WO 03/061555 PCT/US02/23447
WEST NILE VACCINE
This application claims priority from co-pending U.S. application serial number
60/308,334 filed on July 27, 2001.
The instant application is divided out of Indian Patent Application No.
109/KOLNP/2004 (hereinafter referred to as the "parent application").
FIELD OF THE INVENTION
The present invention relates to 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 when feeding on them.
West Nile virus is the causative agent for West Nile Virus disease, particularly
2JD West Nile encephalitis, predominately in humans, other mammals and birds. A major
concern 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
2(5 carriers appears to be the only means of controlling the West Nile virus.
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 akin 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.
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WO 03/061555 PCT/US02/23447

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 infection in equidae is available; and the
only means of controlling the West Nile virus appears to be the prevention of contact
with a carrier.
Therefore, there a need for safe and effective means of controlling the spread
of the West Nile virus in mammals and avians. In particular there is a need for a
vaccine, primarily for administration to equids which is sufficiently safened so as to
be suitable for administration even to pregnant mares without adverse effect. Also
needed is a vaccine capable of the prevention or amelioration of West Nile Virus
disease, particularly West Nile encephalitis, in equids and other mammals.
SUMMARY OF THE INVENTION
The present invention provides a vaccine composition which comprises: an
immunogenically active component selected from the group consisting of a live attenuated,
inactivated or killed whole West Nile virus of strain VM-2,VR-82 or VR 1267 or a subculture
thereof.
The present invention also provides a vaccine composition which comprises: an
immunogenically active component selected from the group consisting of a live
attenui3ted, inactivated or killed whole or subuni'i 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 adjuvant, preferably
comprising an immunogenically stimulating amount of a metabolizable oil; and
optionally a pharmaceutically acceptable carrier..
The present invention discloses a method for the prevention or
amelioration of West Nile encephalitis in equidae which comprises administering to
said equidae a vaccine composition which comprises an immunogenically active
component selected from the group consisting of a live attenuated, inactivated or
killed 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 adjuvant, preferably comprising an
immunogenically stimulating amount of a metabolizable oil; and optionally a
pharmaceutically acceptable carrier.
The present invention further provides a West Nile virus vaccine suitable for
use in horses which comprises an immunogenically active component selected from

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WO 03/061555 PCT/US02/23447
the group consisting of a live attenuated, inactivated or killed 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 adjuvant, preferably comprising an immunogenically stimulating amount
of a metabolizable oil; and optionally a pharmaceutically acceptable carrier.
In a further embodiment of the invention the vaccine composition comprises
at least 1x104 TCIDso per unit dose of inactivated West Nile virus, or a component
thereof, and about 4% to 10% vol/vol of an adjuvant 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 X 108 TClD50 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.
A particularly preferred embodiment of the invention is a vaccine composition
for horses, comprising at least two dosage units of killed or inactivated West Nile
virus, v/herein each said dosage unit comprises about 0.5 to 5 milliliters of a
composition containing at least about 5 X 107 TCID50 of said virus and about 1 to
10% vol/vol of an adjuvant, said adjuvant comprising at least one metabolizable oil
2J0 and at least two nonionic surfactants, and further wherein said dosage unit comprises
a pharmacologically acceptable carrier.
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
In general, the problem with designing a new vaccine is 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
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is often compromised by the addition of an adjuvant. For example, pregnant animals
many limes 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
such as SP oil is used in combination with an immunogenically active component as
described herein, the resultant West Nile viaccine composition is safened for use and
is particularly useful in equidae, particularly horses, and 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.
A safe and effective vaccine composition comprises: an immunogenically
active component selected from the group consisting of a live attenuated, inactivated
or killed 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 adjuvant; and optionally a pharmaceutically
acceptable carrier. This vaccine composition may be administered safely and
effectively to mammals and avians, particularly to equidae, such as horses, donkeys,
asses, etc. The vaccine compositon is particularly suitable for 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.cj. 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 Nos. VR-82, VR-1267 and VR-1267 AF.

