaa
tgacaattgg tggacaggat 2000 ggagacaatg gataccggca ggtattggag
ttacaggcgt 2040 tgtaattgca gttatcgctt tattctgtat atgcaaattt 2080
gtcttttagt ttttcttcag attgcttcat ggaaaagctc 2120 agcctcaaat
caatgaaacc aggatttaat tatatggatt 2160 acttgaatct aagattactt
gacaaatgat aatataatac 2200 actggagctt taaacatagc caatgtgatt
ctaactcctt 2240 taaactcaca gttaatcata aacaaggttt gagtcgacct 2280
gcagccaagc ttatcgat 2298 2 2428 DNA Ebola Zaire 2 atcgataagc
ttggctgcag gtcgactcta gaggatccga 40 gtatggattc tcgtcctcag
aaaatctgga tggcgccgag 80 tctcactgaa tctgacatgg attaccacaa
gatcttgaca 120 gcaggtctgt ccgttcaaca ggggattgtt cggcaaagag 160
tcatcccagt gtatcaagta aacaatcttg aagaaatttg 200 ccaacttatc
atacaggcct ttgaagcagg tgttgatttt 240 caagagagtg cggacagttt
ccttctgatg ctttgtcttc 280 atcatgcgta ccagggagat tacaaacttt
tcttggaaag 320 tggcgcagtc aagtatttgg aagggcacgg gttccgtttt 360
gaagtcaaga agcgtgatgg agtgaagcga cttgaggaat 400 tgctgccagc
agtatctagt ggaaaaaaca ttaagagaac 440 acttgctgcc atgccggaag
aggagacaac tgaagctaat 480 gccggtcagt ttctctcctt tgcaagtcta
ttccttccga 520 aattggtagt aggagaaaag gcttgccttg agaaggttca 560
aaggcaaatt caagtacatg cagagcaagg actgatacaa 600 tatccaacag
cttggcaatc agtaggacac atgatggtga 640 ttttccgttt gatgcgaaca
aattttctga tcaaatttct 680 cctaatacac caagggatgc acatggttgc
cgggcatgat 720 gccaacgatg ctgtgatttc aaattcagtg gctcaagctc 760
gtttttcagg cttattgatt gtcaaaacag tacttgatca 800 tatcctacaa
aagacagaac gaggagttcg tctccatcct 840 cttgcaagga ccgccaaggt
aaaaaatgag gtgaactcct 880 ttaaggctgc actcagctcc ctggccaagc
atggagagta 920 tgctcctttc gcccgacttt tgaacctttc tggagtaaat 960
aatcttgagc atggtctttt ccctcaacta tcggcaattg 1000 cactcggagt
cgccacagca cacgggagta ccctcgcagg 1040 agtaaatgtt ggagaacagt
atcaacaact cagagaggct 1080 gccactgagg ctgagaagca actccaacaa
tatgcagagt 1120 ctcgcgaact tgaccatctt ggacttgatg atcaggaaaa 1160
gaaaattctt atgaacttcc atcagaaaaa gaacgaaatc 1200 agcttccagc
aaacaaacgc tatggtaact ctaagaaaag 1240 agcgcctggc caagctgaca
gaagctatca ctgctgcgtc 1280 actgcccaaa acaagtggac attacgatga
tgatgacgac 1320 attccctttc caggacccat caatgatgac gacaatccta 1360
gccatcaaga tgatgatccg actgactcac aggatacgac 1400 cattcccgat
gtggtggttg atcccgatga tggaagctac 1440 ggcgaatacc agagttactc
ggaaaacggc atgaatgcac 1480 cagatgactt ggtcctattc gatctagacg
aggacgacga 1520 ggacactaag ccagtgccta atagatcgac caagggtgga 1560
caacagaaga acagtcaaaa gggccagcat atagagggca 1600 gacagacaca
atccaggcca attcaaaatg tcccaggccc 1640 tcacagaaca atccaccacg
ccagtgcgcc actcacggac 1680 aatgacagaa gaaatgaacc ctccggctca
accagccctc 1720 gcatgctgac accaattaac gaagaggcag acccactgga 1760
cgatgccgac gacgagacgt ctagccttcc gcccttggag 1800 tcagatgatg
aagagcagga cagggacgga acttccaacc 1840 gcacacccac tgtcgcccca
ccggctcccg tatacagaga 1880 tcactctgaa aagaaagaac tcccgcaaga
cgagcaacaa 1920 gatcaggacc acactcaaga ggccaggaac caggacagtg 1960
acaacaccca gtcagaacac tcttttgagg agatgtatcg 2000 ccacattcta
agatcacagg ggccatttga tgctgttttg 2040 tattatcata tgatgaagga
tgagcctgta gttttcagta 2080 ccagtgatgg caaagagtac acgtatccag
actcccttga 2120 agaggaatat ccaccatggc tcactgaaaa agaggctatg 2160
aatgaagaga atagatttgt tacattggat ggtcaacaat 2200 tttattggcc
ggtgatgaat cacaagaata aattcatggc 2240 aatcctgcaa catcatcagt
