The present invention provides a safe and effective vaccine composition against West Nile virus disease. An immunogenically active component of West Nile virus or plasmid DNA, an adjuvant such as a metabolizable oil, and a pharmacologically acceptable carrier are formulated into an immunizing vaccine. The invention also provides a method for the prevention or amelioration of West Nile disease, such as encephalitis, in equidae by administering the vaccine composition herein set forth.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to safe and effective West Nile Virus vaccines, and to methods of administering same to mammals, in particular horses.

BACKGROUND OF THE INVENTION

Known as a Flavivirus, the West Nile virus was first identified in 1937 in Africa and first found in North America in 1999. Migratory birds are considered the primary means whereby infection is spread within and between countries. The virus is transmitted by mosquitoes that have acquired infection by feeding on viremic birds. The virus is then amplified during periods of adult mosquito blood-feeding. Infected mosquitos then transmit the virus to humans and animals upon feeding thereon.

West Nile virus is the causative agent for West Nile Virus disease, particularly West Nile encephalitis, predominately in humans, other mammals and birds. The chief concern in both the United States and foreign countries is the lack of effective treatment for West Nile virus disease. Anti-inflammatory drugs are used to combat swelling of central nervous system tissues, but beyond that no medical intervention is available. Nor is there believed to be a suitable vaccine known to prevent the infection. To date, preventing contact with carriers appears to be the only means of controlling the West Nile virus.

What is therefore needed in the art is to provide a safe and effective equine West Nile virus vaccine composition. The vaccine composition should be sufficiently safened so as to be suitable for administration even to pregnant mares without adverse effect.

Also needed is a method for the prevention or amelioration of West Nile Virus disease, particularly West Nile encephalitis, in equidae and other mammals.

SUMMARY OF THE INVENTION

The present invention provides a safened vaccine composition which comprises: an effective immunizing amount of an immunogenically active component selected from the group consisting of an inactivated whole or subunit West Nile virus, an antigen derived from said virus, DNA derived from said virus, and a mixture thereof; an immunogenically stimulating amount of a metabolizable oil; and a pharmacologically acceptable carrier.

The present invention also provides a method for the prevention or amelioration of West Nile encephalitis in equidae which comprises administering to said equidae a safened vaccine composition which comprises an effective immunizing amount of an immunogenically active component selected from the group consisting of an inactivated whole or subunit West Nile virus, an antigen derived from said virus, DNA derived from said virus, and a mixture thereof; an immunogenically stimulating amount of a metabolizable oil; and a pharmacologically acceptable carrier.

Also provided as part of the invention is a safe and effective West Nile Virus vaccine suitable for use in horses which comprises an immunogenically active component selected from the group consisting of an inactivated whole or subunit West Nile virus, an antigen derived from said virus, DNA derived from said virus, and a mixture thereof; an immunogenically stimulating amount of a metabolizable oil; and a pharmacologically acceptable carrier.

In a further embodiment, the invention describes a vaccine composition comprising at least about at least about 1.times.10.sup.4 TCID.sub.50 per unit dose of inactivated West Nile virus, or a component thereof, and about 4% to 10% vol/vol of a metabolizable oil comprising about 1 to 3% polyoxyethylene-polyoxypropylene block copolymer, about 2 to 6% of squalane and about 0.1 to 0.5% of polyoxyethylene sorbitan monooleate.

Further provided is a safened and effective West Nile virus vaccine for equidae, comprising at least about 1.times.10.sup.6 TCID.sub.50 per unit dose of killed or inactivated West Nile virus, and at least about 1% vol/vol of an adjuvant comprising at least one metabolizable oil and at least one wetting or dispersing agent.

The invention also sets forth a vaccine regimen for horses, comprising two dosage units of killed or inactivated West Nile virus, wherein each said dosage unit comprises about 0.5 to 5 milliliters of a composition containing at least about 5.times.10.sup.7 TCID.sub.50 of said virus and about 1 to 10% vol/vol of an adjuvant, said adjuvant comprising at least one metabolizable oil and at least two nonionic surfactants, and further wherein said dosage unit comprises a pharmacologically acceptable carrier.

Also provided as part of the invention is an equine vaccine containing West Nile plasmid DNA in an amount of about 50 to 3,000 micrograms per dose, together with one or more adjuvants and a suitable carrier. A vaccine regimen would comprise administering at least about one dose of this composition, and desirably at least about 2 doses, to horses for immunization against West Nile virus disease.