WO 03/061555 PCTAJS02/23447
Whole orsubunit West Nile virus may be used as a live attenuated virus,
however, it is preferably killed, or 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. Attenuation is
achieved by conventional methods.
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, NY)
11) 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 Amos, IA 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" means the ability to
stimulate an immune response, i.e., to stimulate the production of antibodies*.,
2|0 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 amount of the immunogenically active component which is effective and
immunizing may vary and is any amount sufficient to evoke an immune response and
provide immunological protection against West Nile Virus disease. The amount of
immunogenically active component per dosage unit is preferably at least about 1x104
TCIDso, preferably from about 1x104 TCID50 to about 109 TCIDj,Ol more preferably at
least about 1x106 TCID50, particularly 106 to 107 TCIDS0 (preferably at least about 1 x
0 107). These amounts are suitable for killed or inactivated whole or subunit virus or
antigen or DNA derived therefrom or a mixture thereof. It is especially desirable that
at least about 5 X 107 TCID50 of killed or inactivated whole or subunit West Nile virus
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WO 03/061555 ■ PCT/US02/2344;
or antigen or DNA derived therefrom or a mixture thereof be used in the vaccine
composition of the invention.
In a further embodiment of the invention, it is contemplated that about 50 to
3,000 micrograms (pg or 10"* grams) of West Nile plasmid DNA may be utilized in
one dosage unit of the vaccine composition. More preferably, about 100 to 1,000 ug
may be used, with about 100 to 250 pg of plasmid DNA being more preferred.
At least one dosage unit per animal is contemplated herein as a vaccination
regimen. 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
1JD 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
man will recognize that a particular quantity of vaccine composition per dosage unit,
as well as the total number of dosage units per vaccination regimen, may bo
optimized, so long as an effective immunizing amount of the virus or a component
115 thereof is delivered to the animal.
The West Nile virus vaccine composition of the invention contains one or
more adjuvants. As used herein the term "adjuvant" refers to any component which
improves the body's response to a vaccine or an immunogen. The adjuvant will
typically comprise about 0.1 to 50% vol/vol of the vaccine formulation of the
2J0 invention, preferably about 1 to 50% of the vaccine, more preferably about 1 to 20%,
particularly 1 to 10% vol/vol thereof. Amounts of about 4 to 10% are even more
preferred.
Suitable adjuvants 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.
The adjuvant may be a composition comprising in addition to the
metabolizable oil, one or more wetting or dispersing agents in amounts of about 0.1
to 25%, more proferably 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
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WO 03/061555 PCT/US02723447
non-ionic surfactants. Useful non-ionic surfactants include
polyoxyethylene/polyoxypropylene block copolymers, especially those marketed
under the trademark PLURONIC® and available from BASF Corporation (Mt. Olive,
NJ). Other useful nonionic surfactants include polyoxyethylene esters such as
polyoxyethylene sorbitan monooleate, available under the trademark TWEEM 80®. 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 thimerosa! in amounts of up to about 1% vol/vol of the adjuvant.
1 j} A particularly preferred adjuvant is referred to as SP oil. As used in the
description and examples, 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 of the vaccine composition are
suitable, preferably about 4% to 10% vol/vol, and more preferably about 4% 1:o 5%
vol/vol of SP oil.
2|5 Pharmaceuticaly (or 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.
3J0 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.
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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,
1 fy tetanus toxoid, equine influenza virus(EIV),, Eastern, Western and Venezuelan
rhinopneumonitis virus 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, 19713,120,
1109-13. In another embodiment of the invention, the immuncgenically active
component of the invention may be conjugated to suitable biological compounds
such as polysaccharides, peptides, proteins, or the like, or a combination thereof.
20 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
^0 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, intranasatly, or by other available means,
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2to

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 teated 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 prior to potential exposure to the virus
and a second dosage unit at about 3 -5 weeks prior to potential exposure of the
treated animal. As heretofore described, a dosage unit will typically be within the
range of about 0.1 to 10 milliliters of vaccine composition containing the amounts of
active iand percentages of adjuvant and inactive(s) as previously described. A
dosages 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.
For a clearer understanding of the invention, the following examples are set
forth below. These examples are merely illustrative and are not understood to limit
the scope or underlying principles of the invention in any way. Indeed, various
modifications of the invention, in addition to those shown and described herein, will
become apparent to those skilled in the art from the following examples and the
foregoing description. Such modifications are also intended to fall within the scope of
the appended claims.