gaatgagcat ggaacaatgg 2280 gatgattcaa ccgacaaata gctaacatta
agtagtccag 2320 gaacgaaaac aggaagaatt tttgatgtct aaggtgtgaa 2360
ttattatcac aataaaagtg attcttattt ttgaatttgg 2400 gcgagctcga
attcccgagc ttatcgat 2428 3 847 DNA Ebola Zaire 3 atcgatctcc
agacaccaag caagacctga gaaaaaacca 40 tggctaaagc tacgggacga
tacaatctaa tatcgcccaa 80 aaaggacctg gagaaagggg ttgtcttaag
cgacctctgt 120 aacttcttag ttagccaaac tattcagggg tggaaggttt 160
attgggctgg tattgagttt gatgtgactc acaaaggaat 200 ggccctattg
catagactga aaactaatga ctttgcccct 240 gcatggtcaa tgacaaggaa
tctctttcct catttatttc 280 aaaatccgaa ttccacaatt gaatcaccgc
tgtgggcatt 320 gagagtcatc cttgcagcag ggatacagga ccagctgatt 360
gaccagtctt tgattgaacc cttagcagga gcccttggtc 400 tgatctctga
ttggctgcta acaaccaaca ctaaccattt 440 caacatgcga acacaacgtg
tcaaggaaca attgagccta 480 aaaatgctgt cgttgattcg atccaatatt
ctcaagttta 520 ttaacaaatt ggatgctcta catgtcgtga actacaacgg 560
attgttgagc agtattgaaa ttggaactca aaatcataca 600 atcatcataa
ctcgaactaa catgggtttt ctggtggagc 640 tccaagaacc cgacaaatcg
gcaatgaacc gcatgaagcc 680 tgggccggcg aaattttccc tccttcatga
gtccacactg 720 aaagcattta cacaaggatc ctcgacacga atgcaaagtt 760
tgattcttga atttaatagc tctcttgcta tctaactaag 800 gtagaatact
tcatattgag ctaactcata tatgctgact 840 catcgat 847 4 973 DNA Ebola
Zaire 4 atcgatcaga tctgcgaacc ggtagagttt agttgcaacc 40 taacacacat
aaagcattgg tcaaaaagtc aatagaaatt 80 taaacagtga gtggagacaa
cttttaaatg gaagcttcat 120 atgagagagg acgcccacga gctgccagac
agcattcaag 160 ggatggacac gaccaccatg ttcgagcacg atcatcatcc 200
agagagaatt atcgaggtga gtaccgtcaa tcaaggagcg 240 cctcacaagt
gcgcgttcct actgtatttc ataagaagag 280 agttgaacca ttaacagttc
ctccagcacc taaagacata 320 tgtccgacct tgaaaaaagg atttttgtgt
gacagtagtt 360 tttgcaaaaa agatcaccag ttggagagtt taactgatag 400
ggaattactc ctactaatcg cccgtaagac ttgtggatca 440 gtagaacaac
aattaaatat aactgcaccc aaggactcgc 480 gcttagcaaa tccaacggct
gatgatttcc agcaagagga 520 aggtccaaaa attaccttgt tgacactgat
caagacggca 560 gaacactggg cgagacaaga catcagaacc atagaggatt 600
caaaattaag agcattgttg actctatgtg ctgtgatgac 640 gaggaaattc
tcaaaatccc agctgagtct tttatgtgag 680 acacacctaa ggcgcgaggg
gcttgggcaa gatcaggcag 720 aacccgttct cgaagtatat caacgattac
acagtgataa 760 aggaggcagt tttgaagctg cactatggca acaatgggac 800
ctacaatccc taattatgtt tatcactgca ttcttgaata 840 ttgctctcca
gttaccgtgt gaaagttctg ctgtcgttgt 880 ttcagggtta agaacattgg
ttcctcaatc agataatgag 920 gaagcttcaa ccaacccggg gacatgctca
tggtctgatg 960 agggtacatc gat 973 5 1148 DNA Ebola Zaire 5
atcgatagaa aagctggtct aacaagatga caactagaac 40 aaagggcagg
ggccatactg cggccacgac tcaaaacgac 80 agaatgccag gccctgagct
ttcgggctgg atctctgagc 120 agctaatgac cggaagaatt cctgtaagcg
acatcttctg 160 tgatattgag aacaatccag gattatgcta cgcatcccaa 200
atgcaacaaa cgaagccaaa cccgaagacg cgcaacagtc 240 aaacccaaac
ggacccaatt tgcaatcata gttttgagga 280 ggtagtacaa acattggctt
cattggctac tgttgtgcaa 320 caacaaacca tcgcatcaga atcattagaa
caacgcatta 360 cgagtcttga gaatggtcta aagccagttt atgatatggc 400
aaaaacaatc tcctcattga acagggtttg tgctgagatg 440 gttgcaaaat
atgatcttct ggtgatgaca accggtcggg 480 caacagcaac cgctgcggca
actgaggctt attgggccga 520 acatggtcaa ccaccacctg gaccatcact
