Immunization for Ebola virus infection

Abstract

Ebola virus vaccines comprising nucleic acid molecules encoding Ebola viral proteins are provided. In one embodiment, the nucleic acid molecule encodes the transmembrane form of the viral glycoprotein (GP). In another embodiment, the nucleic acid molecule encodes the secreted form of the viral glycoprotein (sGP). In yet another embodiment, the nucleic acid molecule encodes the viral nucleoprotein (NP). Methods for immunizing a subject against disease caused by infection with Ebola virus are also provided.

Description

FIELD OF THE INVENTION

[0001] The present invention relates generally to viral vaccines and, more particularly, to Ebola virus vaccines and methods of protecting against disease caused by infection with Ebola virus.

BACKGROUND OF THE INVENTION

[0002] The Ebola viruses, and the genetically-related Marburg virus, are filoviruses associated with outbreaks of highly lethal hemorrhagic fever in humans and primates in North America, Europe, and Africa. Peters, C. J. et al., Filoviridae: Marburg and Ebola Viruses. in Fields Virology. (eds., Fields, B. N., Knipe, D. M. & Howley, P. M.) 1161-1176 (Philadelphia, Lippincott-Raven, 1996); Peters, C. J. et al, Semin. Virol. 5:147-154 (1994). Ebola viruses are negative-stranded RNA viruses comprised of four subtypes, including those described in the Zaire, Sudan, Reston, and Ivory Coast episodes. Sanchez, A. et al., PNAS (USA) 93:3602-3607 (1996). Although several subtypes have been defined, the genetic organization of these viruses is similar, each containing seven linearly arrayed genes. Among the viral proteins, the envelope glycoprotein exists in two alternative forms, a 50-70 kilodalton (kDa) secreted protein of unknown function encoded by the viral genome and a 130 kDa transmembrane glycoprotein generated by RNA editing that mediates viral entry. Peters, C. J. et al., Filoviridae: Marburg and Ebola Viruses. in Fields Virology. (eds., Fields, B. N., Knipe, D. M. & Howley, P. M.) 1161-1176 (Philadelphia, Lippincott-Raven, 1996); Sanchez, A. et al., PNAS (USA) 93:3602-3607 (1996). Other structural gene products include the nucleoprotein (NP), matrix proteins VP24 and VP40, presumed nonstructural proteins VP30 and VP35, and the viral polymerase (reviewed in Peters, C. J. et al., Filovirdae: Marburg and Ebola Viruses. in Fields Virology. (eds., Fields, B. N., Knipe, D. M. & Howley, P. M.) 1161-1176 (Philadelphia, Lippincott-Raven, 1996)). Although spontaneous variation of its RNA sequence does occur in nature, there appears to be less nucleotide polymorphism within Ebola subtypes than among other RNA viruses (Sanchez, A. et al., PNAS (USA) 93:3602-3607 (1996)), suggesting that immunization may be useful in protecting against this disease. Previous attempts to elicit protective immune responses against Ebola virus using traditional active and passive immunization approaches have, however, not succeeded. Peters, C. J. et al., Filoviridae: Marburg and Ebola Viruses. in Fields Virology. (eds., Fields, B. N., Knipe, D. M. & Howley, P. M.) 1161-1176 (Philadelphia, Lippincott-Raven, 1996); Clegg, J. C. S. et al., New Generation Vaccines. (eds., Levine, M. M., Woodrow, G. C., Kaper, J. B. & Cobon, G. S.) 749-765 (New York, N.Y., Marcel Dekker, Inc. 1997); Jahrling, P. B. et al., Arch. Virol. Suppl. 11:135-140 (1996).

[0003] It would thus be desirable to provide a vaccine to protect against disease caused by infection with Ebola virus. It would further be desirable to provide methods of making and using said vaccine.

SUMMARY OF THE INVENTION

[0004] Ebola virus vaccines comprising nucleic acid molecules encoding Ebola viral proteins are provided. In one embodiment, the nucleic add molecule encodes the transmembrane form of the viral glycoprotein (GP). In another embodiment, the nucleic acid molecule encodes the secreted form of the viral glycoprotein (sGP). In yet another embodiment, the nucleic acid molecule encodes the viral nucleoprotein (NP).

[0005] The present invention also provides methods for immunizing a subject against disease caused by infection with Ebola virus comprising administering to the subject an immunoeffective amount of an Ebola virus vaccine. Administration can be by any of the routes normally used for gene therapy. In a preferred method, the Ebola virus vaccine is administered by intramuscular injection. The genetic immunization methods of the present invention provide protective immunity against disease caused by infection with Ebola virus.

[0006] Additional objects, advantages, and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and subjoined claims and by referencing the following drawings.

[0008] FIGS. 1A and 1B are photographs showing expression of Ebola virus gene products in eukaryotic plasmid expression vectors.

[0009] FIG. 1A. Expression vectors encoding the indicated viral gene products under regulation of the CMV immediate-early region 1 enhancer and promoter were prepared and transfected into 293 cells as previously described. Manthorpe, M. et al. Hum. Gene. Ther. 4:419-431 (1993); Sambrook, J., Fritch, E. F., & Maniatis, T. Cold Spring Harbor, N.Y. Cold Spring Laboratory Harbor Press, 1994. Cell extracts were prepared and analyzed by Western blot analysis for NP (left) or GP (right) using relevant rabbit antisera and a secondary antibody, horseradish peroxidase conjugated donkey anti-rabbit IgG of a dilution of 1:5,000. Incubation with primary antibody was for 30 minutes at mom temperature, and for 30 minutes at room temperature with secondary antibody. Immunocomplexes were then detected by chemiluminescence using super signal substrate reagents (Pierce) according to manufacturer's instructions.

[0010] FIG. 1B. Generation of antibody response in mice immunized with the indicated vectors and analyzed by Western blot for NP, GP, and sGP as shown. Antisera from mice were tested at a dilution of 1:500 (NP), 1:50 (GP), or 1:50 (sGP), respectively, and developed with a secondary antibody (sheep anti-mouse, 1:5,000, Amersham Life Science) and chemiluminescence as in FIG. 1A. The control vector used for immunization represents the expression vector plasmid with no insert. Manthorpe, M. et al., Hum. Gene. Ther. 4:419-431 (1993).

[0011] FIGS. 2A-2D are graphs showing the immune responses to NP and GP after genetic immunization in mice.

[0012] FIG. 2A. Splenic lymphocytes: from vector or NP-plasmid immunized mice were isolated approximately 6 weeks after the initial immunization and sensitized in vitro for 5 days with 10 U/ml hlL-2. Renca-NP cells sensitized splenocytes from vector-immunized or pCMV-NP immunized mice were used to detect CTL activity at the indicated effector target ratios on Renca or Renca-NP cells (left, middle) or with allogeneic effector cells with Renca-NP to show that they are susceptible to lysis (right). Allogeneic effector cells were generated by incubating cells derived from mice with a C57BI/6 background (5.times.10.sup.6/ml) with irradiated Balb/c spleen cells (5.times.10.sup.6/ml) in the presence of IL-2 (20 U/ml) for five days. The chromium release CTL assay with Renca-NP cells was performed in triplicate as previously described. Ohno, T. et al., Gene. Ther. 4:361-366 (1997).

[0013] FIG. 2B. Balb/C female mice were immunized with the sGP plasmid expression vector and analyzed for their ability to lyse the syngeneic Renca cell line stably expressing GP. Isolation of stable transfectants, confirmation of expression, and CTL assay were performed as described (see, Specific Example, II. Methods). Renca-GP or sGP sensitized splenocytes from pCMV-GP or pCMV-sGP immunized mice were used to determine the specific killing of .sup.51chromium labeled Renca-GP cells at the indicated E/T ratios.

[0014] FIG. 2C. Mice immunized with GP were analyzed for their ability to lyse a syngeneic CT26 cell stably expressing GP or CT26 vector control transduced line at the indicated E/T ratios.

[0015] FIG. 2D. Cellular proliferative response in the indicated immunized mice. T cells, enriched or depleted (see, Specific Example, II. Methods), were incubated at 10.sup.5 cells/ml with sGP condition media (25%). Background was determined with cells incubated in media from control transfected 293 cells and subtracted from proliferation seen in sGP-containing supernatants.

[0016] FIGS. 3A-3C are graphs showing immunization with sGP or GP expression plasmids induces T cell responses to sGP in guinea pigs.

[0017] FIGS. 3A-3C. Cell-mediated immunity in guinea pigs was analyzed by performing assays to detect cell proliferation to control or GP antigen (A) or T-cell growth factor production in response to the indicated antigens. The culture supernatants containing these antigens were prepared as previously described (Bottomly, K. et al., Measurement of human and murine interleukin 2 and interleukin 4. in Current Protocols in Immunology. (eds., Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M. & Strober, W.) 6.3.1-6.3.12 (New York, John Wiley & Sons, Inc. 1992); Arai, H. et al., Nat. Med. 3:843-848 (1997)), and included at a final concentration of 10% (volume/volume). In A, cell numbers refer to the concentration of spleen cells per ml in the .sup.3H-thymidine proliferation assay. In B, supernatants from A, harvested at the time of the peak proliferative response to sGP, were incubated with primary guinea pig T cells maintained in 200 U/ml of human IL-2. The percent maximal response refers to the magnitude of stimulation in response to the indicated stimuli relative to supernatants from 24 hour concanaval (in A-stimulated cells (2 .mu.g/ml)). The requirement of T lymphocytes in guinea pig spleen cells for the proliferative response to sGP, performed as described in Specific Example, II. Methods, is shown (C).