EXAMPLE 1
Preparation of Vaccine
A/Formulation of SP oil

Ingredient Description
Polyoxyethylene-polyoxypropylene block copolymer
(Pluronic* L121, BASF, Mt. Olive, NJ)

Volume
20.0 ml

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Squalane (Kodak, Rochester, NY) 40.0 ml
Polyoxyethylenesorbitan monooleate 3.2 ml
(Tweeni® 80, Sigma Chemical, St. Louis, MO)
Buffered salt solution 936.8 ml
(D-V PBS Solution, Ca, Mg free)
The ingredients are mixed and homogenized until a stable mass or emulsion
is formed. Prior to homogenization, the ingredients or mixture may be autoclaved.
The emulsion may be further sterilized by filtration. Formalin may be added up to a
$ final concentration of 0.2%. Thimerosal may be added to a final dilution of 1:10,000.
B/Vaccine Preparation
An equine isolate of West Nile virus, obtained from USDA facilities in Ames,
IA(Lot Mo. VM-2, Equine Origin, 1999 North American isolate, second passage in
VeroM cell culture), was cultivated in multiple cultures of Vero cells in OptiMEM (LTI,
Grand Island, NY) tissue culture medium at 37 °C. The harvests are titrated and then
inactivated by means of addition of a 10% formalin solution to a final concentration of
0.1%. This is allowed to inactivate at 37°C for a period of no less than 144 hours.
Then, another addition of 0.1% formalin is added and incubated at 37CC for another
period of no less than 144 hours.
The vaccines are formulated by suspending the appropriate volume of
inactivated virus fluid in 1-20% by volume of SP oil per 1 mL dose.
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EXAMPLE 2
Evaluation of Antibody Response to the Intramuscular Injection of Test
Vaccine
ii
In this evaluation, horses are randomly divided into four groups: one group of
twenty horses is administered test vaccine at a dose of 1x107 TCID50 (Tissue Culture
Infectious Dose); a second group of twenty horses is administered test vaccine at a '
dose of 5x107 TCIDS0; a third group of five horses is administered test vaccine at a
1 3 dose of 1x108 TCID50; and a fourth group of eight horses are maintained as non-
vaccinated environmental controls. Treated horses are given a first dose of vaccine
according to the group to which they are assigned. At twenty-one days following
administration of the first dose, a second dose of the same vaccine is administered.
All horses are bled for serum at the time of administration of the first and second
15 dose and at weekly intervals through 28 days post second dose administration.
Test Vaccine A

uuiupuiiciii wuiiu.iL/uac vuiuuie/iiiL.West Nile virus-Inactivated 1x10' TCIDM 0.0347 mLMEM1 NA 0.9138 mLSP oil 5% 0.0500 mLPolymyxin B2 30.0 mcg/mL 0.0003 mLNeomycin 30.0 mcg/mL 0.0003 mLThimerosal (5%) 1:20,000 0.0010 mL
Test Vaccine B
Component Cone/Dose Volume/mL

West Nile virus-Inactivated 5x10'TCID50 0.1734 mLMEM NA 0.7752 mLSP oil 5% 0.0500 mLPolymyxin B2 30.0 mcg/mL 0.0002 mLNeomycin3 30.0 mcg/mL 0.0002 mLThimerosal4 (5%) 1:20,000 0.0010 mL


Test Vaccine C
Component Conc./Dose VoIume/mL

West Nile virus-Inactivated 1x10°TCID50 0.3467 mLMEM1 NA 0.6019 mLSP oil 5% 0.0500 mLPolymyxin B2 30.0 mcg/mL 0.0002 mLNeomycin3 30.0 mcg/mL 0.0002 mLThimerosai4 (5%) 1:20,000 0.0010 mL
The serological data obtained is shown in Table I below, wherein: 0 DPV 1
designates day zero, pre vaccination; and 14 DPV 2 designates day 14, post
vaccination. NR designates no results.
As can be seen from the data on Table I, treated horses from all groups
showed significant increases in antb ibodies to West Nile virus while the control
horses maintained a low to non-existent antibody level. The level of response in the
horses that received vaccine was independent of the level of antigen in the vaccine
that they received.
Table I