ttatgaagaa 560 agtgcgattc ggggtaagat tgaatctaga gatgagaccg 600
tccctcaaag tgttagggag gcattcaaca atctaaacag 640 taccacttca
ctaactgagg aaaattttgg gaaacctgac 680 atttcggcaa aggatttgag
aaacattatg tatgatcact 720 tgcctggttt tggaactgct ttccaccaat
tagtacaagt 760 gatttgtaaa ttgggaaaag atagcaactc attggacatc 800
attcatgctg agttccaggc cagcctggct gaaggagact 840 ctcctcaatg
tgccctaatt caaattacaa aaagagttcc 880 aatcttccaa gatgctgctc
cacctgtcat ccacatccgc 920 tctcgaggtg acattccccg agcttgccag
aaaagcttgc 960 gtccagtccc accatcgccc aagattgatc gaggttgggt 1000
atgtgttttt cagcttcaag atggtaaaac acttggactc 1040 aaaatttgag
ccaatctccc ttccctccga aagaggcgaa 1080 taatagcaga ggcttcaact
gctgaactat agggtacgtt 1120 acattaatga tacacttgtg agatcgat 1148 6
1123 DNA Ebola Zaire 6 atcgatccta cctcggctga gagagtgttt tttcattaac
40 cttcatcttg taaacgttga gcaaaattgt taaaaatatg 80 aggcgggtta
tattgcctac tgctcctcct gaatatatgg 120 aggccatata ccctgtcagg
tcaaattcaa caattgctag 160 aggtggcaac agcaatacag gcttcctgac
accggagtca 200 gtcaatgggg acactccatc gaatccactc aggccaattg 240
ccgatgacac catcgaccat gccagccaca caccaggcag 280 tgtgtcatca
gcattcatcc ttgaagctat ggtgaatgtc 320 atatcgggcc ccaaagtgct
aatgaagcaa attccaattt 360 ggcttcctct aggtgtcgct gatcaaaaga
cctacagctt 400 tgactcaact acggccgcca tcatgcttgc ttcatacact 440
atcacccatt tcggcaaggc aaccaatcca cttgtcagag 480 tcaatcggct
gggtcctgga atcccggatc atcccctcag 520 gctcctgcga attggaaacc
aggctttcct ccaggagttc 560 gttcttccgc cagtccaact accccagtat
ttcacctttg 600 atttgacagc actcaaactg atcacccaac cactgcctgc 640
tgcaacatgg accgatgaca ctccaacagg atcaaatgga 680 gcgttgcgtc
caggaatttc atttcatcca aaacttcgcc 720 ccattctttt acccaacaaa
agtgggaaga aggggaacag 760 tgccgatcta acatctccgg agaaaatcca
agcaataatg 800 acttcactcc aggactttaa gatcgttcca attgatccaa 840
ccaaaaatat catgggaatc gaagtgccag aaactctggt 880 ccacaagctg
accggtaaga aggtgacttc taaaaatgga 920 caaccaatca tccctgttct
tttgccaaag tacattgggt 960 tggacccggt ggctccagga gacctcacca
tggtaatcac 1000 acaggattgt gacacgtgtc attctcctgc aagtcttcca 1040
gctgtgattg agaagtaatt gcaataattg actcagatcc 1080 agttttatag
aatcttctca gggatagtgc ataacatatc 1120 gat 1123 7 1165 DNA Ebola
Zaire 7 atcgatcaga tctgcgaacc ggtagagttt agttgcaacc 40 taacacacat
aaagcattgg tcaaaaagtc aatagaaatt 80 taaacagtga gtggagacaa
cttttaaatg gaagcttcat 120 atgagagagg acgcccacga gctgccagac
agcattcaag 160 ggatggacac gaccaccatg ttcgagcacg atcatcatcc 200
agagagaatt atcgaggtga gtaccgtcaa tcaaggagcg 240 cctcacaagt
gcgcgttcct actgtatttc ataagaagag 280 agttgaacca ttaacagttc
ctccagcacc taaagacata 320 tgtccgacct tgaaaaaagg atttttgtgt
gacagtagtt 360 tttgcaaaaa agatcaccag ttggagagtt taactgatag 400
ggaattactc ctactaatcg cccgtaagac ttgtggatca 440 gtagaacaac
aattaaatat aactgcaccc aaggactcgc 480 gcttagcaaa tccaacggct
gatgatttcc agcaagagga 520 aggtccaaaa attaccttgt tgacactgat
caagacggca 560 gaacactggg cgagacaaga catcagaacc atagaggatt 600
caaaattaag agcattgttg actctatgtg ctgtgatgac 640 gaggaaattc
tcaaaatccc agctgagtct tttatgtgag 680 acacacctaa ggcgcgaggg
gcttgggcaa gatcaggcag 720 aacccgttct cgaagtatat caacgattac
acagtgataa 760 aggaggcagt tttgaagctg cactatggca acaatgggac 800
cgacaatccc taatcatgtt tatcactgca ttcttgaata 840 ttgctctcca
