West Nile Virus vaccine

Abstract

The invention provides for immunogenic compositions against West Nile Virus. The immunogenic compositions, in alternate embodiments, also include other equine pathogens. The West Nile Virus composition of the present invention advantageously provides for protection against North American Dominant West Nile Virus strains or isolates.

Claims

1. An immunogenic composition comprising one or more strains of North American Dominant killed or inactivated West Nile Virus or any antigen thereof.

2. The immunogenic composition according to claim 1, wherein said immunogenic composition comprises at least one antigen or one additional strain of Equine Herpes Virus.

3. The immunogenic composition according to claim 1, wherein said immunogenic composition comprises at least one antigen or one additional strain of Equine Influenza Virus.

4. The immunogenic composition according to claim 1, wherein said West Nile Virus is one of the strains selected from the group consisting of Horse Origin 2005, deposited with the ATCC under accession number PTA-9409; NY2002Nassau; NY2002Clinton; NY2002Queens; GA20021; GA20022; TX20021; TX20022; IN2002; NY2003Albany; NY2003Suffolk; NY2003Chatauqua; CO20031; CO20032; TX2003; TX2003Harris4; TX2003Harris6; TX2003Harris7; TX2003Harris10; AZ2004; TX2004Harris4; and combination thereof.

5. The immunogenic composition according to claim 1, wherein any of the strains are present in an amount from about 10.sup.2.0TCID.sub.50/mL-10.sup.10.0TCID.sub.50/mL per dose.

6. The immunogenic composition according to claim 1, wherein said immunogenic composition further comprises suitable excipients or pharmaceutical carrier.

7. The immunogenic composition according to claim 6, wherein said suitable excipients or pharmaceutical carrier is/are selected from the group consisting of a diluent, adjuvant, antimicrobial agent, inactivating agent, and combinations thereof.

8. The immunogenic composition according to claim 7, wherein said adjuvant is selected from the group consisting of, a polymer of acrylic or methacrylic acid, non-metabolized oil, and combinations thereof.

9. The immunogenic composition according to claim 1, wherein one dose of said immunogenic composition is formulated in 0.5 ml to 2.5 ml.

10. The immunogenic composition according to claim 1, wherein said immunogenic composition provides duration of immunity of at least 12 months after the administration of one dose.

11. The immunogenic composition according to claim 1, wherein said immunogenic composition is safe for use in foals or horses 4 months of age or older.

12. A method for reducing the incidence or lessening the severity of clinical symptoms associated with or caused by West Nile Virus in an animal or a herd of animals comprising the step of administering the immunogenic composition according to claim 1 to an animal in need thereof.

13. The method according to claim 12, wherein said immunogenic composition is administered in one or more doses.

14. The immunogenic composition according to claim 1, wherein said immunogenic composition further comprises at least one antigen of one or more additional strains selected from the group consisting of Eastern Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus and Venezuelan Equine Encephalomyelitis Virus, Tetanus Toxoid, and combinations thereof.

15. The immunogenic composition according to claim 14, wherein said Western Equine Encephalomyelitis Virus is the strain deposited with the ATCC under accession number PTA-9410.

16. The immunogenic composition according to claim 14, wherein said Venezuelan Equine Encephalomyelitis Virus is the strain deposited with the ATCC under accession number PTA-9411.

17. The immunogenic composition according to claim 14, wherein said Eastern Equine Encephalomyelitis Virus is the strain deposited with the ATCC under accession number PTA-9412.

18. The immunogenic composition according to claim 14, wherein said Equine Herpes Virus is selected from the group consisting of the strains deposited with the ATCC under accession Nos. PTA-9525 or PTA-9526, and combinations thereof.

19. The immunogenic composition according to claim 14, wherein said Equine Influenza Virus is selected from the group consisting of Influenza/Equine-2/Ohio/03, Influenza/Equine-2/KY/95, Influenza/Equine-2/New Market/2/93, and combinations thereof.

20. The immunogenic composition according to claim 14, wherein said Equine Influenza Virus is selected from the group consisting of the strains deposited with the ATCC under accession Nos. PTA-9522, PTA-9523 or PTA-9524 and combinations thereof.

21. A method for reducing the incidence or lessening the severity of clinical symptoms associated with or caused by one or more of the pathogens selected from the group consisting of West Nile Virus, Eastern Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus, Venezuelan Equine Encephalomyelitis Virus and Clostridium tetani in an animal or a herd of animals comprising the step of administering the immunogenic composition according to claim 14 to an animal in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graphical representation of the Mean Total Clinical Scores;

(2) FIG. 2 is a graphical representation of the Proportion Shedding;

(3) FIG. 3 is a graphical representation of the Nasal Discharge Score;

(4) FIG. 4 is a graphical representation of Proportion Virus Shedding;

(5) FIG. 5 is a graphical representation of Conjunctivitis Score;

(6) FIG. 6 is a graphical representation of Serum Neutralization Titers;

(7) FIG. 7 is a graphical representation of Proportion Positive for EHV-1;

(8) FIG. 8 is a graphical representation of Mean White Blood Cell Count;

(9) FIG. 9 is a graphical representation of Proportion Positive (pyrexic);

(10) FIG. 10A through 10N is a nucleotide alignment of the HE region of WNV isolates (SEQ ID NOS 17-18 and 25-28, respectively, in order of appearance);

(11) FIG. 11A through 110 is a nucleotide alignment of the DE region of WNV isolates (SEQ ID NOS 17-18 and 29-32, respectively, in order of appearance);

(12) FIG. 12A through 12M is a nucleotide alignment of the D NS5 region of WNV isolates (SEQ ID NOS 1-2 and 33-36, respectively, in order of appearance);

(13) FIG. 13A through 13M is a nucleotide alignment of the H NS5 region of WNV isolates (SEQ ID NOS 1-2 and 37-40, respectively, in order of appearance);

(14) FIG. 14A through 14M is a nucleotide alignment of the H WN05 E NS5 region of WNV isolates (SEQ ID NOS 1-6, respectively, in order of appearance);

(15) FIG. 15A through 150 is a nucleotide alignment of the H WN05 region of WNV isolates (SEQ ID NOS 17-22, respectively, in order of appearance);

(16) FIG. 16A through 16K is a nucleotide alignment of the NS5 region of WNV isolates (SEQ ID NOS 12-16, respectively, in order of appearance); and

(17) FIG. 17A through 17Q is a nucleotide alignment of the E region of WNV isolates (SEQ ID NOS 7-11, respectively, in order of appearance).

DETAILED DESCRIPTION

Examples

(18) The following examples are set forth below to illustrate specific embodiments of the present invention. These examples are merely illustrative and are understood not to limit the scope or the underlying principles of the present invention.

Example 1

(19) This example illustrates a preferred vaccine composition in accordance with the present invention.

Materials and Methods

(20) For preparation of working cell stock, the Master Cell Stock (MCS), consisting of the Vero Cell Line known to propagate West Nile Virus, which was tested for purity, identity, and karyology, was thawed and used to inoculate a range of T25 up to T150 cm.sup.2 vessels or 1050 cm.sup.2 roller bottles, or bioreactors or other suitable sterile vessels. Thawed cells were suspended in growth medium at a rate of 0.0015 mL to 5.0 L per vessel, depending on vessel volume. Cells were then incubated at 36-38? C. for up to seven days. Cultures planted from frozen stock were re-fed with medium, if needed, within thirty-six hours after planting to remove residual DMSO. Cultures were re-fed with medium, if needed, during the growth period to remove excessive debris, or to stimulate the growth of cultures which have not reached confluence, or to maintain viability of confluent cultures.

(21) Cells were passaged 1-20 times by decanting the spent medium and then by adding 5-500 mL of 0.25% Trypsin-EDTA Solution to each vessel, depending upon vessel volume. The vessels were agitated gently until the cells slough from the surface. The cells were then removed from the vessels by rinsing with growth medium and pooled together. Prior to inoculation, cell growth medium was decanted from Vero Working Cells that are at least 55% confluent. Virus growth medium described was added to each vessel at 0.15 to 0.4 mL per cm.sup.2 surface area. A Multiplicity of Infection (MOI) of 0.000001-0.0002 was used for infection as determined by performing a cell count of at least two representative vessels. Roller bottle cultures infected were incubated at 36-38? C. for two to five days at 0.1-0.8 rpm.

(22) During the growth period, cultures were checked for typical CPE microscopically and for gross contamination macroscopically. Unsuitable cultures were discarded after sterilization. Cultures may be attenuated using standard techniques or may be used without attenuation.

(23) The microorganisms were then harvested for production purposes. Virus fluids were harvested when CPE reached 85% or greater. Roller bottles were swirled to remove loose cells, and fluids and then pooled into sterile 2-20 L glass, plastic, or PETG bottles, 20 L sterile polypropylene containers or 2-500 L sterile stainless steel tanks containers appropriate for clarification.

(24) Next, the product was prepared. Clarified fluids were inactivated with Formaldehyde Solution, USP, 0.2% by volume, or another effective inactivating agent, transferred to a secondary container, and held at 20-25? C. (room temperature) with agitation for forty-eight hours. A sample of at least 12 mL of the inactivated fluids was taken for inactivation assurance testing (described below) prior to concentration. After inactivation was completed, inactivated lot material was held at 2-7? C. for up to sixty days prior to concentration. A number of suitable adjuvants may be added to the vaccine formulation, most preferably a non-metabolizable oil, preferably mineral oil, and/or a carbomer. Typical processing steps may be employed such as mixing, blending, microfluidization, and emulsification, of the adjuvant and/or the harvested virus antigens with other ingredients.

(25) The product was then standardized. Sufficient volumes of clarified, inactivated, concentrated (optional) lots were combined to provide a calculated titer of at least 10.sup.4.0 TCID.sub.50 per dose of each strain in the final product. Multiple lots may be blended to achieve the titer requirements per dose.

(26) The product was then assembled to final formulation. Based on the desired final serial volume, the amounts of antigenic components, adjuvant, stabilizer and diluent were calculated as follows: a. West Nile Virus, Horse Origin 2005 (ATCC No. PTA-9409): minimum 10.sup.4.0 TCID.sub.50/dose b. Adjuvant: The total adjuvant concentration, preferably a non metabolizable oil, and more preferably mineral oil and/or a carbomer, in a serial is at least 10% v/v and is added at time of serial batching/assembly. c. Diluent: An appropriate volume of phosphate buffered saline (PBS) is added to bring the final volume to the desired volume. d. Additional Formalin: An appropriate volume of 37% Formalin is added to maintain an appropriate level. e. Gentamicin Sulfate

(27) The required amounts of adjuvant and PBS were combined in a sterile vessel. The pH of this mixture was adjusted to approximately 4.9-5.1 with 10N NaOH or 5N HCl if necessary. Clarified, killed, concentrated West Nile Virus, as well as Gentamicin, and Formalin were added and the pH adjusted to 6.9 to 7.1. This was mixed at 2-6? C. for at least 8 hours, not to exceed 48 hours.

(28) The vaccine was given by typical hypodermic injection, with booster vaccinations if desired. Most preferably, the initial dose and the booster doses were 1 mL volume administered intramuscularly at 21-day intervals. The vaccination regimen of initial and booster dose was given at the most preferred 1 mL dose volume to horses, other equidae, and other WNV susceptible species to reduce the incidence of and or severity of clinical signs of WNV infection, and preferably to prevent infection by WNV as well as to prevent disease due to West Nile Virus infection for a sustained period following vaccination.

Results and Discussion

(29) The vaccine was given by various appropriate parenteral routes, dose volumes, and dosing regimens to animals of varying immunological status for WNV, including naive and those with passive antibody, and provided for long duration of immunity up to and exceeding at least 2 years following vaccination. The vaccine was safe for administration in WNV susceptible species, particularly equidae, at any age and at any stage of reproduction, including pregnant females.

Example 2

(30) This investigation was carried out to obtain an efficacy evaluation of a vaccine to protect horses from challenge with West Nile Virus (WNV).