Test Dose Test#1 Test #2Vaccine (TCiDsn^ 0DPV1 14DPV2 0DPV1 14DPV2A . 1x107 <10 160 <10 80A 1x107 <10 20 <10 10A 1x107 <10 >320 <10 40
1 LTl, Grand Island, NY
2 Sigma, St. Louis, MO
3 Sigma, St. Louis, MO
4 Sigma, St. Louis, MO

WO 03/061555 ~ rCT/US02/23447

A 1x107 <10 80 <10 >320
A 1x107 <10 80 <10 40
A 1x107 <10 80 <10 160
A 1x107 <10 40 <10 40
A 1x107 <10 80 <10 80
A 1x107 <10 160 <10 80
A 1x107 <10 40 <10 10
A 1x107 <10 40 <10 10
A 1x107 <10 160 <10 40
A 1x107 <10 >320 <10 NR*
A 1x107 <10 >320 ^10 NR
A 1x107 <10 >320 <10 80
A 1x107 <10 160 <10 20
A 1x107 >20 >320 <10 160
A 1x107 <10 80 <10 80
A 1x107 <10 80 <10 160
A 1x107 <10 160 <10 160
B 5x107 <10 80 <10 40
B 5x107 <10 20 <10 <10
B 5x107 <10 >320 <10 160
B 5x107 <10 80 <10 >320
B 5x107 <10 >320 <10 160
B 5x107 <10 80 <10 80
B 5x107 <10 160 <10 >320
B 5x107 <10 80 <10 40
B 5x107 <10 160 <10 40
B 5x107 <10 40 <10 80
B 5x107 <10 20 <10 20
B 5x107 <10 >320 <10 160
B 5x107 <10 >40 <10 >320
B 5x107 <10 80 <10 40
B 5x107 <10 80 <10 NR
B 5x107 <20 >320 <10 80
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PCT/US02/23H7

E 5x107
B 5x107
B 5x107
B 5x107
C 1x10"
C 1x108
c 1x108
c 1x108
c 1x108
Control 0
Control 0
Control 0
Control 0
Control 0
Control 0
Control 0
Control 0

>20
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10

160
>320
>320
80
>320
>320
160
>320
40
<10
<10
<10
<10
<10
320
160
>320
160
80
NR
40
<10
<10
<10
NR
<10
<10
<10
<10

EXAMPLE 3
Evaluation of Safety of Test Vaccine in Horses Under Field Conditions

13

In this evaluation, 200 healthy male and female horses are vaccinated with a
6x106 TCID50 dose of inactivated test vaccine administered as a 1 mL dose
vaccination by intramuscular administration and followed in three to four weeks by a
second 1 mL dose vaccination. The treated horses are housed and fed using
conventional husbandry practices for farm or stable. All treated horses are observed
by a veterinarian for 30 minutes following vaccination for immediate reactions such
as salivation, labored or irregular breathing, shaking, or anaphylaxis. For two weeks
post-vaccination, the horses are observed daily for any delayed reactions such as
lethargy, anorexia or unusual swelling at the injection site. Blood samples of 5 to 10
mL are taken by venipuncture from treated horses on the day of first vaccination (day

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Zero) and at least once more at two or more weeks post second vaccination (day 36
or greater). Serological assays using PRNT5 testing are perronned.