gttaccgtgt gaaagttctg ctgtcgttgt 880 ttcagggtta agaacattgg
ttcctcaatc agataatgag 920 gaagcttcaa ccaacccggg gacatgctca
tggtctgatg 960 agggtacccc ttaataaggc tgactaaaac actatataac 1000
cttctacttg atcacaatac tccgtatacc tatcatcata 1040 tatttaatca
agacgatatc ctttaaaact tattcagtac 1080 tataatcact ctcgtttcaa
attaataaga tgtgcatgat 1120 tgccctaata tatgaagagg tatgatacaa
ccctaacaga 1160 tcgat 1165 8 30 DNA artificial sequence /note=
"forward primer for VP24" 8 gggatcgatc tccagacacc aagcaagacc 30 9
33 DNA artificial sequence /note= "reverse primer for VP24" 9
gggatcgatg agtcagcata tatgagttag ctc 33 10 30 DNA artificial
sequence /note= "forward primer for VP30" 10 cccatcgatc agatctgcga
accggtagag 30 11 31 DNA artificial sequence /note= "reverse primer
for VP30" 11 cccatcgatg taccctcatc agaccatgag c 31 12 33 DNA
artificial sequence /note= "forward primer for VP35" 12 gggatcgata
gaaaagctgg tctaacaaga tga 33 13 36 DNA artificial sequence /note=
"reverse primer for VP35" 13 cccatcgatc tcacaagtgt atcattaatg
taacgt 36 14 30 DNA artificial sequence /note= "forward primer for
VP40" 14 cccatcgatc ctacctcggc tgagagagtg 30 15 33 DNA artificial
sequence /note= "reverse primer for VP40" 15 cccatcgata tgttatgcac
tatccctgag aag 33 16 30 DNA artificial sequence /note= "reverse
primer for VP30#2" 16 cccatcgatc tgttagggtt gtatcatacc 30 17 676
PRT Ebola Zaire 17 Met Gly Val Thr Gly Ile Leu Gln Leu Pro 1 5 10
Arg Asp Arg Phe Lys Arg Thr Ser Phe Phe 15 20 Leu Trp Val Ile Ile
Leu Phe Gln Arg Thr 25 30 Phe Ser Ile Pro Leu Gly Val Ile His Asn
35 40 Ser Thr Leu Gln Val Ser Asp Val Asp Lys 45 50 Leu Val Cys Arg
Asp Lys Leu Ser Ser Thr 55 60 Asn Gln Leu Arg Ser Val Gly Leu Asn
Leu 65 70 Glu Gly Asn Gly Val Ala Thr Asp Val Pro 75 80 Ser Ala Thr
Lys Arg Trp Gly Phe Arg Ser 85 90 Gly Val Pro Pro Lys Val Val Asn
Tyr Glu 95 100 Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn 105 110 Leu
Glu Ile Lys Lys Pro Asp Gly Ser Glu 115 120 Cys Leu Pro Ala Ala Pro
Asp Gly Ile Arg 125 130 Gly Phe Pro Arg Cys Arg Tyr Val His Lys 135
140 Val Ser Gly Thr Gly Pro Cys Ala Gly Asp 145 150 Phe Ala Phe His
Lys Glu Gly Ala Phe Phe 155 160 Leu Tyr Asp Arg Leu Ala Ser Thr Val
Ile 165 170 Tyr Arg Gly Thr Thr Phe Ala Glu Gly Val 175 180 Val Ala
Phe Leu Ile Leu Pro Gln Ala Lys 185 190 Lys Asp Phe Phe Ser Ser His
Pro Leu Arg 195 200 Glu Pro Val Asn Ala Thr Glu Asp Pro Ser 205 210
Ser Gly Tyr Tyr Ser Thr Thr Ile Arg Tyr 215 220 Gln Ala Thr Gly Phe
Gly Thr Asn Glu Thr 225 230 Glu Tyr Leu Phe Glu Val Asp Asn Leu Thr
235 240 Tyr Val Gln Leu Glu Ser Arg Phe Thr Pro 245 250 Gln Phe Leu
Leu Gln Leu Asn Glu Thr Ile 255 260 Tyr Thr Ser Gly Lys Arg Ser Asn
Thr Thr 265 270 Gly Lys Leu Ile Trp Lys Val Asn Pro Glu 275 280 Ile
Asp Thr Thr Ile Gly Glu Trp Ala Phe 285 290 Trp Glu Thr Lys Lys Asn
Leu Thr Arg Lys 295 300 Ile Arg Ser Glu Glu Leu Ser Phe Thr Val 305
310 Val Ser Asn Gly Ala Lys Asn Ile Ser Gly 315 320 Gln Ser Pro Ala
Arg Thr Ser Ser Asp Pro 325 330 Gly Thr Asn Thr Thr Thr Glu Asp His
Lys 335 340 Ile Met Ala Ser Glu Asn Ser Ser Ala Met 345 350 Val Gln
Val His Ser Gln Gly Arg Glu Ala 355 360 Ala Val Ser His Leu Thr Thr
Leu Ala Thr 365 370 Ile Ser Thr Ser Pro Gln Ser Leu Thr Thr 375 380