Materials and Methods

(31) A total of 30 horses were randomly divided into groups of 15 horses each. A total of 20 horses received 2 doses of vaccine at 21-day intervals and 10 horses were used for control. Each group of horses, Block 1 and Block 2, contained 10 vaccinated horses and 5 control horses. The vaccine was a combination including WNV antigen, specifically an inactivated or killed North American Dominant Strain of WNV, Horse Origin 2005 (ATCC Deposit No. PTA-9409) as well as antigenic components of Venezuelan Equine Encephalomyelitis, TC-83 strain (ATCC Deposit No. PTA-9411) Eastern Equine Encephalomyelitis, NJO strain (ATCC Deposit No. PTA-9412) Western Equine Encephalomyelitis, Fleming strain (ATCC Deposit No. PTA-9410) and Tetanus toxoid formulated approximately as follows:

(32) TABLE-US-00001 Eastern Equine Encephalomyelitis 10.sup.6.7-10.sup.9.2 TCID.sub.50/mL Western Equine Encephalomyelitis 10.sup.6.7-10.sup.9.2 PFU/mL Venezuelan Equine Encephalomyelitis 10.sup.6.7-10.sup.9.2 TCID.sub.50/mL West Nile Virus 10.sup.7.0-10.sup.9. TCID.sub.50/mL Tetanus Toxoid 5-10 CPU/mL Adjuvant 100-200 ?l/mL Diluent - DMEM containing q.s. Gentamycin (30 ?g/mL of diluent volume) Formaldehyde (0.1% of diluent volume)

(33) All groups were challenged with intrathecal inoculation of 1 ml PBS containing approximately 10.sup.5 pfu of a heterologous strain of WNV (NY99, 4132, crow isolate). The challenge was conducted under ketamine-xylazine anesthesia.

(34) Horses were monitored for a maximum of 14 days, then humanely euthanized. Those that developed severe disease prior to 14 days were euthanized prematurely.

(35) The following data were collected to assess the effectiveness of the vaccine: Basic clinical evaluation Body temperature Assay for viremia Histopathology: two sections of brainstem were evaluated by a board-certified veterinary pathologist.

(36) Sera collected on appropriate days were evaluated for characterization of serologic responses to challenge.

Results and Discussion

(37) Viremia after challenge and serum neutralization titers were considered the primary outcome variables in this study. The first block of horses that had been vaccinated were 100% protected from viremia after challenge in this study. In comparison, 4 of the 5 control horses demonstrated viremia for 4-5 days post-challenge and 1 of 5 control horses demonstrated viremia for 1 timepoint. In addition, serum neutralization titers of vaccinated horses were statistically significantly higher than those of control horses at each time point examined after vaccination. Furthermore, the data establish that a WNV vaccine that provides a serum neutralization titer of 1:4 or higher is effective in preventing WNV viremia. The serum titers and viremia after challenge for Block 1 is summarized in Table 1 below:

(38) TABLE-US-00002 TABLE 1 Serum Titers and Viremia for Block 1 Serum Titer Viremia After Challenge Horse Number Treatment Day of Challenge Highest Titer 1 Control <2 390 2 Vaccinate 12 <5 3 Vaccinate 12 <5 4 Control <2 65 5 Control <2 1475 6 Vaccinate 6 <5 7 Vaccinate 97 <5 8 Vaccinate 10 <5 9 Vaccinate 21 <5 10 Vaccinate 35 <5 11 Vaccinate 10 <5 12 Vaccinate 24 <5 13 Vaccinate 4 <5 14 Control <2 235 15 Control <2 165

(39) Viremia after challenge and serum neutralization titers were also considered the primary outcome variables in the second block of horses in this study. In the second block of horses only one vaccinate group horse displayed any timepoints of viremia throughout the challenge period. That horse had 3 separate timepoints on 3 mornings (not those same evenings) with minimal value readings of 5 (where <5 is negative). All control horses in the study (with the exception of one horse which exited the study prematurely but displayed definitive WNV histopathology and was excluded from evaluation) showed high levels of viremia for 1-8 timepoints after challenge.

(40) Since viremia is a prerequisite before virus can cross the blood-brain barrier to cause WNV encephalitis, viremia is well justified as the primary parameter for evaluation of protection in an experimental study of this type.

(41) This study demonstrated that 2 doses of the experimental combination vaccine administered to foals 4 to 5 months of age reliably and effectively stimulated protective serological serum neutralization titers. In addition the data confirm that post vaccination SN titers as low as 1:4 resulting from vaccination using an effectively batched antigen amount of West Nile Virus in this experimental combination vaccine protected vaccinated horses from viremia, clinical disease, and histopathology after a severe intrathecal challenge with a heterologous strain of West Nile Virus.

(42) Histopathology also was different between the two groups with the likelihood of lesions in vaccinates being 40% less in Block 2 and 100% less in Block 1 than the likelihood of lesions in control animals challenged with virulent West Nile virus.

(43) In addition a Control Group horse became weak on his hind legs on Day 9 post-challenge and got progressively worse until he was no longer able to stand. Histopathology of the pons and medulla from this horse showed severe encephalitis and myelitis consistent with WNV pathology that was more prevalent than signs of disease from any other horse in this study.

(44) Two Block 2 control horses in this study displayed 3 days each of clinical signs relating to infection with West Nile Virus. One other control horse had a single timepoint of weakness due to disease. Another control horse did not display any timepoints of clinical signs, although it had multiple days of viremia. Although several vaccinated Block 2 horses in the study had mild to moderate histopathological changes in tissue as a result of the intrathecal challenge of WNV, only very mild clinical disease (mild head tremors) was noted for one vaccinate on one day of the study as compared to multiple days of clinical disease in 2 control horses and a single day of clinical disease in a third control horse.

(45) The results demonstrated that the vaccine is effective and that an immunogenic reaction is induced in the animals that were administered the vaccine. The effectiveness of the vaccine was evidenced in this example by reduction in WNV viremia, by stimulation of high serum neutralization titers to WNV, and by prevention of WNV related clinical signs and histopathology in the brain and meninges. Because this vaccine is comprised of unique constituents, including a long lasting non-metabolizable adjuvant, it was formulated in a low 1 mL dose volume to provide a high degree of safety as a highly immunogenic low passage whole inactivated virus WNV isolate of recent origin and high epidemiological prevalence, and a WNV isolated from the tissues of an infected horse, it provides more comprehensive safety and effectiveness than other vaccines currently available. Additionally, it has the effect of providing a safe vaccine when administered to animals.

Example 3

(46) This example illustrates the efficacy of the immunogenic composition of the present invention against infection by EHV-4

Materials and Methods

(47) Thirty-seven (37) horses, 4-5 months of age, were used in this study. Horses were randomly assigned to either vaccinate or control groups by random number generator and then vaccinated. Twenty-four (24) horses served as vaccinates and thirteen (13) horses were mock-vaccinated control horses. All horses had low (?1:14, avg.=1:7) EHV-4 serum neutralization (SN) titers prior to initiation of the study, indicative of horses susceptible to infection. The vaccine used was an experimental vaccine and had the following components:

(48) The final formulated vaccine contains the following ingredients per 1 mL dose:

(49) TABLE-US-00003 EHV-1 (PTA-9525) 10.sup.7.0-9.0 TCID.sub.50/mL Influenza A2/Ohio/03 (PTA-9522) 10.sup.6.0-9.5 TCID.sub.50/mL Influenza A2/KY/95 (PTA-9523 10.sup.6.0-9.5 TCID.sub.50/mL Influenza A2/NewMarket/2/93 (PTA-9524) 10.sup.6.0-9.5 TCID.sub.50/mL Tetanus Toxoid 5-10 CPU Eastern Equine Encephalomyelitis, 10.sup.6.7-9.2 TCID.sub.50/mL (ATCC Deposit No. PTA-9412) Western Equine Encephalomyelitis, 10.sup.6.7-9.2 PFU/mL (ATCC Deposit No. PTA-9410) Venezuelan Equine Encephalomyelitis, 10.sup.6.7-9.2 TCID.sub.50/mL (ATCC Deposit No. PTA-9411) West Nile Virus, Horse Origin 2005 10.sup.7.0-9.0 TCID.sub.50/mL (ATCC Deposit No. PTA-9409) Adjuvant 100-200 ?l Glycerol 100-200 ?l EDTA 240 mM solution 10-20 ?l Diluent - DMEM containing q.s. Gentamicin (30 ?g/mL of diluent volume) Formaldehyde (0.1-0.2% of diluent volume)

(50) The Experimental Vaccine was administered intramuscularly in a 1 mL dose volume to each of 24 horses in the vaccinate group. Thirteen horses in the control group received a 1 mL dose of adjuvanted DMEM (Lot 004) containing excipients used in the 9-way vaccine (Gentamicin and formaldehyde) but no antigens. Challenge inoculation of virulent EHV-4 HRA005 strain virus was performed 15 days post-booster vaccination.

(51) Each of the vaccinated and control horses was challenged with an EHV-4 strain of virus (HRA005). The titer of the dilute challenge virus was sufficient to provoke disease due to EHV infection in the non-vaccinated horses.

(52) SEDIVET? (romifidine hydrochloride), a sedative and analgesic, was administered intravenously to each horse prior to challenge at a dosage of 50 ?g/kg of body weight. Each horse was then challenged with the EHV-4 strain HRA005 virus. The challenge virus was administered intranasally as an aerosol produced by a nebulizer into an Equine AeroMask (Trudell Medical International, Ontario, Canada).

(53) Daily morning rectal temperatures were recorded for each of the 37 vaccinated and control horses on Day of Challenge and for 14 days post challenge by means of a calibrated, electronic thermometer (GSA Electronics) probe. The daily rectal temperatures were recorded in degrees Fahrenheit (? F.).

Complete Blood Cell Counts

(54) Venous blood from each of the 37 vaccinated and control horses was collected daily on the Day of Challenge and for 14 days post-challenge directly into a Vacutainer Disodium EDTA tube for Complete Blood Counts.

Nasal Exudate Evaluation

(55) All nasal exudate observations were made prior to collection of nasopharyngeal swabs. On the Day of Challenge and for 14 days post challenge, the nasal passages and muzzle of each of the 37 vaccinated and control horses were examined and graded using the grading and scoring description listed below.

(56) The scoring grades of 0 through 6 were assigned on the basis of the severity of the disease indicated by each of the following classifications:

(57) TABLE-US-00004 TABLE 2 Scores for Clinical Symptoms Score sheet Score Description of symptoms designation 0 Essentially normal indicates the horse EN was clean and essentially free of nasal exudate 1 Slight clear serous discharge that may C-1 be frequently observed in both diseased and normal horses 2 Moderate clear serous discharge is C-2 indicative of a definite increase in volume over that normally observed 3 Copious clear serous discharge that is C-3 generally observed only in diseased horses 1.5 Very slight mucopurulent discharge VSM indicates that mucus was definitely present in small amounts in either one or both nostrils 2 Slightly mucopurulent is a discharge SM easily observed in one or both nostrils 4 Moderately mucopurulent indicates that MM mucoid discharges were present in large quantities in both nostrils 6 Heavy mucopurulent indicates that HM copious amounts of a mucoid discharge filled both nostrils

Nasopharyngeal Viral Isolation Methods

(58) On each observation test day each nasal passage of each vaccinated and control was sampled deeply by means of sterile swabs. On collection, each of two swabs were immediately placed in a single tube containing 4 mL of chilled transport medium (Dulbecco's Minimal Essential Medium (DMEM) supplemented with 2% FBS, 2? Pen/Strep, 2? Gentamicin, and 2? Amphotericin B).

(59) For isolation of virus, the tubes were mixed, the swabs aseptically removed, and the medium centrifuged at 1500 rpm for 10 minutes to remove particulates. Medium was filtered through a 0.2? syringe filter prior to inoculation on tissue culture cells. One mL of the clarified transport medium was used to inoculate a 2 cm.sup.2 one day old monolayer of ED cells grown in a 24 well tissue culture plate from which the growth medium had been aseptically removed. Following inoculation, the inoculum was allowed to adsorb on the cell monolayer for one hour at 37? C. in a humidified incubator containing a 5% CO.sub.2 atmosphere. After the adsorption period, an additional 1 mL of re-feed medium (DMEM containing 2-5% fetal bovine serum (FBS), 2 mM L-glutamine and 3? Gentamicin and 2? Amphotericin B) was added to each well. Following addition of re-feed media the plates were then incubated at 37? C. in a CO.sub.2 incubator. Each test and control tissue culture well was examined microscopically for 7 days for signs of cytopathic effect (CPE) typical of the EHV-4 challenge virus. Wells that were negative at the end of the 7 day observation period were subcultured onto fresh cells and observed for an additional 7 days.