Test Vaccine
Component Cone/Dose Volume/mL
West Nile virus-Inactivated 6x10BTCID50 0.21mL
SPoil 5% 0.05mL
MEM N/A 0.74mL
Less than 2% of the horses were observed to have any clinical signs of
debilitation. This evaluation demonstrates that the vaccine of the invention is safe for
use in horses under field conditions.
EXAMPLE 4
Evaluation of Efficacy of Test Vaccine (Muttivalent and Monovalent
Preparations) in Horses Under Field Conditions
The efficacy of a combination vaccine containing killed West Nile virus (WNV)
1J5 against experimental WNV challenge was evaluated.
A total of 30 horses were allotted into one vaccinated group (20 horses) and
one control group (10 horses). Horses in the vaccinated group received
intramuscularly two doses of the test vaccine containing killed West Nile virus (5 X
107 TCID50 per dose with 5% SP oil), influenza virus, encephalomyelitis virus
20 (Eastern, Western and Venezuelan), rhinopneumonitis virus (serotypes 1 and 4), and
tetanus toxoid, three weeks apart. Serum samples were collected periodically for
antibody response measured by plaque reduction neutralization test (PRNT).
Twenty-four (24) days after the second vaccination, all horse were challenged
subcufaneously with WNV. After challenge, horses were monitored for rectal
temperature and any clinical signs twice daily for two weeks and once weekly
Chang, G.J.; Hunt, A.R. and Davis B., Journal of Virology, 74 pp 4244-5422 (2000).
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thereafter for detection of viremia. Horses were euthanized and necropsied on 21
and 22 DPC. Cerebrospinal fluid (CSF), spinal cord (cervical, thoracic, and lumbar)
and brain (frontal, occipital, medulla oblongata, and brain stem) tissue samples were
examined for gross pathology and collected for virus isolation.
Fourteen days after the second vaccination, 75% of vaccinated animals
seroconverted (titer > 5) with a geometric mean titer of 10 while control animals
remained negative (titer < 5). The vaccination conferred a significant protection
against viremia (a precursor to development of full-blown West Nile Virus disease).
Nine out of 10 (90%) controls developed viremia after challenge, while only eight out
of 20 (40%) vaccinates had transient viremia, or viremia lasting only a few days at
most. Importantly, no WNV disease clinical signs were obser 'ed in any of the
challenged vaccinated animals throughout the observation period. (Transient febrile
responses were observed in one control and two vaccinated horses. However, there
was no evidence to suggest these responses were due to WNV infection.) Petechial
hemorrhage in white matter and subdural hemorrhage were found in the brain tissue
from one control animal. WNV was isolated from the brain but not from CSF: and
spinal cord samples collected from this animal. No WNV was isolated from any of
the tissue samples; collected from other challenged horses.
Results from this study demonstrated a significant protection against both
viremia and signs of clinical WNV disease in horses vaccinated with the test
combination vaccine.
A second study has conducted with a protocol similar to that above, except
that a monovalent WNV vaccine (WNV vaccine alone) was utilized, and all horses
were challenged with WNV at 12 months after the second vaccination. Nine out of
2J5 11 (81.8%) of the controls developed viremia after challenge, while only one out of 19
(5.3%) of the vaccinates had transient viremia. No WNV associated clinical signs
were observed in any of the challenged animals throughout the observation period.
No febrile responses were observed in any of the challenged horses. No WNV was
isolated from any of the tissue or CSF samples collected from any of the challenged
horses;. (Prior to challenge at the end of the 12 month period, 17 of the nineteen
vaccinated horses had plaque reduction neutralization test (PRNT) titers of 5 or
greater, while the control group remained negative (<5).)
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^WO 03/061555 PCT/US02/23447
Results from this second study demonstrate a significant protection (94% of
preventable fraction) against viremia in horses vaccinated with the killed monovalent
WNV vaccine. These results also demonstrate a long duration of protective
immunity.
3
EXAMPLE 5
Evaluation of Efficacy of DNA Test Vaccine in Horses Under Field Conditions