Lys Pro Gly Pro Asp Asn Ser Thr His Asn 385 390 Thr Pro Val Tyr Lys
Leu Asp Ile Ser Glu 395 400 Ala Thr Gln Val Glu Gln His His Arg Arg
405 410 Thr Asp Asn Asp Ser Thr Ala Ser Asp Thr 415 420 Pro Ser Ala
Thr Thr Ala Ala Gly Pro Pro 425 430 Lys Ala Glu Asn Thr Asn Thr Ser
Lys Ser 435 440 Thr Asp Phe Leu Asp Pro Ala Thr Thr Thr 445 450 Ser
Pro Gln Asn His Ser Glu Thr Ala Gly 455 460 Asn Asn Asn Thr His His
Gln Asp Thr Gly 465 470 Glu Glu Ser Ala Ser Ser Gly Lys Leu Gly 475
480 Leu Ile Thr Asn Thr Ile Ala Gly Val Ala 485 490 Gly Leu Ile Thr
Gly Gly Arg Arg Thr Arg 495 500 Arg Glu Ala Ile Val Asn Ala Gln Pro
Lys 505 510 Cys Asn Pro Asn Leu His Tyr Trp Thr Thr 515 520 Gln Asp
Glu Gly Ala Ala Ile Gly Leu Ala 525 530 Trp Ile Pro Tyr Phe Gly Pro
Ala Ala Glu 535 540 Gly Ile Tyr Ile Glu Gly Leu Met His Asn 545 550
Gln Asp Gly Leu Ile Cys Gly Leu Arg Gln 555 560 Leu Ala Asn Glu Thr
Thr Gln Ala Leu Gln 565 570 Leu Phe Leu Arg Ala Thr Thr Glu Leu Arg
575 580 Thr Phe Ser Ile Leu Asn Arg Lys Ala Ile 585 590 Asp Phe Leu
Leu Gln Arg Trp Gly Gly Thr 595 600 Cys His Ile Leu Gly Pro Asp Cys
Cys Ile 605 610 Glu Pro His Asp Trp Thr Lys Asn Ile Thr 615 620 Asp
Lys Ile Asp Gln Ile Ile His Asp Phe 625 630 Val Asp Lys Thr Leu Pro
Asp Gln Gly Asp 635 640 Asn Asp Asn Trp Trp Thr Gly Trp Arg Gln 645
650 Trp Ile Pro Ala Gly Ile Gly Val Thr Gly 655 660 Val Val Ile Ala
Val Ile Ala Leu Phe Cys 665 670 Ile Cys Lys Phe Val Phe 675 18 739
PRT Ebola Zaire 18 Met Asp Ser Arg Pro Gln Lys Ile Trp Met 1 5 10
Ala Pro Ser Leu Thr Glu Ser Asp Met Asp 15 20 Tyr His Lys Ile Leu
Thr Ala Gly Leu Ser 25 30 Val Gln Gln Gly Ile Val Arg Gln Arg Val
35 40 Ile Pro Val Tyr Gln Val Asn Asn Leu Glu 45 50 Glu Ile Cys Gln
Leu Ile Ile Gln Ala Phe 55 60 Glu Ala Gly Val Asp Phe Gln Glu Ser
Ala 65 70 Asp Ser Phe Leu Leu Met Leu Cys Leu His 75 80 His Ala Tyr
Gln Gly Asp Tyr Lys Leu Phe 85 90 Leu Glu Ser Gly Ala Val Lys Tyr
Leu Glu 95 100 Gly His Gly Phe Arg Phe Glu Val Lys Lys 105 110 Arg
Asp Gly Val Lys Arg Leu Glu Glu Leu 115 120 Leu Pro Ala Val Ser Ser
Gly Lys Asn Ile 125 130 Lys Arg Thr Leu Ala Ala Met Pro Glu Glu 135
140 Glu Thr Thr Glu Ala Asn Ala Gly Gln Phe 145 150 Leu Ser Phe Ala
Ser Leu Phe Leu Pro Lys 155 160 Leu Val Val Gly Glu Lys Ala Cys Leu
Glu 165 170 Lys Val Gln Arg Gln Ile Gln Val His Ala 175 180 Glu Gln
Gly Leu Ile Gln Tyr Pro Thr Ala 185 190 Trp Gln Ser Val Gly His Met
Met Val Ile 195 200 Phe Arg Leu Met Arg Thr Asn Phe Leu Ile 205 210
Lys Phe Leu Leu Ile His Gln Gly Met His 215 220 Met Val Ala Gly His
Asp Ala Asn Asp Ala 225 230 Val Ile Ser Asn Ser Val Ala Gln Ala Arg
235 240 Phe Ser Gly Leu Leu Ile Val Lys Thr Val 245 250 Leu Asp His
Ile Leu Gln Lys Thr Glu Arg 255 260 Gly Val Arg Leu His Pro Leu Ala
Arg Thr 265 270 Ala Lys Val Lys Asn Glu Val Asn Ser Phe 275 280 Lys
Ala Ala Leu Ser Ser Leu Ala Lys His 285 290 Gly Glu Tyr Ala Pro Phe
Ala Arg Leu Leu 295 300 Asn Leu Ser Gly Val Asn Asn Leu Glu His 305
310 Gly Leu Phe Pro Gln Leu Ser Ala Ile Ala 315 320 Leu Gly Val Ala
Thr Ala His Gly Ser Thr 325 330 Leu Ala Gly Val Asn Val Gly Glu Gln
Tyr 335 340 Gln Gln Leu Arg Glu Ala Ala Thr Glu Ala 345 350 Glu Lys