Serum Neutralization Testing Procedure

(60) A standard microtiter serum neutralization test was employed in this study. All sera were tested in sterile flat bottom microtiter plates using 5 wells per dilution and an 8 well dilution series for each of the 5 test wells. Each of the 5 test wells contained 25 ?l of serum dilution mixed with 25 ?l of the indicator virus and 150 ?l of a freshly planted ED cell suspension containing approximately 5?10.sup.4 cells. The test indicator virus used was EHV-4 HRA005 Lot 033106 SN Stock Virus. In all tests the indicator virus back titration titers ranged between 68-149 TCID.sub.50/25 ?l. Serum neutralizing antibody titers are expressed as Reed-Muench ID.sub.50 titers.

(61) For performance of the test, two-fold dilutions of each test serum was made in a sterile flat bottom microtiter plate using five replicate wells per test serum and an 8 well dilution series. Dilutions were made with an adjustable volume single or multi-channel pipetting instrument using sterile microtiter tips. The volume of serum added each of 5 wells of the first row was 50 ?l. All other wells contained 25 ?l of DMEM (no FBS). Following serial dilution down the plate, 25 ?l was discarded from the last row. 25 ?l of a pre-determined dilution of the indicator virus was added to each test well. Plates were then mixed and incubated for one hour at 37? C. in 5% CO.sub.2. On conclusion of the incubation period, 150 ?l of a suspension containing 5?10.sup.4 ED cells was added to each test and cell control well. The plates were incubated at 37? C. in a CO.sub.2 incubator for 5-7 days, at which time plates were microscopically examined for CPE typical of EHV-4. However, any other commercial available test or any test described in the prior art could be used for this purpose.

Results and Conclusion

Nasal Exudate Evaluation

(62) The vaccination group by day interaction was statistically significant for the nasal discharge scores (P<0.05, Table 1). Statistically significant group effects were seen on Days 6-10 and Day 14 post-challenge (lower nasal scores in the vaccinated group).

(63) When the daily scores were summed over the post-challenge period, horses in the vaccinated group had lower total scores than those in the control group (P<0.05, Table 1). The mitigated fraction was estimated to be 0.824 (95% ASE CI: 0.629, 1.000).

(64) TABLE-US-00005 TABLE 3 Nasal Discharge Score Mitigated fraction Control Vaccinate P-value (95% ASE CI) Cumulative nasal 28.9 13.6 <0.0001 0.824 discharge score (0.629, 1.000)

Conjunctivitis

(65) The vaccination group by day interaction was statistically significant for the conjunctivitis scores. Statistically significant group effects were seen on Days 6, 7, 9, 10, 13 and 14 post-challenge (lower scores in the vaccinated group on 5 of the 6 days, P<0.05, FIG. 2).

Serological Studies

(66) Titers were log transformed prior to the statistical analysis. The vaccination group by day interaction was statistically significant for SN titers. Statistically significant group effects were seen on Day 0 (pre-vaccination; control group titers>vaccinated group titers), Days 35 (the day of challenge) and 7 and 14 days post-challenge (study days 42 and 49). Horses in the vaccinated group had higher titers on Days 35, 42 and 49 than those in the control group (P<0.05, Table 4).

(67) TABLE-US-00006 TABLE 4 Titers Study day Control Vaccinated P-value 0 8.31 5.74 0.0303 21 8.25 6.51 0.1639 35 (day of challenge) 6.12 8.56 0.0495 42 4.57 7.27 0.0069 49 4.87 13.12 <0.0001

White Blood Cell Counts (WBC) and Lymphocyte Counts

(68) The vaccination group by day interaction was statistically significant for WBC and lymphocyte counts. Statistically significant group effects were seen on Days 4-6 (WBC) and Days 4 and 5 (lymphocytes) post-challenge. Horses in the vaccinated group were protected from leucopenia due to EHV4 disease and had higher WBC and lymphocyte counts than those in the control group (P<0.05).

Discussion and Conclusions

(69) In this study, moderate and adequate clinical signs of EHV-4 infection were seen after challenge. Significantly fewer clinical signs of nasal exudate were seen in vaccinated horses on Days 6-10 and Day 14 post-challenge. Conjunctivitis scores were significantly lower in vaccinated horses on Days 7, 9, 10, 13, and 14 post-challenge. Despite the adequate display of clinical signs following challenge, virus shedding in nasal swab samples was infrequent following this EHV-4 challenge. Nasal swabs were examined by virus isolation in cell culture.

(70) Significant group effects for WBCs and lymphocytes were seen on Days 4-6 (WBC) and Days 4-5 (lymphocytes) with vaccinated animals showing higher WBC and lymphocyte counts than control horses. These values establish that control horses did succumb to the immunosuppression brought on by infection with Herpesvirus, and also demonstrate that vaccination with a cross-protective strain of EHV-1 allowed vaccinated horses to be more refractive to the immunosuppressive properties of Herpesvirus infection. Additionally, horses in the vaccinated group had higher serum neutralization titers on Days 35, 42 and 49 than those in the control group

(71) Data from this study confirm that horses vaccinated with a multi-component vaccine containing EHV-1 demonstrate cross-protective immunity when challenged with a heterologous EHV-4 challenge organism.

Example 4

(72) This example is to illustrate the efficacy of the combination vaccine of the present invention as well as duration of immunity.

Materials and Methods

(73) The influenza viral antigen used in the vaccine evaluated in this study was produced on Madin Darby Canine Kidney (MDCK) cells. Following growth, viral fluids were filtered, formalin inactivated, and concentrated. The inactivated viral fluids were tested for residual live virus after inactivation. On completion of satisfactory residual live virus testing the inactivated viral fluids were then used to formulate a vaccine which also contained inactivated Venezuelan, TC-83 strain (ATCC Accession No. PTA-9411), Eastern, NJO strain (ATCC Accession No. PTA-9412), and Western, Fleming strain (ATCC Accession No. PTA-9410), equine encephalomyelitis viruses, inactivated EHV-1 (ATCC Accession No. PTA-9525), inactivated influenza A/equine-2/Kentucky/95 (ATCC Accession No. PTA-9523) and influenza A/equine-2/NewMarket/2/93 (ATCC Accession No. PTA-9524) viruses, inactivated West Nile Virus, Horse Origin 2005 (ATCC Accession No. PTA-9409), and tetanus toxoid.

(74) Vaccine was formulated to appropriate specifications for all antigens included in the product. Influenza A/equi-2/Ohio/03 (ATCC Accession No. PTA-9522) antigen was added to the vaccine at a pre-inactivation titer of 10.sup.6.7 TCID.sub.50/mL.

(75) The final formulated vaccine contains the following ingredients per 1 mL dose:

(76) TABLE-US-00007 EHV-1 10.sup.7.0-9.0 TCID.sub.50/mL Influenza A2/Ohio/03 10.sup.6.7-9.5 TCID.sub.50/mL Influenza A2/KY/95 10.sup.6.7-9.5 TCID.sub.50/mL Influenza A2/NewMarket/2/93 10.sup.6.7-9.5 TCID.sub.50/mL Tetanus Toxoid 5-10 CPU Eastern Equine Encephalomyelitis 10.sup.6.7-9.2 TCID.sub.50/mL Western Equine Encephalomyelitis 10.sup.6.7-9.2 PFU/mL Venezuelan Equine Encephalomyelitis 10.sup.6.7-9.2 TCID.sub.50/mL West Nile Virus 10.sup.7.0--9.0 TCID.sub.50/mL Adjuvant (preferably mineral oil) 100-200 ?l Glycerol 100-200 ?l EDTA 240 mM solution 10-20 ?l Diluent - DMEM containing q.s. Gentamicin (30 ?g/mL of diluent volume) Formaldehyde (0.1-0.2% of diluent volume)

(77) Twenty-six (26) horses, 4-5 months of age, were used in this study. Fifteen horses served as vaccinates and eleven horses were mock-vaccinated control horses.

(78) Vaccine was administered intramuscularly in a 1 mL dose volume to each of 15 horses in the vaccinate group. Eleven horses in the control group received a 1 mL dose of adjuvanted DMEM (Lot 004) containing excipients used in the 9-way vaccine (Gentamicin and formaldehyde) but no antigens. Challenge inoculation of virulent influenza A/equi-2/Ohio/03 strain virus was performed 4 months post-booster vaccination.

(79) Serum samples for serological evaluation were collected from the vaccinated and control horses prior to initial vaccination, at 21 days post first dose vaccination (day of booster vaccination), on the day of challenge, and at 7 and 14 days post challenge. Body temperature, whole blood samples, and nasal swabs were obtained from each horse on the day of challenge, and daily throughout the 10 day post-challenge observation period for a total of 11 observation days. Clinical data was also recorded daily for each horse for the 11-day observation period.

(80) Challenge

(81) The challenge virus seed of Influenza A/equi-2/Ohio/03 was produced in eggs. Challenge virus was diluted on the morning of challenge 1:20 with tissue culture media to affect a titer sufficient to cause clinical influenza in the non-vaccinated challenged horses.

(82) SEDIVET? (romifidine hydrochloride), a sedative and analgesic, was administered intravenously to each horse prior to challenge at a dosage of 50 ?g/kg of body weight. Each horse was then challenged with influenza A/equi-2/Ohio/03 virus. The challenge virus was administered intranasally as an aerosol produced by a nebulizer into an Equine AeroMask (Trudell Medical International, Ontario, Canada) by the following method:

(83) Four milliliters of challenge virus were placed into the nebulizer cup in the AeroMask device. A pressure hose was fitted from an air compressor to the inlet port of the nebulizer. The outlet tube was then inserted into the AeroMask attached to the head of the horse being challenged and air pressure was applied to the inlet port. During this time approximately two milliliters of challenge virus fluid was aerosolized directly into the nostrils of the horse being challenged.

Temperature

(84) Daily rectal temperatures were recorded for each of the 26 vaccinated and control horses on Day of Challenge and for 10 days post challenge by means of a calibrated, electronic thermometer (GSA Electronics) probe. The daily rectal temperatures were recorded in degrees Fahrenheit (? F.).

White Blood Cell Counts

(85) Venous blood from each of the 26 vaccinated and control horses was collected daily on the Day of Challenge and for 10 days post-challenge directly into a vacutainer Disodium EDTA tube for WBC counts.

Nasal Exudate Evaluation

(86) All nasal exudate observations were made prior to collection of nasopharyngeal swabs. On the Day of Challenge and for 10 days post challenge, the nasal passages and muzzle of each of the 26 vaccinated and control horses were examined and graded using the grading and scoring description listed below.

(87) The scoring grades of 0 through 6 were assigned on the basis of the severity of the disease indicated by each of the following classification:

(88) TABLE-US-00008 TABLE 5 Scoring Grades Score sheet Score Description of symptoms designation 0 Essentially normal indicates the horse EN was clean and essentially free of nasal exudate 1 Slight clear serous discharge that may C-1 be frequently observed in both diseased and normal horses 2 Moderate clear serous discharge is C-2 indicative of a definite increase in volume over that normally observed 3 Copious clear serous discharge that is C-3 generally observed only in diseased horses 1.5 Very slight mucopurulent discharge VSM indicates that mucus was definitely present in small amounts in either one or both nostrils 2 Slightly mucopurulent is a discharge SM easily observed in one or both nostrils 4 Moderately mucopurulent indicates that MM mucoid discharges were present in large quantities in both nostrils 6 Heavy mucopurulent indicates that HM copious amounts of a mucoid discharge filled both nostrils

Coughing

(89) Episodes of coughing on each observation day were counted for each horse during the entirety of the observation period, whether or not the individual animal was being examined by the investigator at that time. Observers other than the investigator recorded the number of episodes of coughing of each individual horse during the observation period. Scoring of coughing episodes was actual counts of coughing episodes per horse.

Conjunctivitis

(90) Conjunctivitis was evaluated daily at the time of nasal exudate evaluation. Conjunctivitis scores were recorded as 0=normal; 1=mild to moderate conjunctivitis and 2=severe conjunctivitis.

Nasopharyngeal Viral Isolation/Hemagglutination (Ha) Methods

(91) On each observation test day each nasal passage of each vaccinated and control was sampled deeply by means of sterile swabs. On collection, each of two swabs was immediately placed in a single tube containing 4 mL of chilled transport medium (Dulbecco's Minimal Essential Medium (DMEM) supplemented with 2% FBS, 2? Pen/Strep, 2? Amphotericin B).