1(
This example demonstrates the efficacy of a West Nile Virus (WNV) DNA
vaccine, as part of a further embodiment of the invention. The DNA vaccine
contained 100 ug of purified DNA adjuvanted with 5% SP oil per 2 mL dose, and was
evaluated against experimental WNV challenge.
For the composition of the WNV DNA vaccine, bacterial cells were harvested
1$ from a culture passaged 10 times from a master seed using E. coli DH10B obtained
from Invitrogen (Carlsbad, CA) containing a West Nile plasmid pCBWN obtained
from the Centers for Disease Control (Fort Collins, CO). The bacterial cells were
suspended in glucose-tris-EDTA buffer and lysed with sodium hydroxide and sodium
dodecyl sulfate. The lysate was neutralized with a potassium acetate solution. The
precipitated complex material containing DNA, RNA, cell debris and proteins was
removed by filtration. The filtrate was precipitated with the addition of isopropyl
alcohol. The precipitate was collected by centrifugation and resuspended in buffer.
This process was repeated using ammonium acetate. The precipitate collected was
resuspended in buffer and loaded into a chromatography column packed with
2J5 Polyflo® resin. The column was then washed and the plasmic! DNA was eluted from
the column. The eluate was finally diafiltered extensively against phosphate buffer
saline. The purified plasmid DNA stocks were then shipped for blending. The test
vaccine contained 100 pg of plasmid DNA adjuvanted with 5% SP oil.
The horses used for testing were randomly assigned into two groups: 20
animals received the WNV DNA vaccine, and 10 animals were used as controls. The
first group were vaccinated intramuscularly with two 2.0 mL doses of vaccine three
weeks apart. The control horses received no vaccinations or placebos. One group
of horses (9 vaccinates and 5 controls) were challenged 5 weeks after the second
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•
-WO 03/061S55 ■ PCT/USCI2/23447
vaccination, whereas a second group of horses (11 vaccinates and 5 controls) were
challenged 12 weeks after the second vaccination. Briefly, Aedes albopictus
mosquitoes which had been infected with WNV 12 days prior to the horse challenge,
were allowed to feed on each horse for at least 5 minutes. Following challenge, only
those mosquitoes which were found to have engorged with blood meals from each
horse were frozen at -20°C, and the virus load was titrated as a pool subsequently.
Mosquitoes were then homogenized by vortexing using diluent. The homogenate
was centrifuged and the supernatant was removed for titration on Vero cells.
After challenge, rectal temperatures were taken twice daily for at least 9 days
if) and clinical signs were monitored twice a day for at least 21 days. Serum samples
were taken twice a day for the first 9 days post challenge (DPC); once daily from 10
to 14 DPC and finally at 21 DPC. Virus isolation was performed on serum samples
from 0 DPC to 10 DPC for the first challenge group and from 0 DPC to 11 DPC for
the second challenge group. The first group of 14 horses and the second group of
16 horses were euthanized and necropsied 28 to 37 DPC and 29 to 38 DPC,
respectively. Cerebrospinal fluid (CSF) and tissue samples from the cerebrum,
cerebellum and the brain stem were collected for gross pathology and virus isolation.
At 14 days post second vaccination, 6 of 20 vaccinated had a measurable
titer of 2 or greater, and one horse had a titer of 5. Vaccination had conferred
2\) protection against experimental West Nile challenge using mosquitoes. Viremia was
detected in 5 of 5 control animals and 4 of 9 vaccinates in the first challenge group,
whereas 4 of 5 controls and 2 of 11 vaccinates were viremic in the second challenge
group of horses. The viremia detected was transient, and occurred only within the
first six days after challenge. As a whole, viremia was detected in 9 out of 10 (90%)
control horses, while only 6 out of 20 (30%) vaccinates were detected with viremia.
No neurological signs attributable to WNV infection were observed in any of
the study horses throughout the challenge observation period. No WNV was isolated
from any of the tissue samples collected from any of the study horses. One horse
from the first challenge group which was euthanized on 7 DPC showed no gross or
2J0 microscopic evidence of encephalitis or meningitis.
Results from this study demonstrated a significant protection against viremia
in horses vaccinated with the test vaccine.
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WE CLAIM:
1. A vaccine composition which comprises: an immunogenically active
component selected from the group consisting of a live attenuated, inactivated or killed
whole West Nile viruspi strain VM-2 , VR-82 or VR 1267 or a subculture thereof.
2. A vaccine composition which comprises: an immunogenically active
component selected from the group consisting of a subunit West Nile virus, an antigen
derived from said virus, DMA derived from said virus, plasmid West Nile virus DNA,
plasmid with sequence inserts of said virus, and a mixture thereof; an adjuvant; and
optionally a pharmac'eutically acceptable carrier.
3. The composition according to claim 1 wherein the immunogenically active
component is a killed or inactivated whole West Nile virus.
4. The composition according to ciaim 2 wherein the immunogenically active
component is subunit West Nile virus.
5. The composition according to any of the preceding claims wherein the
adjuvant comprises 0.1 to 50% vol/vol of the vaccine composition.
6. The composition according to any of the preceding claims wherein the
adjuvant comprises a metabolizable oil.
7. The composition according to claim 6 wherein the adjuvant comprises
from 1 to 50% vol/vo! of metabolisable oil.
3. The composition according to claim 6 or 7 wherein the metabolizable oil is
squalane.
9. The composition according to any of claims 6 to 8 wherein the adjuvant
further comprises one or more wetting agents and/or dispersing agents.
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10. The composition according to claim 8 wherein the wetting agents and/or
dispersing agents comprise from 0.1 to 25% vol/vol of the adjuvant.
11. The composition of claims 9 or 10 wherein said wetting or dispersing
agents are selected from the group consisting of non-ionic surfactants.
12. The composition of claim 11, wherein said non-ionic surfactants are
selected from the group consisting of polyoxyethylene/polyoxypropylene block
copolyrners and polyoxyethylene esters.
13. The composition according to any of claims 1, 3 and 5 to 12 wherein said
immunogenically active component is present in sufficient quantity to provide from 1 x
104 TCID50 to 5 x 108 TCID50 per unit dose of the vaccine composition.
14. The composition according to claim 13 wherein said immunogenicaiiy
active component is present in sufficient quantity to provide at least 1 x 10e TIDS0 per
unit dose.
15. The composition of claim 13 or 14, which comprises about 5 X 107 TCIDS0
of said virus

