Patent 6,852,324

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 of the Invention

FIELD OF THE INVENTION

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

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., Filoviridae: 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).

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

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.

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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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).

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.

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.

As referred to herein, the tern "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.

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_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 5xSSC, at 65^oC. in aqueous solution or 42^oC. in formamide, followed by washing with 0.1xSSC, at 65^oC. 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)).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The vaccines of the present invention are also suitable for veterinary immunization. The vaccines of the present invention comprising nucleic add 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.

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.

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.

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. 2:885-892 (1996)).

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.

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.

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.

Claim 1 of 12 Claims

We claim:

1. 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 the transmembrane form of the Ebola virus glycoprotein

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