(92) For isolation of virus, the tubes were mixed, the swabs aseptically removed, and the medium centrifuged at 1500 rpm for 10 to 15 minutes to remove particulates. Medium was filtered through a 0.2? syringe filter prior to inoculation on tissue culture cells. After filtration, 4-6% of sterile 85% sucrose solution was added to each sample for freezing at ?80? C. in order for all samples to be tested concurrently.

(93) All samples were tested in sterile flat bottom microtiter plates using five wells per dilution and a 4 well dilution series for each of the 5 test wells. Upon thawing, 22 ?L of the clarified sample medium was used to inoculate one day old monolayer of MDCK-S cells from which the growth medium had been aseptically removed and replaced with 200 ?l of influenza growth medium (DMEM containing 5-10 units/mL of 10,000 U stock solution Porcine Trypsin, 2 mM L-glutamine, 1? Pen-Strep and 1? Amphotericin B). The plates were then incubated at 35? C. in a CO.sub.2 incubator for 5-7 days. After the 5-7 day incubation period, 500 from all wells of the titration plates were transferred directly into a labeled 96 well vinyl HA plate. Chicken red blood cells were added to each well and allowed to settle for 30-90 minutes at room temperature. Wells were read for positive agglutination as evidence of presence of equine influenza virus.

Hemagglutination Inhibition (Hi) Testing Procedure

(94) Serum samples were prepared by dispensing 0.15 ml of each sample into a test tube and extracting with 0.3 mL of 0.01M Sodium Periodate Solution at room temperature for 15 minutes. Glycerol Solution 3% (0.125 mL) was added to each tube, mixed and incubated at room temperature for 15 minutes. All samples were then heat-inactivated at 56? C. for 30 minutes.

(95) A 0.5% solution of chicken red blood cells was prepared in PBS (SAFC catalog number 59321C) and standardized to an optical density of 0.5 at 550 nm.

(96) Extracted serum samples were tested in duplicate in U bottom polystyrene plates using a 2-fold dilution scheme in PBS ranging from 1:4 to 1:256, 25 ul per well. Influenza A/Equi2/Ohio03 stock virus (25 ?L) was added to serum sample dilution. Plates were gently tapped to mix, and incubated at room temperature for 30 minutes. After incubation, chicken red blood cells were added to each well and incubated undisturbed at room temperature for 1 to 1.5 hours. Results were read by observing plates for presence or absence of agglutinated red blood cells in each well. Antibody titer was determined as the highest dilution of serum at which agglutination did not occur.

Results and Conclusions

(97) When pooled across all timepoints post-challenge, vaccinated animals had lower total clinical scores than the control animals. When the total daily scores were summed over the post-challenge period, horses in the vaccinated group had lower total scores than those in the control group (P<0.05). The mitigated fraction was estimated to be 0.6485 (95% ASE CI: 0.3258, 0.9712).

(98) TABLE-US-00009 TABLE 6 Total Clinical Score Vaccination Group by day Outcome variable group Day interaction Total clinical score.sup.1 <0.0001 <0.0001 0.1321 .sup.1The GLIMMIX procedure would not converge, thus an ANOVA approach was used to evaluate the effect of vaccination over time after challenge. Results were interpreted through the bolded values.

(99) TABLE-US-00010 TABLE 7 Mitigated fraction - total cumulative clinical score Mitigated fraction.sup.2 Control Vaccinate P-value.sup.1 (95% ASE CI) Total cumulative 18.36.sup.3 9.93 0.0055 0.6485 clinical score.sup.2 (0.3258, 0.9712) .sup.1P-value from Wilcoxon's rank sum test .sup.2Nasal discharge score, conjunctivitis score and coughing score were summed with day and across all time points for each animal then ranked for the estimation of the mitigated fraction. .sup.3Mean rank

Nasal Discharge

(100) The main effect of vaccination was statistically significant and reduced nasal discharge due to the influenza challenge. When pooled across all time points post-challenge, vaccinated animals had lower nasal discharge scores than the control animals.

(101) TABLE-US-00011 TABLE 8 Nasal Discharge Score Vaccination Group by day Outcome variable group Day interaction Nasal discharge score.sup.1 0.0012 <0.0001 0.4627 .sup.1The GLIMMIX procedure would not converge, thus an ANOVA approach was used to evaluate the effect of vaccination over time after challenge. Results were interpreted through the bolded values.

Conjunctivitis

(102) For conjunctivitis, the main effect of vaccination was statistically significant. When pooled across all time points post-challenge, vaccinated animals had reduced conjunctivitis due to influenza infection as demonstrated by lower conjunctivitis scores than the control animals.

(103) TABLE-US-00012 TABLE 9 Conjunctivitis Score Vaccination Group by day Outcome variable group Day interaction Conjunctivitis score.sup.1 0.0187 0.0001 0.2498 .sup.1The GLIMMIX procedure would not converge, thus an ANOVA approach was used to evaluate the effect of vaccination over time after challenge. Results were interpreted through the bolded values.

Coughing

(104) Vaccine also protected against the cough resulting from equine influenza infection. Vaccinated animals had lower scores (P<0.05,) on Days 3, 5, 7, 8, and 9 post-challenge than control animals.

(105) TABLE-US-00013 TABLE 10 Coughing Score Vaccination Group by day Outcome variable group Day interaction Coughing score.sup.1 0.0004 0.0009 0.0275 .sup.1The GLIMMIX procedure would not converge, thus an ANOVA approach was used to evaluate the effect of vaccination over time after challenge. Results were interpreted through the bolded values.

Virus Shedding (Nasal Swabs)

(106) The vaccination also reduced the percent of horses shedding virus (P<0.05). The figure below represents that the percentage of vaccinated animals shedding virus was lower (P<0.05) on Days 3, 4, and 5 post-challenge than control animals.

(107) TABLE-US-00014 TABLE 11 Mitigated fraction - days virus shedding Mitigated fraction.sup.2 Control Vaccinate P-value.sup.1 (95% ASE CI) Days virus 2.sup.3 0 0.0004 0.7939 positive.sup.2,3 (0.5343, 1.0000) .sup.1P-value from Wilcoxon's rank sum test .sup.2The number of days of viral shedding was calculated then ranked for the estimation of the mitigated fraction. Asymptotic standard errors (ASE) were used to estimate the 95% confidence intervals (CI). .sup.3The median number of days positive results was obtained from the virus isolation assay.

Hi Titers

(108) The vaccine was also effective in eliciting protective antibody titers to equine influenza virus. Statistically significant higher titers in the vaccinated horses were seen on Day 36 (relative to vaccination), Day 154 (the day of challenge), 159 and 164. Horses in the vaccinated group had higher titers on each of these days than those in the control group (P<0.05).

WBC and Lymphocyte Counts

(109) The vaccination also protected horses from reduction in white blood cell counts seen following influenza virus challenge. (P<0.05). Vaccination with the combination vaccine provided statistically significant protection that was seen on Days 2 and 7 for WBC counts, and Days 2, 6, 7, and 8 post-challenge. Horses in the vaccinated group had higher WBC and lymphocyte counts than those in the control group (P<0.05). A four month Duration of Immunity challenge was performed to demonstrate efficacy of the influenza virus fractions of a multi-component vaccine that included West Nile Virus vaccine (Encephalomyelitis-Rhinopneumonitis-Influenza-West Nile Virus Vaccine, Eastern, Western & Venezuelan, Killed Virus, Tetanus Toxoid) containing 3 Equine influenza A/equi-2 virus strains, ATCC Accession Nos. PTA-9522, PTA-9523, and PTA-9524, each of which is currently relevant in the equine population of the Americas, Europe and Asia. Twenty-six horses (15 vaccinates and 11 controls) were vaccinated twice in 3 week intervals with a 1 mL dose of vaccine, or were mock vaccinated with adjuvanted media components of the vaccine without viral antigen. Four months post-booster vaccination, horses were challenged with a virulent live Equine Influenza A/equi-2/Ohio03 virus. This virulent virus is the current Equine Influenza A/equi-2 strain recommended for inclusion into vaccines by OIE and is currently recognized as the most pertinent strain involved in outbreaks in the United States.

(110) Results from this 4-month DOI challenge study show significant protective effects from challenge by vaccination with the test vaccine, a combination West Nile Virus vaccine with flu and other pertinent equine antigens. Importantly, vaccinated horses displayed statistically lower total clinical signs of influenza virus (nasal discharge, conjunctivitis, and coughing, P=0.0055) with a mitigated fraction estimated to be 0.6485 (95% ASE CI: 0.3258, 0.9712). Additionally, viral shedding was statistically lower in vaccinated horses than control horses (P=0.0004) with a mitigated fraction estimated to be 0.7939 (95% ASE CI: 0.5343, 1.0000). Hemagglutination inhibition titers were significantly higher in vaccinated horses than control horses, and white blood cell and lymphocyte counts remained significantly higher in vaccinated horses on multiple days of the study over those of control horses. No differences in rectal temperature were determined between the two groups.

(111) In conclusion, the data from this study demonstrate that administration of 2?1 mL intramuscular doses of this West Nile Virus combination vaccine administered at a 21 day interval to foals 4 to 5 months of age protected against virulent challenge with the Equine Influenza A/equi-2/Ohio03 virus and provided a duration of immunity of at least 4 months for this product.

Example 5

(112) This example illustrates the efficacy of an immunogenic composition of the present invention when challenged with (Equine Herpes Virus Type 1) EHV-1.

Materials and Methods

(113) The EHV-1 viral antigen used in the vaccine evaluated in this study was produced on Madin Darby Bovine Kidney (MDBK) cells. Following growth, viral fluids were filtered, BPL inactivated, and concentrated. The inactivated viral fluids were tested for residual live virus after inactivation. On completion of satisfactory residual live virus testing, the inactivated viral fluids were then used to formulate a vaccine which also contained inactivated Venezuelan Equine Encephalomyelitis, TC-83 strain (ATCC Accession No. PTA-9411) Eastern Equine Encephalomyelitis, NJO strain (ATCC Accession No. PTA-9412) and Western Equine Encephalomyelitis, Fleming strain (ATCC Accession No. PTA-9410) viruses, inactivated influenza A/equine-2/Kentucky/95 (ATCC Accession No. PTA-9523), influenza A/equine-2/NewMarket/2/93 (ATCC Accession No. PTA-9524) and influenza A/equine-2/Ohio/03 (ATCC Accession No. PTA-9522) viruses, inactivated West Nile Virus (ATCC Accession No. PTA-9409) and tetanus toxoid.

(114) Vaccine was formulated to minimum specifications for all antigens included in the product. EHV-1 antigen was added to the vaccine at a pre-inactivation titer of 10.sup.7.0 TCID.sub.50/mL.

(115) The final formulated vaccine contains the following ingredients per 1 mL dose:

(116) TABLE-US-00015 EHV-1 10.sup.7.0-9.0 TCID.sub.50/mL Influenza A2/Ohio/03 10.sup.6.7-9.5 TCID.sub.50/mL Influenza A2/KY/95 10.sup.6.7-9.5 TCID.sub.50/mL Influenza A2/NewMarket/2/93 10.sup.6.7-9.5 TCID.sub.50/mL Tetanus Toxoid 5-10 CPU Eastern Equine Encephalomyelitis 10.sup.6.7-9.2 TCID.sub.50/mL Western Equine Encephalomyelitis 10.sup.6.7-9.2 PFU/mL Venezuelan Equine Encephalomyelitis 10.sup.6.7-9.2 TCID.sub.50/mL West Nile Virus 10.sup.7.0--9.0 TCID.sub.50/mL Adjuvant (preferably mineral oil) 100-200 ?l Glycerol 100-200 ?l EDTA 240 mM solution 10-20 ?l Diluent - DMEM containing q.s. Gentamicin (30 ?g/mL of diluent volume) Formaldehyde (0.1-0.2% of diluent volume)

(117) Forty (40) horses, 4-5 months of age, were used in this study. Horses were randomly assigned to either vaccinate or control groups and were micro-chipped and then vaccinated. Twenty horses served as vaccinates and twenty horses were mock-vaccinated control horses. All horses had negative to low (<1:6) EHV-1 serum neutralization (SN) titers prior to initiation of the study, indicative of horses susceptible to infection.