Further, objects and features of the invention will become apparent from the detailed description and the claims set forth herein below.

DETAILED DESCRIPTION OF THE INVENTION

Scientists believe that the West Nile virus follows the same pattern of infection found with other mosquito-transmitted viruses. When an infected mosquito bites an equid, the virus enters the skin or tissues just below the bite site, where it is picked up by the circulation. The virus can multiply in the bloodstream, and the equid may develop a fever, which often goes undetected because there are no other signs of illness at the time. However, once the virus has invaded the nervous system, clinical signs appear within one to three days. Most affected equidae, such as horses, first exhibit signs of posterior weakness or paralysis and poor coordination. Depression and related behavior changes may accompany the physical changes. In severe cases, tremors, convulsions, paddling of the limbs and paralysis may develop. Severe neurological problems and mortality have also been observed. To date, no vaccine known to prevent West Nile virus in equidae is available; and the only means of controlling the West Nile virus appears to be the prevention of contact with a carrier.

It has now been found that a safe and effective vaccine composition which comprises an effective immunizing amount of an immunogenically active component selected from the group consisting of an inactivated whole or subunit West Nile virus, an antigen derived from said virus, DNA derived from said virus, plasmid West Nile virus DNA, plasmid with sequence inserts of said virus, and a mixture thereof; an immunogenically stimulating amount of an adjuvant, in particular a metabolizable oil; and a pharmacologically acceptable carrier may be administered to equidae, particularly horses, to prevent or ameliorate West Nile Virus disease such as encephalitis.

DNA derived from the West Nile virus may be obtained via isolation from sources such as the fluids or tissues of equine or avian species diagnosed to have West Nile encephalitis. Such sources include cerebral spinal fluid or sections of spinal cord or brain. DNA may also be obtained using other available techniques such as plasmid technology. For example, suitable cells of an organism, e.g. E. coli, may be transformed with a plasmid containing West Nile protein sequence inserts to obtain a master seed. The master seed may then be cultured and passaged. Transformed cells containing the West Nile DNA may then be harvested, and the DNA isolated and obtained using techniques available to the skilled artisan.

Whole or subunit West Nile virus may be isolated from infected animals using conventional techniques. Samples of the virus may also be obtained from tissue culture collections which maintain a depository for organisms such as West Nile. At the American Type Culture Collection (ATCC), for example, the West Nile virus has been deposited under ATCC No.s VR-82, VR-1267 and VR-1267 AF.

Whole or subunit West Nile virus may be inactivated by conventional inactivating means, for example chemical inactivation using chemical inactivating agents such as binary ethyleneimine, beta-propiolactone, formalin, gluteraldehyde, sodium dodecyl sulfate, or the like or a mixture thereof, preferably formalin. Said virus may also be inactivated by heat or psoralen in the presence of ultraviolet light. (Live, attenuated West Nile virus may also be used in certain embodiments, but this is perhaps much less preferred.)

Whole or subunit West Nile virus may be maintained or grown in mouse brains or in suitable tissue culture media, such as optiMEM (LTI, Grand Island, N.Y.) or MEM media, or in cells known in the art such as African green monkey kidney (Vero) cells or baby hamster (BHK) cells, preferably Vero cells. Said virus may then be separated from the tissue culture or cell media using conventional techniques such as centrifugation, filtration or the like.

A preferred West Nile virus isolate may be obtained from the National Veterinary Services Laboratory (part of the United States Department of Agriculture) in Ames, Iowa as strain VM-2. The virus strain may be plaque purified up to three times, and passaged to X+5 in Vero cells.

As used herein the term "immunogenically active" designates the ability to stimulate an immune response, i.e., to stimulate the production of antibodies, particularly humoral antibodies, or to stimulate a cell-mediated response. For example, the ability to stimulate the production of circulating or secretory antibodies or the production of a cell-mediated response in local mucosal regions, (e.g., intestinal mucosa), peripheral blood, cerebral spinal fluid or the like.