Gln Leu Gln Gln Tyr Ala Glu Ser 355 360 Arg Glu Leu Asp His Leu Gly
Leu Asp Asp 365 370 Gln Glu Lys Lys Ile Leu Met Asn Phe His 375 380
Gln Lys Lys Asn Glu Ile Ser Phe Gln Gln 385 390 Thr Asn Ala Met Val
Thr Leu Arg Lys Glu 395 400 Arg Leu Ala Lys Leu Thr Glu Ala Ile Thr
405 410 Ala Ala Ser Leu Pro Lys Thr Ser Gly His 415 420 Tyr Asp Asp
Asp Asp Asp Ile Pro Phe Pro 425 430 Gly Pro Ile Asn Asp Asp Asp Asn
Pro Gly 435 440 His Gln Asp Asp Asp Pro Thr Asp Ser Gln 445 450 Asp
Thr Thr Ile Pro Asp Val Val Val Asp 455 460 Pro Asp Asp Gly Ser Tyr
Gly Glu Tyr Gln 465 470 Ser Tyr Ser Glu Asn Gly Met Asn Ala Pro 475
480 Asp Asp Leu Val Leu Phe Asp Leu Asp Glu 485 490 Asp Asp Glu Asp
Thr Lys Pro Val Pro Asn 495 500 Arg Ser Thr Lys Gly Gly Gln Gln Lys
Asn 505 510 Ser Gln Lys Gly Gln His Ile Glu Gly Arg 515 520 Gln Thr
Gln Ser Arg Pro Ile Gln Asn Val 525 530 Pro Gly Pro His Arg Thr Ile
His His Ala 535 540 Ser Ala Pro Leu Thr Asp Asn Asp Arg Arg 545 550
Asn Glu Pro Ser Gly Ser Thr Ser Pro Arg 555 560 Met Leu Thr Pro Ile
Asn Glu Glu Ala Asp 565 570 Pro Leu Asp Asp Ala Asp Asp Glu Thr Ser
575 580 Ser Leu Pro Pro Leu Glu Ser Asp Asp Glu 585 590 Glu Gln Asp
Arg Asp Gly Thr Ser Asn Arg 595 600 Thr Pro Thr Val Ala Pro Pro Ala
Pro Val 605 610 Tyr Arg Asp His Ser Glu Lys Lys Glu Leu 615 620 Pro
Gln Asp Glu Gln Gln Asp Gln Asp His 625 630 Thr Gln Glu Ala Arg Asn
Gln Asp Ser Asp 635 640 Asn Thr Gln Ser Glu His Ser Phe Glu Glu 645
650 Met Tyr Arg His Ile Leu Arg Ser Gln Gly 655 660 Pro Phe Asp Ala
Val Leu Tyr Tyr His Met 665 670 Met Lys Asp Glu Pro Val Val Phe Ser
Thr 675 680 Ser Asp Gly Lys Glu Tyr Thr Tyr Pro Asp 685 690 Ser Leu
Glu Glu Glu Tyr Pro Pro Trp Leu 695 700 Thr Glu Lys Glu Ala Met Asn
Glu Glu Asn 705 710 Arg Phe Val Thr Leu Asp Gly Gln Gln Phe 715 720
Tyr Trp Pro Val Met Asn His Lys Asn Lys 725 730 Phe Met Ala Ile Leu
Gln His His Gln 735 19 251 PRT Ebola Zaire 19 Met Ala Lys Ala Thr
Gly Arg Tyr Asn Leu 1 5 10 Ile Ser Pro Lys Lys Asp Leu Glu Lys Gly
15 20 Val Val Leu Ser Asp Leu Cys Asn Phe Leu 25 30 Val Ser Gln Thr
Ile Gln Gly Trp Lys Val 35 40 Tyr Trp Ala Gly Ile Glu Phe Asp Val
Thr 45 50 His Lys Gly Met Ala Leu Leu His Arg Leu 55 60 Lys Thr Asn
Asp Phe Ala Pro Ala Trp Ser 65 70 Met Thr Arg Asn Leu Phe Pro His
Leu Phe 75 80 Gln Asn Pro Asn Ser Thr Ile Glu Ser Pro 85 90 Leu Trp
Ala Leu Arg Val Ile Leu Ala Ala 95 100 Gly Ile Gln Asp Gln Leu Ile
Asp Gln Ser 105 110 Leu Ile Glu Pro Leu Ala Gly Ala Leu Gly 115 120
Leu Ile Ser Asp Trp Leu Leu Thr Thr Asn 125 130 Thr Asn His Phe Asn
Met Arg Thr Gln Arg 135 140 Val Lys Glu Gln Leu Ser Leu Lys Met Leu
145 150 Ser Leu Ile Arg Ser Asn Ile Leu Lys Phe 155 160 Ile Asn Lys
Leu Asp Ala Leu His Val Val 165 170 Asn Tyr Asn Gly Leu Leu Ser Ser
Ile Glu 175 180 Ile Gly Thr Gln Asn His Thr Ile Ile Ile 185 190 Thr
Arg Thr Asn Met Gly Phe Leu Val Glu 195 200 Leu Gln Glu Pro Asp Lys
Ser Ala Met Asn 205 210 Arg Met Lys Pro Gly Pro Ala Lys Phe Ser 215
220 Leu Leu His Glu Ser Thr Leu Lys Ala Phe 225 230 Thr Gln Gly Ser
Ser Thr Arg Met Gln Ser 235 240 Leu Ile Leu Glu Phe Asn Ser Ser Leu
Ala 245 250 Ile 20 324 PRT Ebola Zaire 20 Met Glu Ala Ser Tyr Glu
Arg Gly Arg Pro 1 5 10 Arg Ala Ala Arg Gln His Ser Arg Asp Gly 15