(118) Vaccine was administered intramuscularly in a 1 mL dose volume to each of 20 horses in the vaccinate group. Twenty horses in the control group received a 1 mL dose of adjuvanted DMEM (Lot 004) containing excipients used in the 9-way vaccine (Gentamycin and formaldehyde) but no antigens. Challenge inoculation of virulent EHV-1 A183 strain virus was performed 15 days post-booster vaccination.

(119) Serum samples for serological evaluation were collected from the vaccinated and control horses prior to initial vaccination, at 21 days post first dose vaccination (day of booster vaccination), on the day of challenge, and at 7 and 14 days post challenge. Body temperature, whole blood samples, and nasal swabs were obtained from each horse on the day of challenge, and daily throughout the 14 day post-challenge observation period for a total of 15 observation days. Clinical data was also recorded daily for each horse for the 15-day observation period.

Challenge Procedure

Challenge Virus

(120) The original challenge virus seed used in this challenge study was the first passage of the original seed virus on Equine Dermal (ED) cells. This challenge virus was harvested and frozen at a titer of 10.sup.6.2 TCID.sub.50/mL.

Intranasal Challenge Method

(121) SEDIVET? (romifidine hydrochloride), a sedative and analgesic, was administered intravenously to each horse prior to challenge at a dosage of 50 ?g/kg of body weight. Each horse was then challenged with approximately 10.sup.6.5 TCID.sub.50 of EHV-1 strain. The challenge virus was administered intranasally as an aerosol produced by a nebulizer into an Equine AeroMask (Trudell Medical International, Ontario, Canada) by the following method:

(122) A pressure hose was fitted from an air compressor to the inlet port of the nebulizer. The outlet tube was then inserted into the AeroMask attached to the head of the horse being challenged and approximately 10 psi of air pressure was applied to the inlet port for four minutes. During this time approximately two milliliters of a 10.sup.62 TCID.sub.50/mL challenge virus fluid was aerosolized directly into the nostrils of the horse being challenged.

Pre and Post Challenge Evaluation Parameters

Temperature

(123) Daily morning rectal temperatures were recorded for each of the 40 vaccinated and control horses on Day of Challenge and for 14 days post challenge by means of a calibrated, electronic thermometer (GSA Electronics) probe. The daily rectal temperatures were recorded in degrees Fahrenheit (? F.).

White Blood Cell Counts

(124) Venous blood from each of the 40 vaccinated and control horses was collected daily on the Day of Challenge and for 14 days post-challenge directly into a vacutainer Disodium EDTA tube for WBC counts.

Nasal Exudate Evaluation

(125) All nasal exudate observations were made prior to collection of nasopharyngeal swabs. On the Day of Challenge and for 14 days post challenge, the nasal passages and muzzle of each of the 40 vaccinated and control horses were examined and graded using the grading and scoring description listed below.

(126) The scoring grades of 0 through 6 were assigned on the basis of the severity of the disease indicated by each of the following classification:

(127) (EN) Essentially normal indicates the horse was clean and essentially free of nasal exudate, score, 0;

(128) (C-1) Slight clear serous discharge that may be frequently observed in both diseased and normal horses, score 1;

(129) (C-2) Moderate clear serous discharge is indicative of a definite increase in volume over that normally observed, score 2;

(130) (C-3) Copious clear serous discharge that is generally observed only in diseased horses, score 3;

(131) (VSM) Very slight mucopurulent discharge indicates that mucus was definitely present in small amounts in either one or both nostrils, score 1.5;

(132) (SM) Slightly mucopurulent is a discharge easily observed in one or both nostrils, score 2;

(133) (MM) Moderately mucopurulent indicates that mucoid discharges were present in large quantities in both nostrils, score 4; and

(134) (HM) Heavy mucopurulent indicates that copious amounts of a mucoid discharge filled both nostrils, score 6.

Nasopharyngeal Viral Isolation Methods

(135) On each observation test day each nasal passage of each vaccinated and control was sampled deeply by means of a sterile swabs. On collection, each of two swabs were immediately placed in a single tube containing 4 mL of chilled transport medium (Dulbecco's Minimal Essential Medium (DMEM) supplemented with 2% FBS, 2? Pen/Strep, 2? Gentamicin, and 2? Amphotericin B).

(136) For isolation of virus, the tubes were mixed, the swabs aseptically removed, and the medium centrifuged at 1500 rpm for 10 minutes to remove particulates. Medium was filtered through a 0.2? syringe filter prior to inoculation on tissue culture cells. One mL of the clarified transport medium was used to inoculate a 2 cm.sup.2 one day old monolayer of ED cells grown in a 24 well tissue culture plate from which the growth medium had been aseptically removed. Following inoculation, the inoculum was allowed to adsorb on the cell monolayer for one hour at 37? C. in a humidified incubator containing a 5% CO.sub.2 atmosphere. After the absorption period, an additional 1 mL of re-feed medium (DMEM containing 2-5% fetal bovine serum (FBS), 2 mM L-glutamine and 3? Gentamicin and 2? Amphotericin B) was added to each well. Following addition of re-feed media the plates were then incubated at 37? C. in a CO.sub.2 incubator. Each test and control tissue culture well was examined microscopically for 7 days for signs of cytopathic effect (CPE) typical of the EHV-1 A183 challenge virus. Wells that were negative at the end of the 7 day observation period were subcultured onto fresh cells and observed for an additional 7 days.

WBC Buffy Coat Virus Isolation

(137) Venous blood from each of the 40 vaccinated and control horses was collected on the Day of Challenge and daily for 14 days post-challenge by vacutainer into a Disodium EDTA tube. After permitting gravity sedimentation of the erythrocytes in the tube of EDTA anti-coagulated blood, the plasma and white blood cells were pipetted off and placed in a sterile 5 mL snap-cap tube. The plasma and white blood cell mixture was centrifuged at 1500 RPM for 10-15 minutes to pellet the white blood cells. The pellet was washed twice with 3 mL of phosphate buffered saline (PBS) containing 2? Pen/Strep, 2? Gentamicin, and 2? Amphotericin B. Cells were then suspended in 4 mL of DMEM supplemented with 2% fetal bovine serum (FBS) and 2? Pen/Strep, 2? Gentamicin, and 2? Amphotericin B. One mL buffy coat suspension was used to inoculate a 2 cm.sup.2 one day old monolayer of ED cells grown in a 24 well tissue culture plate from which the growth medium had been aseptically removed. Following inoculation, the inoculum was allowed to adsorb on the cell monolayer for one hour at 37? C. in a humidified incubator containing a 5% CO.sub.2 atmosphere. After the adsorption period, an additional 1 mL of re-feed medium (DMEM containing 5-7% fetal bovine serum (FBS), 2 mM L-glutamine and 1? Gentamicin was added to each well. Following addition of re-feed media the plates were then incubated at 37? C. in a CO.sub.2 incubator. Wells could not be observed microscopically due to large volume of white blood cells settled on the monolayer. Therefore, at the end of 7 days, all wells were subcultured onto fresh ED cells using 0.5 ml of the 1.sup.st passage as inoculum. The subculture was observed for 7 days for CPE typical of challenge virus infection.

Serum Neutralization Testing Procedure

(138) A standard microtiter serum neutralization test was employed in this study. All sera were tested in sterile flat bottom microtiter plates using five wells per dilution and an 8 well dilution series for each of the 5 test wells. Each of the 5 test wells contained 25 ?l of serum dilution mixed with 25 ?l of the indicator virus and 150 ?l of a freshly planted ED cell suspension containing approximately 5?10.sup.4 cells. The test indicator virus used was EHV-1 subtype 1 strain A183. In all tests the indicator virus back titration titers ranged between 109 to 263 TCID.sub.50/25 ?l. Serum neutralizing antibody titers are expressed as Reed-Muench ID.sub.50 titers.

(139) For performance of the test, two-fold dilutions of each test serum was made in a sterile flat bottom microtiter plate using five replicate wells per test serum and an 8 well dilution series. Dilutions were made with an adjustable volume single or multi-channel pipetting instrument using sterile microtiter tips. The volume of serum added each of 5 wells of the first row was 50 ?l. All other wells contained 25 ?l of DMEM (no FBS). Following serial dilution down the plate, 25 ?l was discarded from the last row. 25 ?l of a pre-determined dilution of the indicator virus was added to each test well. Plates were then mixed and incubated for one hour at 37? C. in 5% CO.sub.2. On conclusion of the incubation period, 150 ?l of a suspension containing 5?10.sup.4 ED cells was added to each test and cell control well. The plates were incubated at 37? C. in a CO.sub.2 incubator for 3 days, at which time plates were microscopically examined for CPE typical of EHV-1. Alternatively, any conventional or commercial available assay can be used or those of skill in the art would be able to follow the guidance herein.

Results and Conclusion

(140) Nasal discharge scores, nasal shedding of EHV-1 and conjunctivitis scores were considered the primary outcome variables. All other outcomes were considered secondary.

(141) TABLE-US-00016 TABLE 12 Summary of the statistical analysis (P-values) Vaccination Group by day Outcome variable group Day interaction Nasal discharge score.sup.1 0.0001 <.0001 <0.0001 Virus shedding.sup.1 0.0028 <0.0001 0.0863 Conjunctivitis score.sup.1 0.0020 <0.0001 0.0017 SN Titers <0.0001 <0.0001 <0.0001 WBC 0.3064 <0.0001 <0.0001 .sup.1The GLIMMIX procedure would not converge, thus an ANOVA approach was used to evaluate the effect of vaccination over time after challenge. Results were interpreted through the bolded values.

Nasal Exudate Evaluation

(142) The vaccination group by day interaction was statistically significant for the nasal discharge scores (P<0.05). Statistically significant group effects were seen on Days 4, 5 and on Days 7-11 post-challenge (lower nasal scores in the vaccinated group, P<0.05,). When the daily scores were summed over the post-challenge period, horses in the vaccinated group had lower total scores than those in the control group (P<0.05). The mitigated fraction was estimated to be 0.7250 (95% ASE CI: 0.4886, 0.9614).

(143) TABLE-US-00017 TABLE 13 Mitigated fraction - nasal discharge and conjunctivitis scores, nasal virus shedding (mean ranks) Mitigated fraction.sup.2 Control Vaccinate P-value.sup.1 (95% ASE CI) Nasal discharge 27.75 13.25 <0.0001 0.7250 (0.4886, 0.9614) Days shedding virus.sup.2 24.43 15.78 0.0068 0.4925 (0.1896, 0.7954) Conjunctivitis 25.80 15.20 0.0038 0.5300 (0.2463, 0.8137) .sup.1P-value from Wilcoxon's rank sum test .sup.2Nasal discharge and conjunctivitis scores were summed across all time points then ranked for the estimation of the mitigated fraction. The number of days of viral shedding was calculated then ranked for the estimation of the mitigated fraction. Asymptotic standard errors (ASE) were used to estimate the 95% confidence intervals (CI).

(144) TABLE-US-00018 TABLE 14 Mean nasal discharge score (N = 20 horses per group) Days post-challenge Control Vaccinated P-value.sup.1 0 0.00 0.00 1.0000 1 0.00 0.00 1.0000 2 0.15 0.00 0.6029 3 0.33 0.48 0.6029 4 1.08 0.23 0.0033 5 1.43 0.40 0.0004 6 1.05 0.55 0.0833 7 1.50 0.68 0.0044 8 1.68 0.63 0.0003 9 2.13 0.50 <.0001 10 1.58 0.80 0.0074 11 0.98 0.23 0.0095 12 0.90 0.35 0.0568 13 1.23 0.90 0.2599 14 0.93 0.43 0.0833 .sup.1The GLIMMIX procedure would not converge, thus an ANOVA approach was used to evaluate the effect of vaccination over time on the nasal discharge score.

Conjunctivitis

(145) The vaccination group, by day interaction, was statistically significant for the conjunctivitis scores (P<0.05). Statistically significant group effects were seen on Days 5 and 6, and on Days 9-14 post-challenge (lower scores in the vaccinated group, P<0.05). When the daily scores were summed over the post-challenge period, horses in the vaccinated group had lower total scores than those in the control group (P<0.05). The mitigated fraction was estimated to be 0.5300 (95% ASE CI: 0.2463, 0.8137).