[0018] FIGS. 4A-4F are photographs showing the immunohistochemical analysis of Ebola virus antigens in liver, lung, and spleen from representative protected (GP-animal 3) or infected (vector-animal 2) guinea pigs.

[0019] FIGS. 4A-4F. Magnification: liver, 40.times.; lung, 20.times.; spleen, 20.times..

[0020] FIG. 5 is a schematic of the plasmid pVR 1012-GP(IC) (Ivory Coast strain of GP, SEQ ID NO: 1).

[0021] FIG. 6 is a schematic of the plasmid pVR 1012-GP(S) (Sudan strain of GP, see SEQ ID NO: 2).

[0022] FIG. 7 is a schematic of the plasmid pVR 1012-GP(Z) (Zaire strain of GP, see SEQ ID NO: 3).

[0023] FIG. 8 is a schematic of the plasmid pVR 1012-sGP(Z) (Zaire strain of sGP, see SEQ ID NO: 4).

[0024] FIG. 9 is a schematic of the plasmid pVR 1012-NP.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Ebola virus vaccines are provided comprising a nucleic acid molecule encoding an Ebola viral protein operatively-linked to a control sequence in a pharmaceutically acceptable carrier. In one embodiment, the nucleic acid molecule encodes the transmembrane form of the viral glycoprotein (GP). In another embodiment, the nucleic acid molecule encodes the secreted form of the viral glycoprotein (sGP). In yet another embodiment, the nucleic acid molecule encodes the viral nucleoprotein (NP).

[0026] The present invention further includes vaccines comprising nucleic acid molecules encoding Ebola viral proteins other than GP, sGP, and NP, e.g., other structural gene products which elicit protective immunity from disease caused by infection with Ebola virus. The nucleic acid molecules of the vaccines of the present invention encode structural gene products of any Ebola viral strain including the Zaire, Sudan, Ivory Coast and Reston strains. Nucleic acid molecules encoding structural gene products of the genetically-related Marburg virus strains may also be employed. Moreover, the nucleic acid molecules of the present invention may be modified, e.g., the nucleic acid molecules set forth herein may be mutated, as long as the modified expressed protein elicits protective immunity from disease caused by infection with Ebola virus. For example, the nucleic acid molecule may be mutated so that the expressed protein is less toxic to cells. The present invention also includes vaccines comprising a combination of nucleic acid molecules. For example, and without limitation, nucleic acid molecules encoding GP, sGP and NP of the Zaire, Sudan and Ivory Coast Ebola strains may be combined in any combination, in one vaccine composition.

[0027] The present invention also provides methods for immunizing a subject against disease caused by infection with Ebola virus comprising administering to the subject an immunoeffective amount of an Ebola virus vaccine. Methods of making and using Ebola virus vaccines are also provided by the present invention including the preparation of pharmaceutical compositions.

[0028] As referred to herein, the term "encoding" Is intended to mean that the subject nucleic acid may be transcribed in a cell, e.g., when the subject nucleic acid is linked to appropriate control sequences such as a promoter in a suitable vector (e.g., an expression vector) and the vector is introduced into a cell. The nucleic acid molecules of the present invention may be DNA molecules, cDNA molecules or RNA molecules, and are preferably cDNA molecules. The term "operatively-linked" as used herein refers to functional linkage between a nucleic acid expression control sequence (such as a promoter) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. Expression control sequences are known to those skilled in the art (see, e.g., Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Vectors which contain both a promoter and a cloning site to which an inserted piece of nucleic acid is operatively-linked to the promoter, are well known in the art and are generally referred to herein as "expression vectors" or "expression vector plasmids". Preferably, these vectors are capable of transcribing nucleic acid in vitro and in vivo. A preferred vector is the cytomegalovirus (CMV) expression vector which directs high levels of gene expression in muscle.

[0029] Nucleic acid molecules which hybridize under stringent conditions to the nucleic acid molecules described herein are also within the scope of the present invention. As will be appreciated by those skilled in the art, multiple factors are considered in determining the stringency of hybridization including species of nucleic acid, length of nucleic acid probe, T.sub.m (melting temperature), temperature of hybridization and washes, salt concentration in the hybridization and wash buffers, aqueous or formamide hybridization buffer, and length of time for hybridization and for washes. An example of stringent conditions are DNA-DNA hybridization with a probe greater than 200 nucleotides in 5.times.SSC, at 65.degree. C. in aqueous solution or 42.degree. C. in formamide, followed by washing with 0.1.times.SSC, at 65.degree. C. in aqueous solution. (Other experimental conditions for controlling stringency are described in Maniatis, T. et al., Molecular Cloning: a Laboratory Manual, Cold Springs Harbor Laboratory, Cold Springs, N.Y. (1982) at pages 387-389 and Sambrook, J. et al., Molecular Cloning: a Laboratory Manual, Second Edition, Volume 2, Cold Springs Harbor Laboratory, Cold Springs, N.Y., at pages 8.46-8.47 (1989)).

[0030] It will be appreciated that administration of the vaccines of the present invention can be by any of the routes normally used for gene therapy. In a preferred method, administration is by intramuscular injection, however, other procedures for transfecting cells may also be employed, such as transfection using calcium phosphate coprecipitation, liposome-mediated transfection, plasmid and viral vector-mediated transfection and DNA protein complex-mediated transfection. Viral vector-mediated transfection includes, without limitation, the use of retroviral, replication-deficient retroviral, adenoviral and adeno-associated viral vectors. Cells transfected by the vaccines in the context of ex vivo gene therapy can also be administered.

[0031] It will be appreciated that more than one route of administering the vaccines of the present invention may be employed either simultaneously or sequentially (e.g., boosting). In addition, the vaccines of the present invention may be employed in combination with traditional immunization approaches such as employing protein antigens, vaccinia virus and inactivated virus, as vaccines. Thus, in one embodiment, the vaccines of the present invention are administered to a subject (the subject is "primed" with a vaccine of the present invention) and then a traditional vaccine is administered (the subject is "boosted" with a traditional vaccine). In another embodiment, a traditional vaccine is first administered to the subject followed by administration of a vaccine of the present invention. In yet another embodiment, a traditional vaccine and a vaccine of the present invention are co-administered.

[0032] Immunogenicity may be significantly improved if the vaccines of the present invention are co-administered with an immunostimulatory agent or adjuvant. Adjuvants enhance immunogenicity but are not necessarily immunogenic themselves. Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Adjuvants may thus be employed to enhance the immunogenicity of the vaccines of the present invention, as well as the immunogenicity of traditional vaccines. Suitable adjuvants are well known to those skilled in the art and include, without limitation, aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octodecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, a lipoprotein, ISCOM matrix, DC-Chol, DDA, and other adjuvants and bacterial toxins, components and derivatives thereof.

[0033] The vaccines of the present invention may also be co-administered with cytokines to further enhance immunogenicity. The cytokines may be administered by methods known to those skilled in the art, e.g., as a nucleic acid molecule in plasmid form or as a protein or fusion protein.

[0034] Upon inoculation with a pharmaceutical composition as described herein, the immune system of the host responds to the vaccine by producing antibodies, both secretory and serum, specific for Ebola virus proteins. As a result of the vaccination, the host becomes at least partially or completely immune to Ebola virus infection, or resistant to developing moderate or severe disease caused by Ebola virus infection. Although Ebola virus infection and disease caused thereby are discussed in detail herein, it will be appreciated that the vaccines and methods of the present invention may be employed to immunize a subject against hemorrhagic fever generally, such as that caused by infection by the genetically-related Marburg virus.

[0035] Pharmaceutical compositions comprising the nucleic acid molecules encoding Ebola viral proteins described herein, either alone or in combination, and a pharmaceutically acceptable carrier, are also provided by the present invention. As used herein, the phrase "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as those suitable for parenteral administration, such as, for example, by intramuscular, intraarticular (in the joints), intravenous, intradermal, intraperitoneal, and subcutaneous routes. Examples of such formulations include aqueous and non-aqueous, isotonic sterile injection solutions, which contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0036] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the vaccine dissolved in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the vaccine, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; (d) suitable emulsions; and (e) polysaccharide polymers such as chitians. The vaccine, alone or in combination with other suitable components, may also be made into aerosol formulations to be administered via inhalation, e.g., to the bronchial passageways. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0037] Suitable formulations for rectal administration include, for example, suppositories, which consist of the vaccine with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the vaccine with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

[0038] Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the recipient, e.g., the patient. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules or vials and may be prepared by any method known in the art.

[0039] Pharmaceutical compositions comprising any of the nucleic acid molecules encoding Ebola viral proteins of the present invention are useful to immunize a subject against disease caused by Ebola virus infection. Thus, this invention further provides methods of immunizing a subject against disease caused by Ebola virus infection, e.g., hemorrhagic fever, comprising administering to the subject an immunoeffective amount of a pharmaceutical composition of the invention. This subject may be an animal, for example a mammal, such as a primate or preferably a human.

[0040] The vaccines of the present invention are also suitable for veterinary immunization. The vaccines of the present invention comprising nucleic acid molecules encoding Ebola virus structural gene products from the Reston strain, which is known to infect animals, are particularly useful in such veterinary immunization methods.