20 His Asp His His Val Arg Ala Arg Ser Ser 25 30 Ser Arg Glu Asn
Tyr Arg Gly Glu Tyr Arg 35 40 Gln Ser Arg Ser Ala Ser Gln Val Arg
Val 45 50 Pro Thr Val Phe His Lys Lys Arg Val Glu 55 60 Pro Leu Thr
Val Pro Pro Ala Pro Lys Asp 65 70 Ile Cys Pro Thr Leu Lys Lys Gly
Phe Leu 75 80 Cys Asp Ser Ser Phe Cys Lys Lys Asp His 85 90 Gln Leu
Glu Ser Leu Thr Asp Arg Glu Leu 95 100 Leu Leu Leu Ile Ala Arg Lys
Thr Cys Gly 105 110 Ser Val Glu Gln Gln Leu Asn Ile Thr Ala 115 120
Pro Lys Asp Ser Arg Leu Ala Asn Pro Thr 125 130 Ala Asp Asp Phe Gln
Gln Glu Glu Gly Pro 135 140 Lys Ile Thr Leu Leu Thr Leu Ile Lys Thr
145 150 Ala Glu His Trp Ala Arg Gln Asp Ile Arg 155 160 Thr Ile Glu
Asp Ser Lys Leu Arg Ala Leu 165 170 Leu Thr Leu Cys Ala Val Met Thr
Arg Lys 175 180 Phe Ser Lys Ser Gln Leu Ser Leu Leu Cys 185 190 Glu
Thr His Leu Arg Arg Glu Gly Leu Gly 195 200 Gln Asp Gln Ala Glu Pro
Val Leu Glu Val 205 210 Tyr Gln Arg Leu His Ser Asp Lys Gly Gly 215
220 Ser Phe Glu Ala Ala Leu Trp Gln Gln Trp 225 230 Asp Leu Gln Ser
Leu Ile Met Phe Ile Thr 235 240 Ala Phe Leu Asn Ile Ala Leu Gln Leu
Pro 245 250 Cys Glu Ser Ser Ala Val Val Val Ser Gly 255 260 Leu Arg
Thr Leu Val Pro Gln Ser Asp Asn 265 270 Glu Glu Ala Ser Thr Asn Pro
Gly Thr Cys 275 280 Ser Trp Ser Asp Glu Gly Thr Ser Ile Gln 285 290
Gln Gln Leu Ala Ser Cys Leu His Arg Thr 295 300 Arg Gly Asp Trp His
Ala Ala Leu Lys Phe 305 310 Leu Phe Tyr Phe Ser Phe Leu Phe Arg Ile
315 320 Gly Phe Cys Phe 21 340 PRT Ebola Zaire 21 Met Thr Thr Arg
Thr Lys Gly Arg Gly His 1 5 10 Thr Ala Ala Thr Thr Gln Asn Asp Arg
Met 15 20 Pro Gly Pro Glu Leu Ser Gly Trp Ile Ser 25 30 Glu Gln Leu
Met Thr Gly Arg Ile Pro Val 35 40 Ser Asp Ile Phe Cys Asp Ile Glu
Asn Asn 45 50 Pro Gly Leu Cys Tyr Ala Ser Gln Met Gln 55 60 Gln Thr
Lys Pro Asn Pro Lys Thr Arg Asn 65 70 Ser Gln Thr Gln Thr Asp Pro
Ile Cys Asn
75 80 His Ser Phe Glu Glu Val Val Gln Thr Leu 85 90 Ala Ser Leu Ala
Thr Val Val Gln Gln Gln 95 100 Thr Ile Ala Ser Glu Ser Leu Glu Gln
Arg 105 110 Ile Thr Ser Leu Glu Asn Gly Leu Lys Pro 115 120 Val Tyr
Asp Met Ala Lys Thr Ile Ser Ser 125 130 Leu Asn Arg Val Cys Ala Glu
Met Val Ala 135 140 Lys Tyr Asp Leu Leu Val Met Thr Thr Gly 145 150
Arg Ala Thr Ala Thr Ala Ala Ala Thr Glu 155 160 Ala Tyr Trp Ala Glu
His Gly Gln Pro Pro 165 170 Pro Gly Pro Ser Leu Tyr Glu Glu Ser Ala
175 180 Ile Arg Gly Lys Ile Glu Ser Arg Asp Glu 185 190 Thr Val Pro
Gln Ser Val Arg Glu Ala Phe 195 200 Asn Asn Leu Asn Ser Thr Thr Ser
Leu Thr 205 210 Glu Glu Asn Phe Gly Lys Pro Asp Ile Ser 215 220 Ala
Lys Asp Leu Arg Asn Ile Met Tyr Asp 225 230 His Leu Pro Gly Phe Gly
Thr Ala Phe His 235 240 Gln Leu Val Gln Val Ile Cys Lys Leu Gly 245
250 Lys Asp Ser Asn Ser Leu Asp Ile Ile His 255 260 Ala Glu Phe Gln
Ala Ser Leu Ala Glu Gly 265 270 Asp Ser Pro Gln Cys Ala Leu Ile Gln
Ile 275 280 Thr Lys Arg Val Pro Ile Phe Gln Asp Ala 285 290 Ala Pro
Pro Val Ile His Ile Arg Ser Arg 295 300 Gly Asp Ile Pro Arg Ala Cys
Gln Lys Ser 305 310 Leu Arg Pro Val Pro Pro Ser Pro Lys Ile 315 320
Asp Arg Gly Trp Val Cys Val Phe Gln Leu 325 330 Gln Asp Gly Lys Thr
Leu Gly Leu Lys Ile 335 340 22 326 PRT Ebola Zaire 22 Met Arg Arg