(146) TABLE-US-00019 TABLE 15 Mean conjunctivitis score (N = 20 horses per group) Days post-challenge Control Vaccinated P-value.sup.1 0 0.00 0.00 1.0000 1 0.00 0.00 1.0000 2 0.00 0.00 1.0000 3 0.05 0.00 0.7321 4 0.15 0.15 1.0000 5 0.70 0.25 0.0022 6 0.85 0.25 <.0001 7 0.75 0.65 0.4936 8 0.45 0.35 0.4936 9 0.50 0.15 0.0168 10 0.45 0.15 0.0403 11 0.50 0.05 0.0022 12 0.45 0.00 0.0022 13 0.45 0.05 0.0063 14 0.35 0.05 0.0403 .sup.1The GLIMMIX procedure would not converge, thus an ANOVA approach was used to evaluate the effect of vaccination over time.

Virus Isolation from Nasopharyngeal Swabs

(147) The main effect of vaccination group was statistically significant (fewer animals shedding in the vaccinated group, P<0.05). When the number of days shedding was evaluated, horses in the vaccinated group had fewer days of virus shedding than those in the control group (P<0.05, Table 2). The mitigated fraction was estimated to be 0.4925 (95% ASE CI: 0.1896, 0.7954).

(148) TABLE-US-00020 TABLE 16 Proportion virus shedding (nasal swab, N = 20 horses per group) Days post-challenge Control Vaccinated 0 0.00 0.00 1 0.00 0.00 2 0.05 0.05 3 0.05 0.10 4 0.20 0.05 5 0.25 0.15 6 0.45 0.25 7 0.45 0.35 8 0.50 0.10 9 0.45 0.15 10 0.25 0.00 11 0.35 0.10 12 0.30 0.10 13 0.15 0.00 14 0.05 0.00

White Blood Cell Counts

(149) The vaccination group by day interaction was statistically significant for WBC counts (P<0.05, Table 1). Statistically significant group effects were seen on Days 2 and 3 post-challenge. Horses in the vaccinated group had higher WBC counts than those in the control group, indicating the vaccine prevented the horses from suffering the leucopenia caused by infection with EHV 1 (P<0.05).

(150) TABLE-US-00021 TABLE 17 Mean WBC counts (N = 20 horses per group) Days post-challenge Control Vaccinated P-value.sup.1 1 14.0413 14.4887 0.6081 2 10.7963 14.1287 0.0001 3 11.1263 14.0687 0.0008 4 11.5013 13.1037 0.0667 5 10.7413 11.1987 0.6001 6 9.1063 9.4187 0.7203 7 10.1563 9.9037 0.7721 8 10.7813 10.6037 0.8386 9 11.1813 12.0737 0.3065 10 11.9713 12.4187 0.6081 11 12.6713 13.2137 0.5341 12 13.2913 13.5637 0.7549 13 14.7063 14.1737 0.5415 14 15.8463 14.4587 0.1121 .sup.1P-values from the ANOVA

Serological Studies

(151) Titers were log transformed prior to the statistical analysis. The vaccination group by day interaction was statistically significant for SN titers (P<0.05). Statistically significant group effects were seen on Days 35 (the day of challenge) and 7 and 14 days post-challenge (study days 42 and 49). Horses in the vaccinated group had higher titers than those in the control group (P<0.05).

(152) TABLE-US-00022 TABLE 18 Geometric mean - serum neutralization titers (N = 20 horses per group) Study day Control Vaccinated P-value.sup.1 0 3.987 3.384 0.4005 21 3.190 2.624 0.3168 35 (day of challenge) 3.480 6.863 0.0006 42 3.519 19.252 <0.0001 49 33.153 187.417 <0.0001 .sup.1P-values from the ANOVA. Serum neutralization titers were log (natural) transformed prior to the statistical analysis.

Results and Discussion

(153) Respiratory disease caused by equine herpesvirus type 1 is usually an epidemic disease of na?ve weanling and yearling horses that occurs in the first year of life, usually in the fall and winter months. Signs of acute infection include fever up to 106? F., viremia and leucopenia and/or neutropenia. Nasal discharge is usually evident during febrile periods of this first exposure. Natural infection by EHV-1 does not result in permanent immunity of the respiratory tract. Indeed, horses may be re-infected naturally every 3 to 6 months throughout life. After the first experience with this virus, re-infection results in production of virus, but usually without clinical signs of disease, resulting in carrier animals that act as natural reservoirs of the virus.

(154) The equine herpesvirus-1 multi-component vaccine described in this report has been shown to be efficacious in reducing the respiratory manifestations, clinical symptoms and virus shedding from nasal exudate of horses challenged with a virulent heterologous strain of Equine Herpesvirus type 1. Reduction in shedding of virus from the respiratory route is important epidemiologically due to this being the natural route of exposure to naive animals as well as for re-infection of herd mates from those experiencing a natural infection. It was also a safe vaccine with no adverse reactions, either systemic or at the site of vaccine administration, observed following vaccine use in the study horses.

(155) In this study, vaccination group by day interaction showed statistical significance for the primary outcome variables nasal discharge scores and conjunctivitis. Statistically significant group effects were seen in the vaccinate group for nasal discharge on Days 4, 5 and on Days 7-11 post-challenge. Group effects for conjunctivitis were also statistically significant on Days 5 and 6 and 9-14 with lower scores in the vaccinate group (P<0.05). This is significant epidemiologically because the EHV-1 virus is delicate and does not survive in the environment readily. Close contact is important for transmission of disease through nasal secretions containing virulent EHV-1 virus (Campbell and Studdert, 1983).

(156) Importantly, another primary outcome variable in this study, virus shedding in nasal exudates, showed a main effect of vaccination as statistically significant (P<0.05). Horses in the vaccinated group also had statistically fewer days of virus shedding than those in the control group (P<0.05).

(157) Serum neutralization titers were statistically significant after vaccination and throughout the challenge period in vaccinates versus control horses (P<0.05). Humoral immunity and mucosal antibodies may be important in determining whether an EHV-1 infection becomes a productive or limited infection event (Kidd, Smith, Hannant, et. al, 1994).

Example 6

(158) This example illustrates the efficacy and 6 month duration of immunity of an immunogenic composition of the present invention when challenged with West Nile Virus.

Materials and Methods

(159) The WNV viral antigen used in the vaccine evaluated in this study was produced on E vero cells as described in Example 1. A total of 15 horses were randomly divided into groups, one being a control group of 5 horses. The vaccinated group of 10 horses received 2 doses of vaccine at 21-day intervals cells. On completion of satisfactory residual live virus testing the inactivated viral fluids were then used to formulate a vaccine which also contained inactivated Venezuelan Equine Encephalomyelitis, TC-83 strain (ATCC Accession No. PTA-9411), Eastern Equine Encephalomyelitis, NJO strain (ATCC Accession No. PTA-9412), and Western Equine Encephalomyelitis, Fleming strain (ATCC Accession No. PTA-9410) viruses, inactivated influenza A/equine-2/Kentucky/95 (ATCC Accession No. PTA-9523), influenza A/equine-2/NewMarket/2/93 (ATCC Accession No. PTA-9524) and influenza A/equine-2/Ohio/03 (ATCC Accession No. PTA-9522) viruses, inactivated West Nile Virus (ATCC Accession No. 9409) and tetanus toxoid. Vaccine was formulated to minimum specifications for all antigens included in the product.

(160) The final formulated vaccine contains the following ingredients per 1 mL dose:

(161) TABLE-US-00023 EHV-1 10.sup.7.0-9.0 TCID.sub.50/mL Influenza A2/Ohio/03 10.sup.6.7-9.5 TCID.sub.50/mL Influenza A2/KY/95 10.sup.6.7-9.5 TCID.sub.50/mL Influenza A2/NewMarket/2/93 10.sup.6.7-9.5 TCID.sub.50/mL Tetanus Toxoid 5-10 CPU Eastern Equine Encephalomyelitis 10.sup.6.7-9.2 TCID.sub.50/mL Western Equine Encephalomyelitis 10.sup.6.7-9.2 L PFU/mL Venezuelan Equine Encephalomyelitis 10.sup.6.7-9.2 TCID.sub.50/mL West Nile Virus 10.sup.7.0-9.0 TCID.sub.50/mL Adjuvant (preferably mineral oil) 100-200 ?l Glycerol 100-200 ?l EDTA 240 mM solution 10-20 ?l Diluent - DMEM containing q.s. Gentamicin (30 ?g/mL of diluent volume) Formaldehyde (0.1-0.2% of diluent volume)

(162) Fifteen horses were used in this study. Horses were randomly assigned to either vaccinate or control groups and then vaccinated. Ten horses served as vaccinates and five horses were mock-vaccinated control horses.

(163) The vaccine was administered intramuscularly in a 1 mL dose volume to each of the horses in the vaccinate group. Each control received a 1 mL dose of adjuvanted DMEM containing excipients used in the 9-way vaccine (gentamycin and formaldehyde) but no antigens.

(164) All groups were challenged approximately 6 months following vaccination with intrathecal inoculation of 1 ml PBS containing approximately 10.sup.5 pfu of a heterologous strain of WNV (NY99, 4132, crow isolate). The challenge was conducted under ketamine-xylazine anesthesia.

(165) Horses were monitored for a maximum of 14 days.

Results and Discussion

(166) Viremia after challenge was considered the primary outcome variable in this study. The horses that had been vaccinated were 90% protected from viremia after challenge in this study. In comparison, all of the 5 control horses demonstrated viremia for 3-5 days post-challenge.

(167) In addition, serum neutralization titers of vaccinated horses were significantly higher than those of control horses after vaccination. All the vaccinated horses developed measurable serum neutralization titers following vaccination, whereas none of the controls displayed any titer to WNV. This study demonstrated that 2 doses of the experimental combination vaccine reliably and effectively stimulated protective serological serum neutralization titers.

(168) Since viremia is a prerequisite before virus can cross the blood-brain barrier to cause WNV encephalitis, viremia is well justified as the primary parameter for evaluation of protection in an experimental study of this type.

(169) The results demonstrated that an immunogenic reaction is induced in the animals that were administered the vaccine, and that the vaccine is effective at providing protection for at least 6 months following vaccination. The effectiveness of the vaccine was evidenced in this example by reduction in WNV viremia and by stimulation of high serum neutralization titers to WNV. Because this vaccine is comprised of unique constituents including a long lasting non-metabolizable adjuvant, is formulated in a low 1 mL dose volume to provide a high degree of safety as a highly immunogenic low passage whole inactivated virus WNV isolate of recent origin and high epidemiological prevalence (a North American Dominant WNV strain), and a WNV isolated from the tissues of an infected horse, it provides more comprehensive safety and long lasting effectiveness of at least 6 months duration than other vaccines currently available. Additionally, it has the effect of providing a safe vaccine when administered to animals, and in particular to horses.

Example 7

(170) This example illustrates the efficacy of one embodiment of the immunogenic composition of the present invention including encephalomyelitis antigens with tetanus toxoid antigen.

Materials and Methods

(171) Host animal and laboratory animal immunization/serology were evaluated to demonstrate efficacy of encephalomyelitis antigens and the tetanus toxoid antigen fraction in an Encephalomyelitis-Rhinopneumonitis-Influenza-West Nile Virus Vaccine, including Eastern, Western, and Venezuelan Encephalomyelitis, Killed Virus, and Tetanus Toxoid. The efficacy and lack of interference on equine encephalitis virus vaccines and tetanus toxoid fractions can be unequivocally demonstrated by laboratory animal potency testing of the combination vaccine. Demonstration of serological response following vaccination of horses is also indicative of vaccine-toxoid efficacy. Hence, both lab animal potency and host animal serology were used in this study to confirm the efficacy of the experimental vaccine. The vaccine was also evaluated for safety in animals including horses.

(172) Horses 4-5 months of age, from non-vaccinated mares, were vaccinated with an efficacy serial of WNV combination vaccine containing inactivated Venezuelan Equine Encephalomyelitis Virus, TC-83 strain (ATCC Accession No. PTA-9411) Eastern Equine Encephalomyelitis Virus, NJO strain (ATCC Accession No. PTA-9412) Western Equine Encephalomyelitis Virus, Fleming strain (ATCC Accession No. PTA-9410) West Nile Virus (WNV), Horse Origin 2005 (ATCC Accession No. PTA-9409) Equine Herpesvirus Type 1 (ATCC Accession No. PTA-9525) (EHV-1), Influenza A/equine-2/Ohio/03 (ATCC Accession No. PTA-9522), Influenza A/equine-2/Kentucky/95 (ATCC Accession No. PTA-9523), Influenza A/equine-2/NewMarket/2/93 (ATCC Accession No. PTA-9524) and Tetanus Toxoid. Horses were vaccinated on Day 0 and Day 21 of the study. Blood samples were collected at Day 0, Day 21 and Day 35. Day 0 and Day 35 serological results are reported herein.