[0041] The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, immunogenic and protective. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the immune system of the individual to synthesize antibodies, and, if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be monitored on a patient-by-patient basis. However, suitable dosage ranges are readily determinable by one skilled in the art and generally range from about 300 .mu.g to about 4-5 mg. The dosage may also depend, without limitation, on the route of administration, the patient's state of health and weight, and the nature of the formulation.

[0042] Methods of immunizing a subject against multiple strains of Ebola virus are further provided herein. The nucleic acid molecules encoding Ebola viral proteins of the present invention may be combined with nucleic acid molecules encoding other viral proteins of other virus strains to achieve protection against multiple strains of Ebola virus. Typically the vaccines will be in an admixture and administered simultaneously, but may also be administered separately.

[0043] In some instances it may be desirable to combine the Ebola virus vaccines of the present invention with vaccines which induce protective responses to other agents, particularly other viruses. For example, the vaccine compositions of the present invention can be administered simultaneously, separately or sequentially with other genetic immunization vaccines such as those for influenza (Ulmer, J. B. et al., Science 259:1745-1749 (1993); Raz, E. et al., PNAS (USA) 91:9519-9523 (1994)), malaria (Doolan, D. L. et al., J. Exp. Med. 183:1739-1746 (1996); Sedegah, M. et al., PNAS (USA) 91:9866-9870 (1994)), and tuberculosis (Tascon, R. C. et al., Nat. Med. 2:888-892 (1996)).

[0044] It will also be appreciated that single or multiple administrations of the vaccine compositions of the present invention may be carried out. For example, subjects who are particularly susceptible to Ebola virus infection may require multiple immunizations to establish and/or maintain protective immune responses. Levels of induced immunity can be monitored by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to maintain desired levels of protection.

[0045] This invention also provides kits comprising the vaccines of the present invention. For example, kits comprising a vaccine and instructions for use are within the scope of this invention.

[0046] The vaccines and methods of the present invention evoke a protective immune response and do not lead to immunopotentiation or exacerbated disease. The vaccines lack transmissibility, are genetically stable and induce protective levels of humoral and cell-mediated immunity.

[0047] In order to more fully demonstrate the advantages arising from the present invention, the following example is set forth. It is to be understood that the following is by way of example only and is not intended as a limitation on the scope of the invention.

SPECIFIC EXAMPLE

[0048] I. Results

[0049] Immune response to viral gene products in mice. To characterize immune responses to selected Ebola virus proteins, eukaryotic expression vector plasmids were injected into mice. The cytomegalovirus (CMV) immediate early region 1 enhancer was used to stimulate transcription because ft directs high levels of gene expression in muscle. Manthorpe, M. et al., Hum. Gene. Ther. 4:419-431 (1993). cDNAs encoding an abundant structural protein, the major viral nucleocapsid phosphoprotein (NP), the secreted glycoprotein (sGP), or the membrane-associated glycoprotein (GP) were inserted. Alternative forms of GP were chosen because it had been postulated that the transmembrane protein contained a protein sequence motif also found in oncogenic retroviruses that might suppress immune responses. Burkreyev, A. A. et al., FEBS. Lett. 323:183-187 (1993); Cianciolo, G. J. et al., Science 230:453-455 (1985); Harris, D. T. et al., J. Immunol. 138:889-894 (1987); Volchkov, V. E. et al., FEBS. Lett. 305:181-184 (1992); Sanchez, A. et al., Virus. Res. 29:215-240 (1993). Expression of the relevant proteins was confirmed after transfection of the human renal epithelial cell line, 293, by immunoblotting with antisera to these gene products (FIG. 1A). For NP, the expected full-length 104 kDa protein normally produced by the virus was seen, together with lower molecular weight species likely generated from truncated protein or degradation products described previously. Sanchez, A. et al., Virology 170:81-91 (1989). Similarly, expression of sGP and GP revealed a heterogeneous pattern whose sizes correlated with the expected products of cleavage or post-translational carbohydrate modification. Sanchez, A. et al., PNAS (USA) 93:3602-3607 (1996).

[0050] These plasmids were injected into mice to characterize their ability to induce humoral and cellular immune responses to the relevant viral proteins. Three injections, each with 50 .mu.g of plasmid DNA in saline (100 .mu.l), were performed at two-week intervals in Balb/C female mice (6-8 week old, Charles River). Serum from immunized recipients were examined for antibody responses. An antibody response to the viral NP gene product was readily detectable (FIG. 1B), with titers of .gtoreq.1:16,000 by Western blot analysis. The titer of antibody induced in response to injection with plasmids encoding the viral glycoproteins was lower. After immunization with GP, no antibody was detectable by Western blotting, while immunization with sGP induced an antibody response (FIG. 1B). The more sensitive ELISA (Ksiazek, T. G., Lab. Anim. 20:34-46 (1991); Ksiazek, T. G. et al., J. Clin. Microbiol. 30:947-950 (1992)) did allow detection of antibodies to both GP and sGP at titers of 1:400 and 1:1,200, respectively. Cytolytic T cell (CTL) responses to these viral proteins were analyzed next. Despite the substantial humoral immune response to NP, minimal CTL activity was detected against syngeneic cells expressing this viral protein (FIG. 2A). In contrast, genetic immunization with sGP, which elicited a weaker antibody response, induced a marked cytolytic T cell response to cells expressing GP (FIG. 2B). Immunization with the GP plasmid also induced a significant CTL response to GP (FIG. 2C). These data suggested that both the secreted and transmembrane form of the protein could be processed for antigen presentation and the transmembrane form was a target for recognition by these cytolytic T cells. Finally, antigen-specific T cell proliferation to sGP was also observed in GP and sGP but not plasmid control injected mice (FIG. 2D).

[0051] The ability of antibodies detected in mouse sera after immunization to neutralize virus was tested in an in vitro infection assay. McCormick, J. B. et al., J. Infect. Dis. 147:264-267 (1983). In no case was neutralization of infectivity observed, even at titers of 1:10 (data not shown), despite the documented presence of antibody after NP and sGP immunization by Western blot analysis. Infectivity in vitro was thus not inhibited by Ebola-specific antibodies.

[0052] Immune function and viral challenge in guinea pigs. To determine whether the in vivo immune responses could protect against viral infection, virus was adapted to growing guinea pigs. Though this species is not well-suited to analysis of immune function, infection in adult mice has not been successful. Moreover, infection in guinea pigs, used originally to propagate virus from infected humans, is a well-established animal model for the human disease. Infection gives rise to a syndrome of hemorrhagic fever with levels of virus, organ pathology, and infection of reticuloendothelial and mononudear cells comparable to humans. Bowen, E. T. W. et al., Lancet 1:571-573 (1977).

[0053] Two groups of immunized guinea pigs were studied. Animals were injected intramuscularly with the relevant expression vector plasmids, and the response to infection in groups immunized with either sGP, GP, NP, or control plasmids was observed. In the first group, animals were challenged within 2 months after the initial immunization, at which time the antibody titers were high, ranging from 1:1,600 to >1:25,000 (Table 1A). In these animals, nearly complete protection from lethal challenge was observed in GP (6/6), sGP (5/6), and NP (4/4) immunized subjects, in contrast to controls (0/6). In a second group, guinea pigs were challenged four months after the initial immunization (Table 1B). As in the first group, all animals immunized with the control vector succumbed to infection within a week after virus challenge (n=4). In this group, antibody titers were lower, and three of the four guinea pigs immunized with NP succumbed to infection, with the single survivor appearing severely ill after 1 week, in contrast to the protective response with NP at the earlier time point after immunization in Group I. More effective protection was seen in animals immunized with vector expressing GP, protection was noted in four of five animals challenged, with one survivor appearing weak but surviving the viral challenge. Similarly, three of the five animals immunized with sGP showed no ill effects following viral challenge. Protection in this group again correlated with the ability to sustain an effective immune response to GP or sGP. Together, all guinea pigs immunized with vectors expressing GP or sGP which had titers greater than 1:5,120 were resistant to infection (11/11) compared to 0/10 controls (p=0, by Fisher's exact test). Twelve of fourteen animals with antibody titers .gtoreq.2,560 survived viral challenge compared to controls (p=0.00003, by Fisher's exact test). Similar to immunized mice, guinea pigs injected with GP or sGP plasmids were able to generate cell-mediated immune responses to the viral glycoprotein in addition to the antibody response. These responses were antigen-specific and T cell-dependent, as detected in sGP antigen-dependent spleen cell proliferation and T-cell growth factor assays (FIG. 3A-C). Thus, the ability to generate and sustain significant cellular immune responses in vivo correlated with protection from infection. Though antibody titer correlated with protection, cell-mediated immunity appeared necessary for protection since passive transfer of antibody to GP does not confer protection (Peters, C. J. et al., Filovirdae: Marburg and Ebola Viruses. in Fields Virology. (eds., Fields, B. N., Knipe, D. M. & Howley, P. M.) 1161-1176 (Philadelphia, Lippincott-Raven, 1996); Jahrling, P. B. et al., Arch. Virol. Suppl. 11:135-140 (1996)) and antisera from protected guinea pigs did not inhibit virus replication in vivo (n=3) or at a 1:10 dilution in vitro (data not shown). Since the Hartley guinea pig to which the virus has been adapted for growth is outbred, cellular adoptive transfer studies could not be performed.