The effective immunizing amount of the immunogenically active component may vary and may be any amount sufficient to evoke an immune response and provide immunological protection against West Nile Virus disease. Amounts wherein a dosage unit comprises at least about 1.times.10.sup.4 TCID.sub.50 of killed or inactivated whole or subunit virus cells or antigen or DNA cells derived therefrom or a mixture thereof, preferably at least about 1.times.10.sup.6 TCID.sub.50, are suitable. Even more preferably, at least about 1.times.10.sup.7 TCID.sub.50 per dosage unit may be utilized. It is especially desirable that at least about 5.times.10.sup.7 TCID.sub.50 of killed or inactivated whole or subunit West Nile virus cells or antigen or DNA cells derived therefrom or a mixture thereof be used in the vaccine composition of the invention. In certain embodiments, as much as 1.times.10.sup.9 TCID.sub.50 and more may be utilized. A range of about 1.times.10.sup.4 TCID.sub.50 to about 1.times.10.sup.8 TCID.sub.50 may also be utilized.

In a further embodiment of the invention, it is contemplated that about 50 to 3,000 micrograms (.mu.g or 10.sup.-6 grams) of West Nile plasmid DNA may be utilized in one dosage unit of the vaccine composition. More preferably, about 100 to 1,000 .mu.g may be used, with about 100 to 250 .mu.g of plasmid DNA being more preferred.

At least one dosage unit per animal is contemplated herein as a vaccination regimen. In some embodiments, two or more dosage units may be especially useful. A dosage unit may typically be about 0.1 to 10 milliliters of vaccine composition, preferably about 0.5 to 5 milliliters, and even more preferably about 1 to 2 milliliters, with each dosage unit containing the heretofore described quantity of virus or virus component. The skilled artisan will quickly recognize that a particular quantity of vaccine composition per dosage unit, as well as the total number of dosage units per vaccination regimen, may be optimized, so long as an effective immunizing amount of the virus or a component thereof is ultimately delivered to the animal.

The West Nile virus vaccine composition of the invention may also contain one or more adjuvants. As used herein the term "adjuvant" refers to any component which improves the body's response to a vaccine. The adjuvant will typically comprise about 0.1 to 50% vol/vol of the vaccine formulation of the invention, more preferably about 1 to 50% of the vaccine, and even more desirably about 1 to 20% thereof. Amounts of about 4 to 10% may be even more preferred.

Suitable adjuvants can include immunostimulating oils such as certain metabolizable oils. Metabolizable oils suitable for use in the composition of the invention include oil emulsions, e.g., SP oil (hereinafter described), Emulsigen (MPV Laboratories, Ralston, NZ), Montanide 264,266,26(Seppic SA, Paris, France), as well as peanut oil and other vegetable-based oils, squalane (shark liver oil) or other metabolizable oil which can be shown to be suitable as an adjuvant in veterinary vaccine practice.

In addition, the adjuvant may include one or more wetting or dispersing agents in amounts of about 0.1 to 25%, more preferably about 1 to 10%, and even more preferably about 1 to 3% by volume of the adjuvant. Particularly preferred as wetting or dispersing agents are non-ionic surfactants. Useful non-ionic surfactants include polyoxyethylene/polyoxypropylene block copolymers, especially those marketed under the trademark PLURONIC.RTM. and available from BASF Corporation (Mt. Olive, N.J.). Other useful nonionic surfactants include polyoxyethylene esters such as polyoxyethylene sorbitan monooleate, available under the trademark TWEEN 80.RTM.. It may be desirable to include more than one, e.g. at least two, wetting or dispersing agents in the adjuvant as part of the vaccine composition of the invention.

Other components of the adjuvant may include such preservative compounds as formalin and thimerosal in amounts of up to about 1% vol/vol of the adjuvant.

As an adjuvant, SP oil is preferred. As used in the specification and claims, the term "SP oil" designates an oil emulsion comprising a polyoxyethylene-polyoxypropylene block copolymer, squalane, polyoxyethylene sorbitan monooleate and a buffered salt solution. In general, the SP oil emulsion will comprise about 1 to 3% vol/vol of block copolymer, about 2 to 6% vol/vol of squalane, more particularly about 3 to 6% of squalane, and about 0.1 to 0.5% vol/vol of polyoxyethylene sorbitan monooleate, with the remainder being a buffered salt solution.

When utilized, immunogenically stimulating amounts of SP oil as adjuvant in the vaccine composition of the invention may vary according to the immunogenically active component, the degree of potential infectious exposure, method of administration of the vaccine composition, the age and size of the equid, or the like. In general, amounts of about 1% to 50% vol/vol, preferably about 4% to 10% vol/vol, and more preferably about 4% to 5% vol/vol of SP oil are suitable.