(173) In addition, the same WNV combination vaccine used to vaccinate horses was tested for potency in guinea pigs. Data presented in this report collectively and definitively establish the efficacy of each antigen tested (EEE, VEE, WEE, tetanus) in this study and also confirm the safety of a WNV combination vaccine.

(174) Bulk lots of EEE, WEE, and VEE viruses and tetanus toxoid were produced. Following growth, viral fluids were filtered, formalin inactivated, and concentrated. The inactivated viral fluids were tested for residual live virus after inactivation.

(175) Inactivated viral and toxoid fluids described above were used to formulate a vaccine that also contained inactivated Equine Herpesvirus Type 1, inactivated influenza A/equine-2/Kentucky/95, influenza A/equine-2/NewMarket/2/93 and influenza A/equine-2/Ohio/03 viruses.

(176) The vaccine was formulated to specifications for all antigens included in the product.

(177) The final formulated vaccine contained the following ingredients per 1 mL dose:

(178) TABLE-US-00024 EHV-1 10.sup.7.0-9.0 TCID.sub.50/mL Influenza A2/Ohio/03 10.sup.6.7-9.5 TCID.sub.50/mL Influenza A2/KY/95 10.sup.6.7-9.5 TCID.sub.50/mL Influenza A2/NewMarket/2/93 10.sup.6.7-9.5 TCID.sub.50/mL Tetanus Toxoid 5-10 CPU Eastern Equine Encephalomyelitis 10.sup.6.7-9.2 TCID.sub.50/mL Western Equine Encephalomyelitis 10.sup.6.7-9.2 PFU/mL Venezuelan Equine Encephalomyelitis 10.sup.6.7-9.2 TCID.sub.50/mL West Nile Virus 10.sup.7.0-9.0 TCID.sub.50/mL Adjuvant (preferably mineral oil) 100-200 ?l Glycerol 100-200 ?l EDTA 240 mM solution 10-20 ?l Diluent - DMEM containing q.s. Gentamicin (30 ?g/mL of diluent volume) Formaldehyde (0.1-0.2% of diluent volume)

(179) Forty horses, four to 5 months of age, were used in this study. Horses remained with their dams on pasture throughout the vaccination period and were weaned from their dams when the 2-week post-booster sera were collected. Horses were assigned to either one of the two treatment groups randomly as they were vaccinated intramuscularly (IM) with a 1.0 ml dose. The primary immunization was followed three weeks later by a 1.0 ml IM booster vaccination. Twenty horses received vaccine. Twenty horses received placebo.

(180) Guinea pigs were also vaccinated with the same combination WNV vaccine.

(181) Horses were vaccinated and serum samples collected using the following schedule:

(182) TABLE-US-00025 TABLE 19 Vaccination and Serum Sampling Schedule Day of Test Activity 0 Collect pre-vaccination blood and give primary vaccination 21 Collect blood and give booster vaccination 35 Collect blood for final serology

(183) Guinea pigs were vaccinated and serum collected using the schedule outlined by 9 CFR, 113.207(b) and 113.114(c.).

(184) Sera from horses in this study were tested following general guidelines. The assay was modified to determine titers by testing at 1:2 and 1:10 dilutions for Day 0 samples and at 1:10 and 1:40 dilutions for the 2-week post-booster serum samples. Sera were tested for EEE, WEE and VEE antibody and were tested for Tetanus toxoid antibody.

Results and Discussion

(185) Horse Serological Evaluation for EEE, WEE and VEE

(186) At Day 0 of the Study, not all foals were sero-negative to encephalomyelitis viruses. Five of the vaccinated foals had significant (>1:10) residual maternal antibody to EEE virus. In addition, two of the vaccinated foals had residual maternal antibody (>1:10) to WEE virus. Despite existing and potentially interfering passively acquired, maternal antibody at the time of administration of the first dose of the WNV combination vaccine, titers for all three fractions increased substantially (>4 fold in 80% of horses tested for EEE, >4 fold in 90% of horses tested for WEE and >4 fold in 100% of horses tested for VEE) following vaccination, yet remained negative or low for the non-vaccinated foals. Individual foal data are presented below.

(187) EEE, WEE and VEE Equine Serological Titers

(188) TABLE-US-00026 TABLE 20 Plaque Reduction Neutralization Titration Horse Test Day 0 Day 35 Day 0 Day 35 Day 0 Day 35 ID Article EEE EEE WEE WEE VEE VEE 1 V >10 >40 2 >40 <2 >40 2 V <2 >40 <2 10 <2 40 3 V <2 >40 <2 <10 <2 >40 4 V <2 >40 <2 10 <2 >40 5 V >10 >40 >10 >40 <2 >40 6 V >10 >40 <2 >40 NS 10 7 V <2 >40 <2 >40 <2 >40 8 V >10 >40 2 >40 <2 >40 9 V <2 >40 <2 >40 <2 >40 10 V <2 >40 <2 >40 <2 10 11 V <2 >40 <2 >40 <2 >40 12 V <2 >40 <2 >40 <2 40 13 V <2 >40 <2 >40 <2 >40 14 V <2 >40 <2 10 <2 >40 15 V >10 >40 >10 >40 2 >40 16 V 2 >40 <2 10 <2 >40 17 V <2 >40 <2 10 <2 >40 18 V <2 >40 <2 >40 <2 40 19 V <2 10 <2 >40 <2 >40 20 V 2 >40 <2 >40 <2 10 21 C <2 <10 <2 <10 <2 10 22 C <2 <10 <2 <10 <2 <10 23 C <2 <10 <2 10 <2 <10 24 C <2 <10 NS <10 <2 <10 25 C <2 <10 <2 <10 <2 <10 26 C <2 <10 <2 10 <2 <10 27 C <2 <10 <2 <10 <2 <10 28 C 2 <10 <2 <10 <2 10 29 C 2 <10 <2 10 <2 <10 30 C <2 <10 <2 <10 <2 <10 31 C <2 <10 <2 <10 <2 <10 32 C <2 <10 <2 <10 <2 <10 33 C >10 >40 <2 10 <2 <10 34 C <2 <10 <2 <10 <2 <10 35 C >10 10 2 <10 <2 10 36 C 2 <10 <2 <10 <2 <10 37 C <2 <10 <2 <10 <2 <10 38 C <2 <10 2 <10 <2 <10 39 C <2 10 <2 <10 <2 <10 40 C <2 >40 <2 <10 <2 <10

(189) Guinea Pig Serological Evaluation for EEE, WEE, VEE and Tetanus Toxoid

(190) Nine of ten guinea pigs vaccinated with the combination vaccine seroconverted satisfactorily at (?1:40) to EEE virus. Ten of ten guinea pigs had satisfactory titers for VEE virus (?1:4) and ten of ten guinea pigs seroconverted satisfactorily to WEE virus (?1:40). Also a serum pool from 10 vaccinated guinea pigs was tested for tetanus antibody and was shown to be satisfactory with a value of 4.3 anti-toxin units/ml (AU/ml).

(191) Guinea pig potency tests were completed and found to be satisfactory for all four antigens including tetanus toxoid, EEE, VEE, and WEE.

(192) The vaccine was also administered to horses (20 vaccinates and 20 controls) via primary immunization followed by booster immunization 3 weeks later. Fourteen days post-booster vaccination, horses were bled and serum collected for all serological testing. Equine response to encephalomyelitis antigens was tested utilizing 2 dilutions (1:2 and 1:10 for Day 0 samples and 1:10 and 1:40 for Day 35 samples) in 24-well plates to determine antibody titers.

(193) The satisfactory guinea pig potency testing conclusively establishes the efficacy of 4 antigens (VEE, EEE, WEE and tetanus toxoid) in the West Nile Virus combination vaccine as a 9-antigen-containing vaccine-toxoid. Furthermore, satisfactory potency results are substantiated and confirmed by host animal horse serology data in which vaccinated horses demonstrated a substantial rise in titer to each encephalitis virus fraction following vaccination. Additionally, the absence of observation of any adverse reactions in any of the vaccinated horses or guinea pigs confirms the safety of the WNV combination vaccine in animals.

Example 8

(194) This example illustrates that a vaccine or immunogenic composition in accordance with the present invention has a duration of immunity of at least one year.

Materials and Methods

(195) Host animal vaccination and challenge at least 1 year post-booster vaccination was used to confirm duration of immunity for the West Nile Virus antigen fraction in an Encephalomyelitis-Rhinopneumonitis-Influenza-West Nile Virus Vaccine, Eastern, Western & Venezuelan, Killed Virus, Tetanus Toxoid prepared from a North American Dominant isolate of WNV designated North American Equine E159 (NAEE159).

(196) TABLE-US-00027 TABLE 21 The final formulated vaccine contains the following ingredients per 1 mL dose: Ingredients 1 mL Dose Eastern Equine Encephalomyelitis 10.sup.7.5-9.2 TCID.sub.50/mL Western Equine Encephalomyelitis 10.sup.8.2-9.2 PFU/mL Venezuelan Equine 10.sup.7.7-9.2 TCID.sub.50/mL Encephalomyelitis West Nile Virus (North American 10.sup.8.0-9.2 TCID.sub.50/mL Dominant prepared from NAEE159) EHV-1 10.sup.7.0-9.0 TCID.sub.50/mL Equine Influenza A2/Ohio/2003 10.sup.7.3-9.5 TCID.sub.50/mL Equine Influenza A2/Kentucky/95 10.sup.7.3-9.5 TCID.sub.50/mL Equine Influenza A2/NewMarket/ 10.sup.7.3-9.5 TCID.sub.50/mL 2/93 Tetanus Toxoid 5-10 CPU Non-metabolizable Oil Adjuvant 100-200 ?L Diluent - DMEM containing q.s. Gentamicin 30 ?g/mL of diluent volume Formaldehyde 0.1% of diluent volume

(197) Thirty horses (20 vaccinates and 10 controls), 4-5 months of age were used in this study. Horses were randomly assigned to one of two treatments and vaccinated intramuscularly (IM) with a 1.0 mL dose of the assigned vaccine or control product. The primary immunization was followed three weeks later by a 1.0 mL IM booster vaccination.

(198) Horses were vaccinated once and then again about 30 days later. Horses were randomly assigned to either vaccine or control groups. Twenty horses received the vaccine group receiving VEWT/WNV/EHV-1/Influenza vaccine. Ten horses received adjuvanted DMEM containing excipients used in the vaccine (Gentamicin and formaldehyde) but no antigens. The non-metabolizable oil adjuvant used for all administrations was preferably mineral oil.

(199) Challenge inoculation of the virulent heterologous WNV NY99 strain virus was performed 380 days post-booster vaccination. The second cohort of horses were challenged 408 days post-booster inoculation in a similar manner.

(200) Serum samples for serological evaluation were collected from the vaccinated and control horses prior to initial vaccination, at 21 days post first dose vaccination (day of booster vaccination), monthly post-booster, on the day of challenge, and at 7 and 14 days post-challenge. Body temperature and serum samples were obtained from each horse on the day of challenge, twice daily on Days 1 through 6 post-challenge, and daily on Days 7-10 and Day 14 post-challenge. Clinical data was also recorded during those same time periods for the 15-day observation period.

(201) The heterologous challenge virus, designated WNV NY99, was originally isolated from the brain of an infected crow (CDC, Ft. Collins, Colo.). On the day of challenge, the stock virus was thawed on ice and virus was diluted to the desired concentration in phosphate-buffered saline immediately prior to inoculation of horses.

(202) Rectal temperatures were recorded for each of the vaccinated and control horses on the day before challenge, day of challenge and twice daily on days 1-14, then daily on Days 14-21 post challenge by means of a calibrated, electronic thermometer (GSA Electronics) probe. The daily rectal temperatures were recorded in degrees Fahrenheit (? F.).

(203) Venous blood from each of the vaccinated and control horses was collected on the Day of Challenge, twice daily on Days 1-6, and daily on Days 7-10 and Day 14 days post-challenge by Vacutainer into an SST tube. After centrifugation, serum was aliquoted and frozen immediately.