1TABLE 1 Group I Plasmid Subject ELISA(Pre) ELISA(Post) Viral Ag Survival GP 1 >1:25,600 1:12,800 - Yes GP 2 >1:25,600 1:25,600 - Yes GP 3 >1:25,600 1:25,600 - Yes GP 4 1:25,600 1:6,400 - Yes GP 5 1:25,600 1:12,800 - Yes GP 6 1:25,600 1:25,600 - Yes SGP 1 1:12,800 1:25,600 - Yes SGP 2 1:6,400 1:25,600 - Yes SGP 3 1:6,400 1:25,600 - Yes SGP 4 1:25,600 1:25,600 - Yes SGP 5 >1:25,600 1:12,800 - Yes SGP 6 1:1,600 Negative + No NP 1 1:12,800 >1:25,600 - Yes NP 2 >1:25,600 1:25,600 - Yes NP 3 1:12,800 1:12,800 - Yes NP 4 1:25,600 1:25,600 - Yes Vector alone 1 Negative Negative + No Vector alone 2 Negative n.d. + No Vector alone 3 Negative Negative + No Vector alone 4 Negative Negative + No Vector alone 5 Negative n.d. + No Vector alone 6 Negative n.d. + No Guinea pigs were immunized on days 1, 14, 28, 42, and challenged on day 62.

[0054]

2TABLE 1 Group II Plasmid Subject ELISA(Pre) ELISA(Post) Viral Ag Survival GP 1 1:2,560 n.d. +/f No GP 2 1:5,120 1:10,240 - Yes GP 3 1:10,240 1:10,240 - Yes GP 4 1:1,280 n.d. +/f No GP 5 1:5,120 1:20,480 - Yes (ill) SGP 1 1:2,560 n.d. + No SGP 2 1:10,240 1:5,120 +/f Yes SGP 3 1:10,240 1:81,920 - Yes SGP 4 1:2,560 1:5,120 - Yes SGP 5 1:640 n.d. + No NP 1 n.d. n.d. + No NP 2 n.d. n.d. + No NP 3 n.d. n.d. + No NP 4 n.d. Negative + Yes (ill) Vector alone 1 Negative n.d. + No Vector alone 2 Negative n.d. + No Vector alone 3 Negative n.d. + No Vector alone 4 Negative n.d. + No Guinea pigs were immunized on days 1, 14, 42, and 112 and challenged on day 122. n.d. = not done. Viral ag denotes presence of virus determined by immunohistochemistry (30) performed on spleen, liver, lung, kidney, and heart tissues; "+" = widespread systemic involvement of the mononuclear phagocyte system and to a lesser extent endothelial and parenchymal cells; "+/f" = focal involvement (seen in the spleen of SGP #2, the liver and spleen of GP #1, and the lung of GP#4) where rare sites of anti-Ebola antibody staining were detected.; "-" = no Ebola virus antigen detected in tissues. ELISA determinations made prior to viral challenge (Pre) or at least 7 days after (Post) infection, respectively. The surviving NP immunized animal (4) was found to have significant levels of virus in major organs by immunohistochemistry, and more than 5 logs of virus was detected in the serum and spleen, in contrast to GP and sGP animals where no virus was detected.

[0055] Histopathologic analysis of infection in immunized guinea pigs. Pathologic analysis revealed widespread tissue necrosis and dissemination of virus by immunohistochemistry, similar to human disease. Virus load correlated with susceptibility to infection with titers of .gtoreq.10.sup.5 in infected animals compared to undetectable levels in immunized survivors. In infected animals, the liver, lung, and spleen showed evidence of significant viral antigen by immunohistochemistry (FIG. 4, Table 1), and both reticuloendothelial and mononuclear phagotic involvement was observed.

[0056] Determination of antibody response in animals which survived virus challenge revealed increases in the immune response to viral proteins when initial titers were lower (Table 1). Less consistent increases in antibody titers were observed in the NP immunized animals. These data suggest that Ebola virus infection may stimulate immunity in survivors of a viral challenge when immune responses are not optimal.

[0057] II. Methods

[0058] Plasmids. Plasmids containing the GP, sGP, or NP cDNAs (Sanchez, A. et al., Virus. Res. 29:215-240 (1993), Genbank) were used to subclone the relevant inserts into CMV expression vectors which utilized the bovine growth hormone polyadenylation sequence. Manthorpe, M. et al., Hum. Gene. Ther. 4:419-431 (1993). (see FIGS. 5-9 and SEQ ID NOS: 1-4). Briefly, for GP, plasmid pGEM-3Zf(-)-GP was digested with EcoR I, treated with the Klenow fragment of E. coli DNA polymerase, and digested with BamH I. The GP fragment was then inserted into the pCMV expression vector plasmid digested with BamH I, Klenow fragment and Bgl II. For sGP, the plasmid pCRII-sGP was digested with EcoR I, treated with Klenow enzyme, and the resulting fragment inserted into the BamH I/Bgl II CMV plasmid which had been incubated with Klenow fragment, calf intestinal phosphatase (CIP), then phenol chloroform extract. For the NP expression vector, plasmid pSP64-NP2 (Sanchez, A. et al., Virology 170:81-91 (1989)) was digested with EcoR I, treated with Klenow enzyme, and digested with BamH I. The NP insert was cloned into CMV treated with BamH I, Klenow enzyme, followed by heat inactivation and Bgl II digestion.

[0059] Cell lines and transfectants. For stable transfectants, the relevant cDNAs were inserted into a CMV expression plasmid containing a neomycin resistant gene, pCMV-neo (H. Arai, unpublished data), which was digested with Xba I, and treated with CIP and Klenow enzyme. The EcoR I/BamH I GP fragment from pGEM-3Zf(-)-GP, the EcoR I sGP fragment from pCRII-SGP, or the EcoR I/BamH I NP fragment from pSP64-NP2 was treated with Klenow enzyme and ligated to this plasmid backbone. These vectors were transfected into Renca or CT26 which was syngeneic to Balb/C mice using calcium phosphate and selected in 0.7 or 1 mg/ml G418 for 2-6 weeks. Expression of GP, sGP, or NP from these vectors in Renca or CT26 cells was also confirmed by Western blot analysis (data not shown).

[0060] Cell proliferation assay. Spleen cells from male Hartley guinea pigs or Balb/C female mice (8-10 weeks) immunized with the indicated plasmid expression vectors were incubated with sGP or vector control supernatants (25% volume:volume) from transfected 293 cells at the indicated cell concentrations. T cell depletion was performed using the CT5 monoclonal antibody (Tan, B. T. G. et al., Hybridoma 4:115-124 (1985)) (Biosource, Camarillo, Calif.) for guinea pigs or anti-Thy 1.2 antibody in the mouse using immunomagnetic microbeads (Miltenyl Biotec, Inc., Auburn, Calif.).

[0061] Viral challenge in guinea pigs. Animals were immunized by injection of 100 .mu.l (0.5 mg/ml) in each hind leg (two injections at each time point) with the indicated plasmid expression vectors. Animals were challenged by inoculation with a stock of Ebola virus (Zaire, 1976) that had been passaged once in vero E6 cells and serially passaged by intraperitoneal injection of spleen homogenates in Hartley guinea pigs seven times. Immunized guinea pigs were injected intraperitoneally with 0.5 ml of a 1:1,000 dilution of spleen cell homogenate in Hank's balanced salt solution 122 days after the initial plasmid DNA injection (1000 pfu). Survival was determined 10 days later at which times animals were sacrificed for serologic and pathologic analysis. ELISA, enzyme-linked immunosorbent assay (Volchkov, V. E. et al., FEBS. Lett. 305:181-184 (1992); Sanchez, A. et al., Virus. Res. 29:215-240 (1993)) on infected cell supernatants and enriched viral extracts containing GP, sGP, or NP were performed as previously described.

[0062] III. Discussion

[0063] Following the initial report that injection of plasmid DNA into muscle could direct the synthesis of recombinant proteins (Wolff, J. A. et al., Science 247:1465-1468 (1990)), the suggestion was made that this gene transfer approach may be useful for vaccination and was termed genetic immunization. Tang, D. C. et al., Nature 356:152-154 (1992). This approach has been applied to different infectious diseases, including influenza (Ulmer, J. B. et al., Science 259:1745-1749 (1993); Raz, E. et al., PNAS (USA) 91:9519-9523 (1994)), malaria (Doolan, D. L. et al., J. Exp. Med. 183:1739-1746 (1996); Sedegah, M. et al., PNAS(USA) 91:9866-9870 (1994)), and tuberculosis (Tascon, R. C. et al., Nat Med. 2:888-892 (1996)) and has also been used to modulate antibody and cell-mediated immune responses in autoimmune and allergic diseases. Raz, E. et al., PNAS (USA) 90:4523-4527 (1993); Waisman, A. et al., Nat. Med. 2:899-905 (1996); McCormick, J. B. et al., J. Infect. Dis. 147:264-267 (1983); Border, W. A. et al., Nat. Med. 1:1000-1001 (1995).