16. The composition according to any of the preceding claims further
comprising a vaccine component directed against one or more of 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), Eastern, Western and
Venezuelan rhinopneumonitis virus.
17. The composition according to claim 1 or 3 comprising: at least 1X104
TCID50 per unit dose of killed or inactivated West Nile virus; and about 4% to 10% vol/vol
of an adjuvant comprising about 1 to 3% polyoxyethylene-polyoxypropylene block
copoiymer, about 2 to 6% of squalane and about 0-1 to 0.5% of polyoxyethylene sorbitan
monooleate.
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18. The compositon of any of the preceding claims, which is formulated into at
least two dosage units.
19. A composition according to any of the preceding claims in which the
composition is provided in one or more dosage units of 0.5 to 5 ml of the composition.
20. A composition according to any of claims 2, 5 to 12 and 16 to 18
comprising West Nile plasmid DNA in an amount of about 50 to 3,000 micrograms per
dose.
21. The composition of claim 20 wherein said composition comprises about
100 to 1,000 micrograms per dose.
22. The composition of claim 21, wherein said composition comprises about
100 to 250 micrograms per dose.
23. A vaccine composition comprising at least about 1 x 104 TCIDS0 per unit
dose of killed or inactivated West Nile virus of strain VM-2 , VR-82 or VR 12S7 or a
subculture thereof; and about 4 to 10% vol/vol of a metabolizable oil adjuvant comprising
about 1 to 3% polyoxyethylene-polyoxypropylene block copolymer, about 2 to 6% of
squalane and about 0.1 to 0.5% of polyoxyethylene sorblt.an monooleate.
24. A safened and effective West Nile virus vaccine for equidae comprising at
least about 1 x 106 TCID50 per unit dose of killed or inactivated West Nile virus and at
least about 1% vol/vol of an adjuvant comprising at least one rnetabolozable oil and at
least one wetting or dispersing agent.
25. A method for the prevention or amelioration of West Nile encephalitis in
equidae which comprises administering to said equidae a vaccine composition as
defined in any of claims 1 to 24.
26. The method according to claim 25 wherein said equidae are horses.
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27. The method according to claim 26 wherein said horses are pregnant
mares.
28. The method according to claim 27 wherein said vaccine composition is
administered parenterally. ■
29. The method according to claim 28 wherein said vaccine composition is |
administered intramuscularly. ;
30. The use of an immunogenically active component selected from the group i
consisting of a live attenuated, inactivated or killed 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; in the [
•xs^aration of a vacc-ins composition as defined in any of claims 1 to 24 for use in the prevention or amelioration of diseases in mammals or avians caused by West Nile virus. ',
31. The use according to claim 29 where in. the vaccine composition is for use j
i
in the prevention or amelioration of diseases in equidae caused by West Nile virus. i
i
32. The use according to claim 30 wherein the equidae is horse. i
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i

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.