In general, it is believed that a live virus vaccine may potentially lack sufficient safety in a given target host, and that a killed or inactivated virus vaccine may potentially lack the ability to stimulate a sufficiently effective immunologic response. Commonly, an adjuvant or immunogenically stimulating compound is used in combination with a killed or inactivated virus in a vaccine composition to obtain acceptable efficacy. However, safety to the target host is often compromised by the addition of an adjuvant. For example, pregnant animals many times have been known to have a significantly higher rate of miscarriage after being administered a killed or inactivated virus vaccine that contains an adjuvant.

It has now been found that when a suitable adjuvant, e.g. a metabolizable oil preferably such as SP oil, is used in combination with an immunogenically active component as described hereinabove, the resultant West Nile vaccine composition is safened for use in equidae, particularly horses, even for use in pregnant mares, while demonstrating important efficacy as well. Thus, the invention achieves the concomitant goals of effective immunization and safety, especially for pregnant animals. This combination of active immunogen and adjuvant is unheralded in the art.

Pharmacologically acceptable carriers suitable for use in the vaccine composition of the invention may be any conventional liquid carrier suitable for veterinary pharmaceutical compositions, preferably a balanced salt solution or other water-based solution suitable for use in tissue culture media. Other available carriers may also be utilized.

Additional excipients available in the art may also be included in the vaccine composition according to the various embodiments heretofore described. For example, pH modifiers may be utilized.

The components of the vaccine composition of the invention as heretofore described, including the carrier, may be combined together using available techniques.

In addition to the immunogenically active component of West Nile virus as described hereinabove as active ingredient, it is contemplated that the vaccine composition of the invention may also contain other active components such as an antipathogenic component directed against rabies virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus, equine herpes virus such as EHV-1 or EHV-4, Ehrlichia risticii, Streptococcus equi, tetanus toxoid, equine influenza virus (EIV), or the like or a combination thereof. The quantities of one or more of these viruses may be determined from efficacy literature in the art, or determined using available techniques.

In one embodiment of the invention the immunogenically active component of the invention may be incorporated into liposomes using known technology such as that described in Nature, 1974, 252, 252-254 or Journal of Immunology, 1978, 120, 1109-13. In another embodiment of the invention, the immunogenically active component of the invention may be conjugated to suitable biological compounds such as polysaccharides, peptides, proteins, or the like, or a combination thereof.

In a preferred embodiment of the invention, the inventive vaccine composition may be formulated in dosage unit form as heretofore described to facilitate administration and ensure uniformity of dosage. Formulation may be effected using available techniques, such as those applicable to preparations of emulsions.

The inventive vaccine composition may be administered parenterally, for example, intramuscularly, subcutaneously, intraperitoneally, intradermally or the like, preferably intramuscularly; or said composition may be administered orally or intranasally.

Accordingly, the present invention also provides a method for the prevention or amelioration of West Nile encephalitis in equidae, preferably horses, which comprises administering to said equidae a safened vaccine composition as described hereinabove.

In actual practice, the vaccine composition of the invention is administered parenterally, subcutaneously, orally, intranasally, or by other available means, preferably parenterally, more preferably intramuscularly, in effective amounts according to a schedule which may be determined by the time of anticipated potential exposure to a carrier of the West Nile virus. In this way, the treated animal may have time to build immunity prior to the natural exposure. By way of non-limiting example, a typical treatment schedule or dosing regimen may include parenteral administration, preferably intramuscular injection of one dosage unit, at least about 2-8 weeks prior to potential exposure. At least two administrations are preferred, for example one dosage unit at about 8 weeks and a second dosage unit at about 3-5 weeks prior to potential exposure of the treated animal. As heretofore set forth, a dosage unit will typically be within the range of about 0.1 to 10 milliliters of vaccine composition containing the previously described amounts of active and percentages of adjuvant and inactive(s) as set forth. A dosage unit within the range of about 0.5 to 5 milliliters is perhaps more preferred, with about 1 to 2 milliliter(s) being particularly preferred.

Claim 1 of 20 Claims

1. A vaccine composition which comprises: an effective immunizing amount of West Nile plasmid DNA, wherein said plasmid DNA is pCBWN; an immunogenically stimulating amount of a metabolizable oil; and a pharmacologically acceptable carrier.

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