[0064] The immune response to selected Ebola virus proteins after genetic immunization in mice was analyzed and their ability to protect against lethal infection in a susceptible animal model, the guinea pig, was tested. The immune analyses performed in different species suggest similar patterns of response, though the specific peptides which may be recognized by the immune system to confer protection in the guinea pig could differ from the mouse. Because the principles of MHC antigen presentation and recognition apply broadly across species (Monaco, J. J., Immunol. Today 13:173-179 (1992); Jorgensen, J. L. et al., Annu. Rev. Immunol. 10:835-873 (1992); Zinkemagel, R. M. et al., Immunol. Today 18:14-17 (1997)), the finding that protection was observed in different members of an outbred strain and that similar immune responses were seen in different species is not unexpected and suggests that this approach may be applicable to humans.

[0065] Immunization with plasmids encoding distinct viral proteins induced different antibody and cytolytic T cell responses. The broadest immune response was conferred by GP and sGP, which induced both cellular and humoral immunity to the membrane-associated GP. In guinea pigs challenged with doses of virus that are otherwise lethal, sGP provided nearly equivalent protection to GP, with no significant difference between these groups. The ability of vectors expressing GP to confer immunity may be explained by the generation of lower molecular weight degradation products (FIG. 1B) which could provide sufficient protein for antigen presentation to induce detectable, cellular, and humoral immune responses in guinea pigs.

[0066] Despite the fact that plasmid DNA injection has been shown to affect the immune response to different antigens in infectious and autoimmune diseases, the ability of individual gene products to protect against disease in vivo is not readily predictable. In particular, the rapid rates of Ebola virus replication and the poor immunogenicity of its proteins had previously rendered it resistant to immune interventions. Several attempts to confer protection with passive transfer of immunoglobulin were unsuccessful (Peters, C. J. et al., Filoviridae: Marburg and Ebola Viruses. in Fields Virology. (eds., Fields, B. N., Knipe, D. M. & Howley, P. M.) 1161-1176 (Philadelphia, Lippincott-Raven, 1996); Jahrling, P. B. et al., Arch. Virol. Suppl. 11:135-140 (1996)), in agreement with the finding set forth herein that antisera from protected animals fails to neutralize virus replication in vitro. Previous studies using formalin-fixed virus or purified viral proteins for immunization have also not proven effective. Peters, C. J. et al., Filoviridae: Marburg and Ebola Viruses. in Fields Virology. (eds., Fields, B. N., Knipe, D. M. & Howley, P. M.) 1161-1176 (Philadelphia, Lippincott-Raven, 1996); Clegg, J. C. S. & Sanchez, A. Vaccines against arenaviruses and filoviruses. In New Generation Vaccines. (eds., Levine, M. M., Woodrow, G. C., Kaper, J. B. & Cobon, G. S.) 749-765 (New York, N.Y., Marcel Dekker, Inc. 1997).

[0067] It is likely that traditional immunization approaches using protein antigens, vaccinia virus, or inactivated virus do not allow for appropriate uptake and presentation of viral antigens by dendritic or other antigen-presenting cells to induce protective immune responses. It has been shown recently that genetic immunization leads to production of recombinant protein(s) in muscle which are delivered to bone marrow-derived antigen-presenting cells. Iwasaki, A. et al., J. Immunol. 159:11-14 (1997); Doe, B. et al., PNAS (USA) 93:8578-8583 (1996); Corr, M. et al., J. Exp. Med. 184:1555-1560 (1996). Synthesis of Ebola glycoprotein after gene transfer apparently allows more efficient processing and presentation and the generation of immune responses not seen with virus or with viral vectors. GP is a large molecule which contains both T and B cell epitopes. Although antibody levels provide a surrogate marker of protection, the fact that passive transfer of antibody did not confer protection implies that immunoglobulin switching and synthesis is reflective of the T helper response to GP. Genetic immunization stimulates T helper cells to generate both CTL and B cell antibody responses to the virus. Although antibody production confirms effective immunization, a productive T cell response, likely involving T.sub.H1 cell stimulation, as shown by the T cell proliferation and CTL assays (FIG. 3), is needed for effective immunity. Taken together, these studies suggest that transcription and translation of viral genes in host cells by genetic immunization induces alternative, more effective, processing and antigen presentation which better stimulates immunity to Ebola virus. Since there are yet no effective antiviral agents, the ability to generate protective immunity by vaccination may prove useful in selected high risk populations, particularly in regions of ongoing outbreaks, and among medical and laboratory personnel exposed to the virus. Although it remains important to identify agents which treat acute infection, genetic immunization may help to limit the spread of this highly lethal infectious disease.

[0068] The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying drawings and claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims.

[0069] All references cited herein are incorporated by reference as if fully set forth.