Val Ile Leu Pro Thr Ala Pro 1 5 10 Pro Glu Tyr Met Glu Ala Ile Tyr
Pro Val 15 20 Arg Ser Asn Ser Thr Ile Ala Arg Gly Gly 25 30 Asn Ser
Asn Thr Gly Phe Leu Thr Pro Glu 35 40 Ser Val Asn Gly Asp Thr Pro
Ser Asn Pro 45 50 Leu Arg Pro Ile Ala Asp Asp Thr Ile Asp 55 60 His
Ala Ser His Thr Pro Gly Ser Val Ser 65 70 Ser Ala Phe Ile Leu Glu
Ala Met Val Asn 75 80 Val Ile Ser Gly Pro Lys Val Leu Met Lys 85 90
Gln Ile Pro Ile Trp Leu Pro Leu Gly Val 95 100 Ala Asp Gln Lys Thr
Tyr Ser Phe Asp Ser 105 110 Thr Thr Ala Ala Ile Met Leu Ala Ser Tyr
115 120 Thr Ile Thr His Phe Gly Lys Ala Thr Asn 125 130 Pro Leu Val
Arg Val Asn Arg Leu Gly Pro 135 140 Gly Ile Pro Asp His Pro Leu Arg
Leu Leu 145 150 Arg Ile Gly Asn Gln Ala Phe Leu Gln Glu 155 160 Phe
Val Leu Pro Pro Val Gln Leu Pro Gln 165 170 Tyr Phe Thr Phe Asp Leu
Thr Ala Leu Lys 175 180 Leu Ile Thr Gln Pro Leu Pro Ala Ala Thr 185
190 Trp Thr Asp Asp Thr Pro Thr Gly Ser Asn 195 200 Gly Ala Leu Arg
Pro Gly Ile Ser Phe His 205 210 Pro Lys Leu Arg Pro Ile Leu Leu Pro
Asn 215 220 Lys Ser Gly Lys Lys Gly Asn Ser Ala Asp 225 230 Leu Thr
Ser Pro Glu Lys Ile Gln Ala Ile 235 240 Met Thr Ser Leu Gln Asp Phe
Lys Ile Val 245 250 Pro Ile Asp Pro Thr Lys Asn Ile Met Gly 255 260
Ile Glu Val Pro Glu Thr Leu Val His Lys 265 270 Leu Thr Gly Lys Lys
Val Thr Ser Lys Asn 275 280 Gly Gln Pro Ile Ile Pro Val Leu Leu Pro
285 290 Lys Tyr Ile Gly Leu Asp Pro Val Ala Pro 295 300 Gly Asp Leu
Thr Met Val Ile Thr Gln Asp 305 310 Cys Asp Thr Cys His Ser Pro Ala
Ser Leu 315 320 Pro Ala Val Ile Glu Lys 325 23 288 PRT Ebola Zaire
23 Met Glu Ala Ser Tyr Glu Arg Gly Arg Pro 1 5 10 Arg Ala Ala Arg
Gln His Ser Arg Asp Gly 15 20 His Asp His His Val Arg Ala Arg Ser
Ser 25 30 Ser Arg Glu Asn Tyr Arg Gly Glu Tyr Arg 35 40 Gln Ser Arg
Ser Ala Ser Gln Val Arg Val 45 50 Pro Thr Val Phe His Lys Lys Arg
Val Glu 55 60 Pro Leu Thr Val Pro Pro Ala Pro Lys Asp 65 70 Ile Cys
Pro Thr Leu Lys Lys Gly Phe Leu 75 80 Cys Asp Ser Ser Phe Cys Lys
Lys Asp His 85 90 Gln Leu Glu Ser Leu Thr Asp Arg Glu Leu 95 100
Leu Leu Leu Ile Ala Arg Lys Thr Cys Gly 105 110 Ser Val Glu Gln Gln
Leu Asn Ile Thr Ala 115 120 Pro Lys Asp Ser Arg Leu Ala Asn Pro Thr
125 130 Ala Asp Asp Phe Gln Gln Glu Glu Gly Pro 135 140 Lys Ile Thr
Leu Leu Thr Leu Ile Lys Thr 145 150 Ala Glu His Trp Ala Arg Gln Asp
Ile Arg 155 160 Thr Ile Glu Asp Ser Lys Leu Arg Ala Leu 165 170 Leu
Thr Leu Cys Ala Val Met Thr Arg Lys 175 180 Phe Ser Lys Ser Gln Leu
Ser Leu Leu Cys 185 190 Glu Thr His Leu Arg Arg Glu Gly Leu Gly 195
200 Gln Asp Gln Ala Glu Pro Val Leu Glu Val 205 210 Tyr Gln Arg Leu
His Ser Asp Lys Gly Gly 215 220 Ser Phe Glu Ala Ala Leu Trp Gln Gln
Trp 225 230 Asp Arg Gln Ser Leu Ile Met Phe Ile Thr 235 240 Ala Phe
Leu Asn Ile Ala Leu Gln Leu Pro 245 250 Cys Glu Ser Ser Ala Val Val
Val Ser Gly 255 260 Leu Arg Thr Leu Val Pro Gln Ser Asp Asn 265 270
Glu Glu Ala Ser Thr Asn Pro Gly Thr Cys 275 280 Ser Trp Ser Asp Glu
Gly Thr Pro 285 24 11 PRT Ebola Zaire 24 Val Tyr Gln Val Asn Asn
Leu Glu Glu Ile 1 5 10 Cys 25 23 PRT Ebola Zaire 25 Leu Lys Phe Ile
Asn Lys Leu Asp Ala Leu 1 5 10 Leu Val Val Asn Tyr Asn Gly Leu Leu
Ser 15 20 Ser Ile Phe
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