Sequence CWU 1

1

4 1 7003 DNA Artificial Sequence Description of Artificial SequencePlasmid containing DNA forGP of Ebola Virus, Ivory Coast strain 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat 1020 tccccgtgcc aagagtgacg taagtaccgc ctatagactc tataggcaca cccctttggc 1080 tcttatgcat gctatactgt ttttggcttg gggcctatac acccccgctt ccttatgcta 1140 taggtgatgg tatagcttag cctataggtg tgggttattg accattattg accactcccc 1200 tattggtgac gatactttcc attactaatc cataacatgg ctctttgcca caactatctc 1260 tattggctat atgccaatac tctgtccttc agagactgac acggactctg tatttttaca 1320 ggatggggtc ccatttatta tttacaaatt cacatataca acaacgccgt cccccgtgcc 1380 cgcagttttt attaaacata gcgtgggatc tccacgcgaa tctcgggtac gtgttccgga 1440 catgggctct tctccggtag cggcggagct tccacatccg agccctggtc ccatgcctcc 1500 agcggctcat ggtcgctcgg cagctccttg ctcctaacag tggaggccag acttaggcac 1560 agcacaatgc ccaccaccac cagtgtgccg cacaaggccg tggcggtagg gtatgtgtct 1620 gaaaatgagc gtggagattg ggctcgcacg gctgacgcag atggaagact taaggcagcg 1680 gcagaagaag atgcaggcag ctgagttgtt gtattctgat aagagtcaga ggtaactccc 1740 gttgcggtgc tgttaacggt ggagggcagt gtagtctgag cagtactcgt tgctgccgcg 1800 cgcgccacca gacataatag ctgacagact aacagactgt tcctttccat gggtcttttc 1860 tgcagtcacc gtcgtcgaca cgtgtgatca gatatcgcgg ccgcgcggcc gctctagaat 1920 tctctaatca cagtcatcat gggagcgtca gggattctgc aattgccccg tgagcgcttc 1980 aggaaaacat ctttctttgt ttgggtaata atcctattcc ataaagtctt ttcaatcccg 2040 ttgggggttg tacacaacaa taccctacaa gtgagtgata ttgacaagtt tgtgtgccga 2100 gacaaactct cttcaactag ccaattgaag tcagtcgggt tgaacttgga gggcaatgga 2160 gtagcaactg atgtaccaac ggcaaccaaa agatggggtt ttcgagctgg tgttccacca 2220 aaggtggtaa attacgaagc tggagaatgg gctgagaact gttataacct ggctataaag 2280 aaagttgatg gtagtgagtg cctaccagaa gcccctgagg gagtgaggga ttttccccgt 2340 tgccgctatg tacacaaagt ctcaggaact ggaccatgcc caggaggact cgcctttcac 2400 aaagaaggag ccttcttcct gtatgaccga ctcgcatcaa caatcattta tcggggtaca 2460 acctttgccg aaggagttat tgcatttctg atcttgccta aggcgcgaaa ggattttttc 2520 cagtctcctc cattgcatga gcctgccaac atgaccacgg atccctccag ttactatcac 2580 acgacaacaa taaactacgt ggttgataat tttggaacca acaccacaga gtttctgttc 2640 caagtcgatc atttgacgta tgtgcagctc gaggcaagat tcacaccaca attccttgtc 2700 ctcctaaatg aaaccatcta ctctgataac cgcagaagta acacaacagg aaaactaatc 2760 tggaaaataa atcccactgt tgataccagc atgggtgagt gggctttctg ggaaaataaa 2820 aaaacttcac aaaaaccctt tcaagtgaag agttgtcttt cgtacctgta ccagaaaccc 2880 agaaccaggt ccttgacacg acagcgacgg tctctcctcc catctccgcc cacaaccacg 2940 caggcgaaga ccacaaagaa ttggtttcag aggattccac tccagtggtt cagatgcaaa 3000 acatcaaggg aaaggacaca atgccaacca cagtgacggg tgtaccaaca accacaccct 3060 ctccatttcc aatcaatgct cgcaacactg atcataccaa atcatttatc ggcctggagg 3120 ggccccaaga agaccacagc accacacagc ctgccaagac caccagccaa ccaaccaaca 3180 gcacagaatc gacgacacta aacccaacat cagagccctc cagtagaggc acgggaccat 3240 ccagccccac ggtccccaac accacagaaa gccacgccga acttggcaag acaaccccaa 3300 ccacactccc agaacagcac actgccgcca gtgccattcc aagagccgtg caccccgacg 3360 aactcagtgg acctggcttc ctgacgaaca caatacgggg ggtgacaaat ctcctgacag 3420 gatccagaag aaagcgaagg gatgtcactc ccaatacaca acccaaatgc aacccaaacc 3480 tgcactattg gacagccttg gatgagggtg ctgccatagg tttagcctgg ataccatact 3540 tcgggccagc agctgaggga atttacactg aaggcataat ggagaatcaa aatggattga 3600 tctgtggatt gaggcagctg gccaacgaaa cgacacaagc tcttcaattg ttcttaaggg 3660 caactactga gttgcgtaca ttctctatac taaatcggaa agcaatagac ttcttgctcc 3720 aaagatgggg aggaacatgt cacattctag ggcctgattg ttgcattgaa ccccaagatt 3780 ggaccaaaaa tatcactgat aaaattgatc aaataatcca tgactttgtc gataataatc 3840 ttccaaatca gaatgatggc agcaactggt ggactggatg gaaacaatgg gttcctgctg 3900 gaataggaat cacaggagta atcattgcta ttattgcttt gctgtgcatt tgcaaattca 3960 tgctttgaac taatatagca tcatacttta gaattctaga ccaggcgcct ggatccagat 4020 ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 4080 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 4140 gtctgagtag gtgtcattct attctggggg gtggggtggg gcagcacagc aagggggagg 4200 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgggt acccaggtgc 4260 tgaagaattg acccggttcc tcctgggcca gaaagaagca ggcacatccc cttctctgtg 4320 acacaccctg tccacgcccc tggttcttag ttccagcccc actcatagga cactcatagc 4380 tcaggagggc tccgccttca atcccacccg ctaaagtact tggagcggtc tctccctccc 4440 tcatcagccc accaaaccaa acctagcctc caagagtggg aagaaattaa agcaagatag 4500 gctattaagt gcagagggag agaaaatgcc tccaacatgt gaggaagtaa tgagagaaat 4560 catagaattt cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 4620 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 4680 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4740 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 4800 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 4860 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 4920 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag 4980 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 5040 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 5100 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 5160 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 5220 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 5280 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 5340 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 5400 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 5460 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 5520 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 5580 ctccgggggg ggggggcgct gaggtctgcc tcgtgaagaa ggtgttgctg actcatacca 5640 ggcctgaatc gccccatcat ccagccagaa agtgagggag ccacggttga tgagagcttt 5700 gttgtaggtg gaccagttgg tgattttgaa cttttgcttt gccacggaac ggtctgcgtt 5760 gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa gttcgattta ttcaacaaag 5820 ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt acaaccaatt aaccaattct 5880 gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca 5940 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc 6000 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa 6060 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg 6120 actgaatccg gtgagaatgg caaaagctta tgcatttctt tccagacttg ttcaacaggc 6180 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat 6240 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc 6300 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga 6360 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca 6420 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag 6480 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga 6540 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg 6600 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc 6660 ggcctcgagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt 6720 atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc aatgtaacat 6780 cagagatttt gagacacaac gtggctttcc cccccccccc attattgaag catttatcag 6840 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 6900 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg 6960 acattaacct ataaaaatag gcgtatcacg aggccctttc gtc 7003 2 7073 DNA Artificial Sequence Description of Artificial SequencePlasmid containing DNA for GP of Ebola Virus, Sudan strain 2 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat 1020 tccccgtgcc aagagtgacg taagtaccgc ctatagactc tataggcaca cccctttggc 1080 tcttatgcat gctatactgt ttttggcttg gggcctatac acccccgctt ccttatgcta 1140 taggtgatgg tatagcttag cctataggtg tgggttattg accattattg accactcccc 1200 tattggtgac gatactttcc attactaatc cataacatgg ctctttgcca caactatctc 1260 tattggctat atgccaatac tctgtccttc agagactgac acggactctg tatttttaca 1320 ggatggggtc ccatttatta tttacaaatt cacatataca acaacgccgt cccccgtgcc 1380 cgcagttttt attaaacata gcgtgggatc tccacgcgaa tctcgggtac gtgttccgga 1440 catgggctct tctccggtag cggcggagct tccacatccg agccctggtc ccatgcctcc 1500 agcggctcat ggtcgctcgg cagctccttg ctcctaacag tggaggccag acttaggcac 1560 agcacaatgc ccaccaccac cagtgtgccg cacaaggccg tggcggtagg gtatgtgtct 1620 gaaaatgagc gtggagattg ggctcgcacg gctgacgcag atggaagact taaggcagcg 1680 gcagaagaag atgcaggcag ctgagttgtt gtattctgat aagagtcaga ggtaactccc 1740 gttgcggtgc tgttaacggt ggagggcagt gtagtctgag cagtactcgt tgctgccgcg 1800 cgcgccacca gacataatag ctgacagact aacagactgt tcctttccat gggtcttttc 1860 tgcagtcacc gtcgtcgaca cgtgtgatca gatatcgcgg ccgctctagc tagatgcatg 1920 ctcgagcggc cgccagtgtg atggatatct gcagaattct atcttcagga tctcgccatg 1980 gagggtctta gcctactcca attgcccaga gataaatttc gaaaaagctc tttctttgtt 2040 tgggtcatca tcttatttca aaaggccttt tccatgcctt tgggtgttgt gaccaacagc 2100 actttagaag taacagagat tgaccagcta gtctgcaagg atcatcttgc atcaactgac 2160 cagctgaaat cagttggtct caacctcgag gggagcggag tatctactga tatcccatct 2220 gcgacaaagc gttggggctt cagatctggt gtgcctcccc aagtggtcag ctatgaagca 2280 ggagaatggg ctgaaaattg ctacaatctt gaaataaaga aaccggacgg gagcgaatgc 2340 ttacccccac cgccggatgg tgtcagaggc tttccaaggt gccgctatgt tcacaaagcc 2400 caaggaaccg ggccctgccc gggtgactat gcctttcaca aggatggagc tttcttcctc 2460 tatgacaggc tggcttcaac tgtaatttac agaggagtca attttgctga gggggtaatc 2520 gcattcttga tattggctaa accaaaggaa acgttccttc aatcaccccc cattcgagag 2580 gcagcaaact acactgaaaa tacatcaagt tactatgcca catcctactt ggagtacgaa 2640 atcgaaaatt ttggtgctca acactccacg acccttttca aaattaacaa taatactttt 2700 gttcttctgg acaggcccca cacgcctcag ttccttttcc agctgaatga taccattcaa 2760 cttcaccaac agttgagcaa cacaactggg aaactaattt ggacactaga tgctaatatc 2820 aatgctgata ttggtgaatg ggctttttgg gaaaataaaa aaatctctcc gaacaactac 2880 gtggagaaga gctgtctttc gaaactttat cgctcaacga gacagaagac gatgatgcga 2940 catcgtcgag aactacaaag ggaagaatct ccgaccgggc caccaggaag tattcggacc 3000 tggttccaaa ggattcccct gggatggttt cattgcacgt accagaaggg gaaacaacat 3060 tgccgtctca gaattcgaca gaaggtcgaa gagtagatgt gaatactcag gaaactatca 3120 cagagacaac tgcaacaatc ataggcacta acggtaacaa catgcagatc tccaccatcg 3180 ggacaggact gagctccagc caaatcctga gttcctcacc gaccatggca ccaagccctg 3240 agactcagac ctccacaacc tacacaccaa aactaccagt gatgaccacc gaggaaccaa 3300 caacaccacc gagaaactct cctggctcaa caacagaagc acccactctc accaccccag 3360 agaatataac aacagcggtt aaaactgttt gggcacaaga gtccacaagc aacggtctaa 3420 taacttcaac agtaacaggt attcttggga gccttggact tcgaaaacgc agcagaagac 3480 aagttaacac cagggccacg ggtaaatgca atcccaactt acactactgg actgcacaag 3540 aacaacataa tgctgctggg attgcctgga tcccgtactt tggaccgggt gcagaaggca 3600 tatacactga aggccttatg cacaaccaaa atgccttagt ctgtggactc agacaacttg 3660 caaatgaaac aactcaagct ctgcagcttt tcttaagggc cacgacggag ctgcggacat 3720 ataccatact caataggaag gccatagatt tccttctgcg acgatggggc gggacatgta 3780 ggatcctggg accagattgt tgcattgagc cacatgattg gaccaaaaac atcactgata 3840 aaatcaacca aatcatccat gatttcatcg acaacccttt acccaatcag gataatgatg 3900 ataattggtg gacgggctgg agacagtgga tccctgcagg aataggcatt actggaatta 3960 ttattgcaat cattgctctt ctttgcgtct gcaagctgct ttgttgaata tcagaattcc 4020 agcactggcg gccgttacta gtggatccga gctcggatcc aagctctaga ccaggcgcct 4080 ggatccagat ctgctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 4140 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 4200 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcagcacagc 4260 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgggt 4320 acccaggtgc tgaagaattg acccggttcc tcctgggcca gaaagaagca ggcacatccc 4380 cttctctgtg acacaccctg tccacgcccc tggttcttag ttccagcccc actcatagga 4440 cactcatagc tcaggagggc tccgccttca atcccacccg ctaaagtact tggagcggtc 4500 tctccctccc tcatcagccc accaaaccaa acctagcctc caagagtggg aagaaattaa 4560 agcaagatag gctattaagt gcagagggag agaaaatgcc tccaacatgt gaggaagtaa 4620 tgagagaaat catagaattt cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 4680 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 4740 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 4800 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 4860 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 4920 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 4980 cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag 5040 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 5100 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 5160 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 5220 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg 5280 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 5340 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 5400 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 5460 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 5520 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 5580 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 5640 agttgcctga ctccgggggg ggggggcgct gaggtctgcc tcgtgaagaa ggtgttgctg 5700 actcatacca ggcctgaatc gccccatcat ccagccagaa agtgagggag ccacggttga 5760 tgagagcttt gttgtaggtg gaccagttgg tgattttgaa cttttgcttt gccacggaac 5820 ggtctgcgtt gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa gttcgattta 5880 ttcaacaaag ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt acaaccaatt 5940 aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc 6000 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc 6060 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac 6120 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc 6180 atgagtgacg actgaatccg gtgagaatgg caaaagctta tgcatttctt tccagacttg 6240 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt 6300 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca 6360 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc 6420 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag 6480 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc 6540 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc 6600 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc 6660 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga 6720 atttaatcgc ggcctcgagc aagacgtttc ccgttgaata tggctcataa caccccttgt 6780 attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc 6840 aatgtaacat cagagatttt gagacacaac gtggctttcc cccccccccc attattgaag 6900 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 6960 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat 7020 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtc 7073 3 7285 DNA Artificial Sequence Description of Artificial SequencePlasmid containing DNA of GP of Ebola Virus, Zaire strain 3 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa

tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat 1020 tccccgtgcc aagagtgacg taagtaccgc ctatagactc tataggcaca cccctttggc 1080 tcttatgcat gctatactgt ttttggcttg gggcctatac acccccgctt ccttatgcta 1140 taggtgatgg tatagcttag cctataggtg tgggttattg accattattg accactcccc 1200 tattggtgac gatactttcc attactaatc cataacatgg ctctttgcca caactatctc 1260 tattggctat atgccaatac tctgtccttc agagactgac acggactctg tatttttaca 1320 ggatggggtc ccatttatta tttacaaatt cacatataca acaacgccgt cccccgtgcc 1380 cgcagttttt attaaacata gcgtgggatc tccacgcgaa tctcgggtac gtgttccgga 1440 catgggctct tctccggtag cggcggagct tccacatccg agccctggtc ccatgcctcc 1500 agcggctcat ggtcgctcgg cagctccttg ctcctaacag tggaggccag acttaggcac 1560 agcacaatgc ccaccaccac cagtgtgccg cacaaggccg tggcggtagg gtatgtgtct 1620 gaaaatgagc gtggagattg ggctcgcacg gctgacgcag atggaagact taaggcagcg 1680 gcagaagaag atgcaggcag ctgagttgtt gtattctgat aagagtcaga ggtaactccc 1740 gttgcggtgc tgttaacggt ggagggcagt gtagtctgag cagtactcgt tgctgccgcg 1800 cgcgccacca gacataatag ctgacagact aacagactgt tcctttccat gggtcttttc 1860 tgcagtcacc gtcgtcgaca cgtgtgatca gatatcgcgg ccgctctaga ccaggcgcct 1920 ggatcgatcc gcgatgaaga ttaagccgac agtgagcgta atcttcatct ctcttagatt 1980 atttgttttc cagagtaggg gtcgtcaggt ccttttcaat cgtgtaacca aaataaactc 2040 cactagaagg atattgtggg gcaacaacac aatgggcgtt acaggaatat tgcagttacc 2100 tcgtgatcga ttcaagagga catcattctt tctttgggta attatccttt tccaaagaac 2160 attttccatc ccacttggag tcatccacaa tagcacatta caggttagtg atgtcgacaa 2220 actagtttgt cgtgacaaac tgtcatccac aaatcaattg agatcagttg gactgaatct 2280 cgaagggaat ggagtggcaa ctgacgtgcc atctgcaact aaaagatggg gcttcaggtc 2340 cggtgtccca ccaaaggtgg tcaattatga agctggtgaa tgggctgaaa actgctacaa 2400 tcttgaaatc aaaaaacctg acgggagtga gtgtctacca gcagcgccag acgggattcg 2460 gggcttcccc cggtgccggt atgtgcacaa agtatcagga acgggaccgt gtgccggaga 2520 ctttgccttc cataaagagg gtgctttctt cctgtatgat cgacttgctt ccacagttat 2580 ctaccgagga acgactttcg ctgaaggtgt cgttgcattt ctgatactgc cccaagctaa 2640 gaaggacttc ttcagctcac accccttgag agagccggtc aatgcaacgg aggacccgtc 2700 tagtggctac tattctacca caattagata tcaggctacc ggttttggaa ccaatgagac 2760 agagtacttg ttcgaggttg acaatttgac ctacgtccaa cttgaatcaa gattcacacc 2820 acagtttctg ctccagctga atgagacaat atatacaagt gggaaaagga gcaataccac 2880 gggaaaacta atttggaagg tcaaccccga aattgataca acaatcgggg agtgggcctt 2940 ctgggaaact aaaaaaaacc tcactagaaa aattcgcagt gaagagttgt ctttcacagt 3000 tgtatcaaac ggagccaaaa acatcagtgg tcagagtccg gcgcgaactt cttccgaccc 3060 agggaccaac acaacaactg aagaccacaa aatcatggct tcagaaaatt cctctgcaat 3120 ggttcaagtg cacagtcaag gaagggaagc tgcagtgtcg catctaacaa cccttgccac 3180 aatctccacg agtccccaat ccctcacaac caaaccaggt ccggacaaca gcacccataa 3240 tacacccgtg tataaacttg acatctctga ggcaactcaa gttgaacaac atcaccgcag 3300 aacagacaac gacagcacag cctccgacac tccctctgcc acgaccgcag ccggaccccc 3360 aaaagcagag aacaccaaca cgagcaagag cactgacttc ctggaccccg ccaccacaac 3420 aagtccccaa aaccacagcg agaccgctgg caacaacaac actcatcacc aagataccgg 3480 agaagagagt gccagcagcg ggaagctagg cttaattacc aatactattg ctggagtcgc 3540 aggactgatc acaggcggga gaagaactcg aagagaagca attgtcaatg ctcaacccaa 3600 atgcaaccct aatttacatt actggactac tcaggatgaa ggtgctgcaa tcggactggc 3660 ctggatacca tatttcgggc cagcagccga gggaatttac atagaggggc taatgcacaa 3720 tcaagatggt ttaatctgtg ggttgagaca gctggccaac gagacgactc aagctcttca 3780 actgttcctg agagccacaa ctgagctacg caccttttca atcctcaacc gtaaggcaat 3840 tgatttcttg ctgcagcgat ggggcggcac atgccacatt ctgggaccgg actgctgtat 3900 cgaaccacat gattggacca agaacataac agacaaaatt gatcagatta ttcatgattt 3960 tgttgataaa acccttccgg accaggggga caatgacaat tggtggacag gatggagaca 4020 atggataccg gcaggtattg gagttacagg cgttataatt gcagttatcg ctttattctg 4080 tatatgcaaa tttgtctttt agtttttctt cagattgctt catggaaaag ctcagcctca 4140 aatcaatgaa accaggattt aattatatgg attacttgaa tctaagatta cttgacaaat 4200 gataatataa tacactggag ctttaaacat agccaatgtg attctaactc ctttaaactc 4260 acagttaatc ataaacaagg tttggtaccg agctcgaatt atctgctgtg ccttctagtt 4320 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 4380 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 4440 ctattctggg gggtggggtg gggcagcaca gcaaggggga ggattgggaa gacaatagca 4500 ggcatgctgg ggatgcggtg ggctctatgg gtacccaggt gctgaagaat tgacccggtt 4560 cctcctgggc cagaaagaag caggcacatc cccttctctg tgacacaccc tgtccacgcc 4620 cctggttctt agttccagcc ccactcatag gacactcata gctcaggagg gctccgcctt 4680 caatcccacc cgctaaagta cttggagcgg tctctccctc cctcatcagc ccaccaaacc 4740 aaacctagcc tccaagagtg ggaagaaatt aaagcaagat aggctattaa gtgcagaggg 4800 agagaaaatg cctccaacat gtgaggaagt aatgagagaa atcatagaat ttcttccgct 4860 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 4920 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 4980 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 5040 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 5100 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 5160 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 5220 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 5280 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 5340 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 5400 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 5460 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 5520 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 5580 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 5640 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 5700 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 5760 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 5820 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccgggg ggggggggcg 5880 ctgaggtctg cctcgtgaag aaggtgttgc tgactcatac caggcctgaa tcgccccatc 5940 atccagccag aaagtgaggg agccacggtt gatgagagct ttgttgtagg tggaccagtt 6000 ggtgattttg aacttttgct ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat 6060 ctgatccttc aactcagcaa aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc 6120 agcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg 6180 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 6240 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 6300 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 6360 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 6420 ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 6480 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 6540 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg 6600 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 6660 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata 6720 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 6780 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 6840 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 6900 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt 6960 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt 7020 attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca 7080 acgtggcttt cccccccccc ccattattga agcatttatc agggttattg tctcatgagc 7140 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 7200 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 7260 aggcgtatca cgaggccctt tcgtc 7285 4 7272 DNA Artificial Sequence Description of Artificial SequencePlasmid containing DNA for soluble GP of Ebola Virus, Zaire strain 4 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240 ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300 tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960 tagaagacac cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat 1020 tccccgtgcc aagagtgacg taagtaccgc ctatagactc tataggcaca cccctttggc 1080 tcttatgcat gctatactgt ttttggcttg gggcctatac acccccgctt ccttatgcta 1140 taggtgatgg tatagcttag cctataggtg tgggttattg accattattg accactcccc 1200 tattggtgac gatactttcc attactaatc cataacatgg ctctttgcca caactatctc 1260 tattggctat atgccaatac tctgtccttc agagactgac acggactctg tatttttaca 1320 ggatggggtc ccatttatta tttacaaatt cacatataca acaacgccgt cccccgtgcc 1380 cgcagttttt attaaacata gcgtgggatc tccacgcgaa tctcgggtac gtgttccgga 1440 catgggctct tctccggtag cggcggagct tccacatccg agccctggtc ccatgcctcc 1500 agcggctcat ggtcgctcgg cagctccttg ctcctaacag tggaggccag acttaggcac 1560 agcacaatgc ccaccaccac cagtgtgccg cacaaggccg tggcggtagg gtatgtgtct 1620 gaaaatgagc gtggagattg ggctcgcacg gctgacgcag atggaagact taaggcagcg 1680 gcagaagaag atgcaggcag ctgagttgtt gtattctgat aagagtcaga ggtaactccc 1740 gttgcggtgc tgttaacggt ggagggcagt gtagtctgag cagtactcgt tgctgccgcg 1800 cgcgccacca gacataatag ctgacagact aacagactgt tcctttccat gggtcttttc 1860 tgcagtcacc gtcgtcgaca cgtgtgatca gatatcgcgg ccgctctaga ccaggcgcct 1920 ggatcgaatt gatgaagatt aagccgacag tgagcgtaat cttcatctct cttagattat 1980 ttgttttcca gagtaggggt cgtcaggtcc ttttcaatcg tgtaaccaaa ataaactcca 2040 ctagaaggat attgtggggc aacaacacaa tgggcgttac aggaatattg cagttacctc 2100 gtgatcgatt caagaggaca tcattctttc tttgggtaat tatccttttc caaagaacat 2160 tttccatccc acttggagtc atccacaata gcacattaca ggttagtgat gtcgacaaac 2220 tagtttgtcg tgacaaactg tcatccacaa atcaattgag atcagttgga ctgaatctcg 2280 aagggaatgg agtggcaact gacgtgccat ctgcaactaa aagatggggc ttcaggtccg 2340 gtgtcccacc aaaggtggtc aattatgaag ctggtgaatg ggctgaaaac tgctacaatc 2400 ttgaaatcaa aaaacctgac gggagtgagt gtctaccagc agcgccagac gggattcggg 2460 gcttcccccg gtgccggtat gtgcacaaag tatcaggaac gggaccgtgt gccggagact 2520 ttgccttcca taaagagggt gctttcttcc tgtatgatcg acttgcttcc acagttatct 2580 accgaggaac gactttcgct gaaggtgtcg ttgcatttct gatactgccc caagctaaga 2640 aggacttctt cagctcacac cccttgagag agccggtcaa tgcaacggag gacccgtcta 2700 gtggctacta ttctaccaca attagatatc aggctaccgg ttttggaacc aatgagacag 2760 agtacttgtt cgaggttgac aatttgacct acgtccaact tgaatcaaga ttcacaccac 2820 agtttctgct ccagctgaat gagacaatat atacaagtgg gaaaaggagc aataccacgg 2880 gaaaactaat ttggaaggtc aaccccgaaa ttgatacaac aatcggggag tgggccttct 2940 gggaaactaa aaaaacctca ctagaaaaat tcgcagtgaa gagttgtctt tcacagttgt 3000 atcaaacgga gccaaaaaca tcagtggtca gagtccggcg cgaacttctt ccgacccagg 3060 gaccaacaca acaactgaag accacaaaat catggcttca gaaaattcct ctgcaatggt 3120 tcaagtgcac agtcaaggaa gggaagctgc agtgtcgcat ctaacaaccc ttgccacaat 3180 ctccacgagt ccccaatccc tcacaaccaa accaggtccg gacaacagca cccataatac 3240 acccgtgtat aaacttgaca tctctgaggc aactcaagtt gaacaacatc accgcagaac 3300 agacaacgac agcacagcct ccgacactcc ctctgccacg accgcagccg gacccccaaa 3360 agcagagaac accaacacga gcaagagcac tgacttcctg gaccccgcca ccacaacaag 3420 tccccaaaac cacagcgaga ccgctggcaa caacaacact catcaccaag ataccggaga 3480 agagagtgcc agcagcggga agctaggctt aattaccaat actattgctg gagtcgcagg 3540 actgatcaca ggcgggagaa gaactcgaag agaagcaatt gtcaatgctc aacccaaatg 3600 caaccctaat ttacattact ggactactca ggatgaaggt gctgcaatcg gactggcctg 3660 gataccatat ttcgggccag cagccgaggg aatttacata gaggggctaa tgcacaatca 3720 agatggttta atctgtgggt tgagacagct ggccaacgag acgactcaag ctcttcaact 3780 gttcctgaga gccacaactg agctacgcac cttttcaatc ctcaaccgta aggcaattga 3840 tttcttgctg cagcgatggg gcggcacatg ccacattctg ggaccggact gctgtatcga 3900 accacatgat tggaccaaga acataacaga caaaattgat cagattattc atgattttgt 3960 tgataaaacc cttccggacc agggggacaa tgacaattgg tggacaggat ggagacaatg 4020 gataccggca ggtattggag ttacaggcgt tataattgca gttatcgctt tattctgtat 4080 atgcaaattt gtcttttagt ttttcttcag attgcttcat ggaaaagctc agcctcaaat 4140 caatgaaacc aggatttaat tatatggatt acttgaatct aagattactt gacaaatgat 4200 aatataatac actggagctt taaacatagc caatgtgatt ctaactcctt taaactcaca 4260 gttaatcata aacaaggttt ggaattgatc tgctgtgcct tctagttgcc agccatctgt 4320 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 4380 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 4440 tggggtgggg cagcacagca agggggagga ttgggaagac aatagcaggc atgctgggga 4500 tgcggtgggc tctatgggta cccaggtgct gaagaattga cccggttcct cctgggccag 4560 aaagaagcag gcacatcccc ttctctgtga cacaccctgt ccacgcccct ggttcttagt 4620 tccagcccca ctcataggac actcatagct caggagggct ccgccttcaa tcccacccgc 4680 taaagtactt ggagcggtct ctccctccct catcagccca ccaaaccaaa cctagcctcc 4740 aagagtggga agaaattaaa gcaagatagg ctattaagtg cagagggaga gaaaatgcct 4800 ccaacatgtg aggaagtaat gagagaaatc atagaatttc ttccgcttcc tcgctcactg 4860 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 4920 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 4980 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5040 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5100 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5160 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct 5220 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5280 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 5340 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 5400 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 5460 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 5520 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 5580 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 5640 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 5700 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 5760 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 5820 gtctatttcg ttcatccata gttgcctgac tccggggggg gggggcgctg aggtctgcct 5880 cgtgaagaag gtgttgctga ctcataccag gcctgaatcg ccccatcatc cagccagaaa 5940 gtgagggagc cacggttgat gagagctttg ttgtaggtgg accagttggt gattttgaac 6000 ttttgctttg ccacggaacg gtctgcgttg tcgggaagat gcgtgatctg atccttcaac 6060 tcagcaaaag ttcgatttat tcaacaaagc cgccgtcccg tcaagtcagc gtaatgctct 6120 gccagtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa 6180 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta 6240 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg 6300 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt 6360 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat 6420 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg 6480 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc 6540 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg 6600 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc 6660 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg 6720 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat 6780 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca 6840 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata 6900 aatcagcatc catgttggaa tttaatcgcg gcctcgagca agacgtttcc cgttgaatat 6960 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg 7020 atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg tggctttccc 7080 ccccccccca ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 7140 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 7200 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 7260 ggccctttcg tc 7272

Claims

1. (canceled)

2. (canceled)

3. The pharmaceutical composition of claim 8, wherein the control sequence is a promoter.

4. The pharmaceutical composition of claim 3, wherein the promoter is the CMV immediate-early region 1 promoter.

5. The pharmaceutical composition of claim 8, further comprising an adjuvant.

6. (canceled)

7. (canceled)

8. A pharmaceutical composition comprising a nucleic acid molecule encoding an Ebola virus structural gene product operatively-linked to a heterologous control sequence, in a pharmaceutically acceptable carrier, wherein the Ebola virus structural gene product is virus nucleoprotein.

9. A method of producing a vaccine against disease caused by infection by Ebola virus, comprising the steps of: a) administering the pharmaceutical composition of claim 8 to a test host to determine an amount and a frequency of administration thereof to elicit a protective immune response in said host; and b) formulating said pharmaceutical composition in a form suitable for administration to a treatable host in accordance with said determined amount and frequency of administration.

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. A vaccine comprising a nucleic acid molecule encoding the Ebola virus nucleoprotein operatively-linked to a heterologous control sequence, in a pharmaceutically acceptable carrier.

19. The vaccine of claim 18, wherein the control sequence is a promoter.

20. The vaccine of claim 19, wherein the promoter is the CMV immediate-early region 1 promoter.

21. The vaccine of claim 18, further comprising an adjuvant.

22. (canceled)

23. A method of immunizing a subject against hemorrhagic fever comprising the step of administering to the host an immunoeffective amount of the vaccine of any of claims 18 to 21, wherein the hemorrhagic fever is caused by infection with Ebola virus.

24. (canceled)

25. The method of claim 23, wherein the host is a human and administration is by intramuscular injection.

26. The method of claim 23, wherein the subject receives a second administration of an immunoeffective amount of a vaccine against disease caused by infection by Ebola virus.