(204) Vero Cells were grown in 6-well plates to confluency. To perform the plaque assay, serial 10-fold dilutions of serum were prepared in 96-well plates in BA-1 medium (MEM salts containing 1% BSA, 250 mg/L sodium bicarbonate, 500 gentamicin and 2.5 ?g amphotericin B/mL in 50 mM Tris, pH 7.6). Serum dilutions (0.1 mL) were inoculated into each well of the 6-well plate and incubated for 45-60 minutes with rocking every 15 minutes. After the incubation period, 2 mL of overlay (2? medium containing MEM without phenol red prepared at twice the normal concentration and supplemented with 4% FBS, 200 IU penicillin G/mL and 100 ?g streptomycin/mLwarmed to 45? C.) was added to each well. Plates were incubated at 37? C.

(205) Two days after inoculation, 2 mL of a second overlay containing 2? agarose prepared by mixing equal volumes of 2? medium and 2? agarose was added to each well. Plates were examined and plaque numbers recorded in each well on days 3, 4 and 5 following inoculation. The virus titer per mL of original material is calculated as the number of plaques in a well (or average of multiple wells inoculated with the same dilution) times the dilution for the well being counted multiplied by 10.

(206) A standard microtiter serum neutralization test was employed in this study. All sera were tested in sterile flat bottom 96 well microtiter plates using five wells per dilution and an 8 well dilution series for each of the 5 test wells. Each of the 5 test wells contained 25 ?L of serum dilution mixed with 25 ?L of the indicator virus and 150 ?L of a freshly planted Vero cell suspension containing approximately 4?10.sup.4 cells. The test indicator virus used was WNV NY99. Serum neutralizing antibody titers are expressed as Reed-Muench ID.sub.50 titers.

(207) For performance of the test, two-fold dilutions of each test serum were made in a sterile flat bottom microtiter plate using five replicate wells per test serum and an 8 well dilution series. Dilutions were made with an adjustable volume single or multi-channel pipetting instrument using sterile microtiter tips. The volume of serum added to each of 5 wells of the first row was 50 ?L. All other wells contained 25 ?L of DMEM (no FBS). Following serial dilution down the plate, 25 ?L was discarded from the last row. 25 ?L of a pre-determined dilution of the indicator virus was added to each test well. Plates were then mixed and incubated for one hour at 37? C. in 5% CO.sub.2. On conclusion of the incubation period, 150 ?L of a suspension containing 4?104 Vero cells were added to each test and cell control well. The plates were incubated at 37? C. in a CO.sub.2 incubator for 5-7 days, at which time plates were microscopically examined for CPE typical of WNV.

(208) Histopathology was evaluated by a Board Certified Veterinary Pathologist. The scoring system used to describe defects in the pons or medulla was as follows:

(209) Score:

(210) 0=no significant lesions in section

(211) 0.5=rare, small, multifocal glial nodules scattered throughout the parenchyma

(212) 1=mild, nonsuppurative encephalitis. This is characterized by mild multifocal perivascular cuffs with lymphocytes and plasma cells and a rare neutrophil and scattered multifocal glial nodules composed of glial cells with a few mononuclear inflammatory cells. Occasionally within this grade, there may be minimal perivascular cuffing and more moderate scattered glial nodules.
2=moderate nonsuppurative encephalitis characterized by moderate lymphoplasmacytic perivascular cuffs around many vessels and multifocal accumulations of glial nodules scattered throughout the parenchyma
3=severe nonsuppurative encephalitis characterized by severe and thick lymphoplasmacytic perivascular cuffing with multiple scattered glial nodules throughout the parenchyma

Results and Discussion

(213) There were no adverse reactions to vaccine administration at either dosing time point. All 4 to 5 month old foals receiving the experimental vaccine were free of either systemic or injection site adverse reactions in the study. This confirms the excellent safety of the vaccine of the present invention against WNV containing North American Dominant WNV antigen prepared from isolate NAEE159.

Horse Challenge with Heterologous West Nile Virus

Viremia

(214) Each of the 10 control horses (100%) were viremic for at least 1 day post-challenge, while only 2 of 20 horses (10%) in the WNV vaccine group were viremic.

Clinical Signs

(215) Seven of the 10 horses (70%) in the control group developed signs of encephalomyelitis consistent with West Nile Virus infection. Each of these animals was viremic for at least one day during the challenge period. In the WNV vaccine group, 1 of the 20 horses (5%) developed signs consistent with West Nile Virus infection. Notably clinical signs progressed to death or euthanasia in 70% of the controls and only 5% of the vaccinates. All control mortalities were viremic, confirming fatal encephalitis due to WNV, whereas only one of two vaccinated animals that died was viremic during the challenge period.

Serum Neutralization Titers

(216) All vaccinated horses responded favorably to the WNV vaccine by developing protective levels of serum neutralizing (SN) antibody following vaccination. Over one year following vaccination, 17 of 20 (85%) of vaccinated horses maintained protective SN titers. By contrast, none of the control horses developed rising SN titers prior to virulent WNV challenge. Also, all vaccinated horses displayed an anamnestic rise in SN titers following virulent WNV challenge

Histopathology

(217) Severity scores were provided for both the medulla and pons. Also with regard to this efficacy parameter, WNV vaccine containing North American Dominant WNV antigen prepared from isolate NAEE159 proved highly effective. Among the control horses, 50% displayed severe lesions of WNV encephalitis whereas only 10% of vaccinates were similarly affected.

Discussion and Conclusions

(218) The WNV vaccine was prepared from a viral isolate (North American Equine E159) obtained from a horse in 2005 during the North American pandemic when a specific dominant WNV genotype emerged. This genotype is characterized by a specific valine to alanine amino acid change at the 159.sup.th amino acid in the envelope (E) protein of the virus (when compared to the publicly available sequence for the WNV-NY99 isolate having the ATCC Accession No. AF196835), which has made all such isolates more robust and prolific, thereby displacing other WNV isolates, and making this genotype dominant among disease-causing WNV isolates in North America. Because it was prepared from the dominant genotype, the vaccine used in this study is indicative of the unique safety and efficacy achievable with vaccine prepared from all such North American Dominant isolates with this E protein profile and resulting prolificacy. Notably, all previously tested WNV vaccines have been prepared from a less prolific isolate of differing genotype and E protein amino acid sequence, namely WNV NY99. Based on this difference in nucleic acid sequence, E protein amino acid sequence, viral prolificacy, and unique ability to cause a pandemic, the North American Dominant isolates are displacing or have displaced NY99 from the environment. The unique genotype and phenotype (prolificacy), and, most importantly, the overwhelming environmental presence of North American Dominant WNV isolates and the absence of WNV NY99 is compelling evidence for the superiority of the North American Dominant West Nile Virus vaccine. Such superiority is confirmed by the safety and efficacy of the vaccine as demonstrated in this challenge study using vaccine prepared from North American Dominant isolate North American Equine E159 (NAEE159) (ATCC Accession No PTA-9409).

(219) In this study, 4 to 5 month old horses were safely and effectively vaccinated with a multi-component VEWT/WNV/EHV-1/Equine Influenza vaccine batched at an appropriate antigen amount with the WNV component being North American Dominant WNV antigen prepared from isolate NAEE159c(ATCC Accession No. PTA-9409).

(220) Study horses were intrathecally challenged at least 380 days post-booster vaccination with 10.sup.5 PFU of a virulent heterologous West Nile Virus strain. Horses were evaluated for 14 days post-challenge for clinical signs (including temperature and mortality), viremia, serum neutralization titers, and histopathology scores from sections of the pons and medulla taken after euthanasia and necropsy.

(221) Viremia after challenge and serum neutralization titers were key outcome variables in this study that were highly indicative of vaccine efficacy. Horses that had been vaccinated more than one year earlier with VEWT/WNV/EHV-1/Influenza Lot 916 were 90% protected from viremia after challenge in this study. In comparison, 100% of control horses demonstrated viremia post-challenge. Additionally, serum neutralization titers of vaccinated horses were significantly higher than those of control horses at 14 days post-challenge, and displayed an anamnestic response typical of an effective vaccine following heterologous, virulent WNV challenge.

(222) In addition the vaccine containing North American Dominant WNV antigen prepared from isolate NAEE159 reduced clinical signs and mortality resulting from encephalomyelitis following heterologous challenge with virulent WNV. Vaccine efficacy at least one year following vaccination was also confirmed by reduction in lesions typical of WNV infection.

(223) This study demonstrated for the first time that 2 doses of the experimental combination vaccine prepared at appropriate doses of antigen including North American Dominant WNV antigen prepared from isolate NAEE159 administered to foals 4 to 5 months of age safely, reliably and effectively stimulated protective serological serum neutralization titers that resulted in duration of immunity of at least one year with protection from viremia, clinical signs, mortality, and encephalitic lesions after virulent heterologous challenge with West Nile Virus.

Example 9

(224) In this study, a combination vaccine was prepared using a North American Dominant isolate of WNV, North American Equine E159(NAEE159) (ATCC Accession No. PTA-9409). The 14-day post second-vaccination sera samples from the guinea pigs vaccinated with this Encephalomyelitis-Rhinopneumonitis-Influenza-West Nile Virus Vaccine, Eastern, Western & Venezuelan, Killed Virus, Tetanus Toxoid were collected and tested for West Nile Virus plaque reduction neutralization (PRN). The sera from the vaccinated guinea pigs were tested for neutralizing antibody to both a North American Dominant isolate of WNV and to WNV isolate NY99. Notably, the vaccine displayed superior activity in stimulating neutralizing antibodies to North American Dominant WNV, as opposed to NY99 WNV. These data support the conclusion of the superior efficacy of WNV vaccines prepared from North American Dominant WNV isolates as contrasted with earlier less effective vaccines prepared from or based on the NY99 WNV isolate.

(225) Furthermore, a vaccine prepared from an additional North American Dominant isolate of WNV, North American Donkey E159 (NADE159) will similarly demonstrate, as described above, the superior efficacy of such vaccines over the former NY99 based vaccines. Hence, data from multiple North American Dominant isolates cultivated from different host species, originating from unique North American locations, and obtained at different times in North America will confirm the unexpected but superior efficacy of North American Dominant isolates of WNV for vaccine preparation.

(226) The data from the plaque reduction neutralization assay also established that a vaccine prepared from a North American Dominant isolate of WNV that stimulates a titer of 1:12 or higher in vaccinated guinea pigs that provides 50% viral plaque reduction in at least 90% of vaccinated guinea pigs, correlates to vaccine protection against WNV challenge in the horse and provides for a duration of immunity of at least one year. West Nile Virus vaccination/challenge data in the horse at an antigen inclusion level of 10.sup.7.6-9.0 TCID.sub.50 or higher per dose correlated with these guinea pig PRN titer results and confirmed the WNV immunizing dose that provides 1 year or longer duration of immunity in the horse. The corresponding dose in guinea pigs also stimulates serum neutralizing antibodies to a titer of at least 1:12 against North American Dominant WNV in guinea pigs.

(227) Data presented in this report collectively demonstrate the unexpected efficacy of vaccines prepared from North American Dominant isolates of WNV, define the correlation between vaccine efficacy in the horse and guinea pig serum levels of neutralizing antibody, confirm that a 1:12 titer or higher in guinea pigs identifies an effective equine vaccine providing at least one year duration of immunity, and quite notably, demonstrate the superior efficacy of vaccines prepared from North American Dominant WNV as contrasted with NY99 WNV.

Materials and Methods

(228) In order to demonstrate efficacy of the West Nile Virus antigen, prepared using a North American Dominant isolate of WNV, North American Equine E159(NAEE159) (ATCC Accession No. PTA-9409), in an Encephalomyelitis-Rhinopneumonitis-Influenza-West Nile Virus Vaccine, Eastern, Western & Venezuelan, Killed Virus, Tetanus Toxoid and to establish an effective dose measurable in horses or guinea pigs, host animal vaccination/challenge studies were performed in conjunction with guinea pig vaccination/serology studies. In this study, 14-day post second-vaccination sera samples from guinea pigs vaccinated with Encephalomyelitis-Rhinopneumonitis-Influenza-West Nile Virus Vaccine, Eastern, Western & Venezuelan, Killed Virus, Tetanus Toxoid were collected and tested. Additionally, a plaque reduction neutralization assay was developed to measure the titer correlated to protection against challenge in the host animal. This titer was determined to be 1:12 or higher in the guinea pig.

Vaccine Formulations

(229) Experimental Serials (Protective Dose Vaccine)

(230) Experimental Serials were formulated to confirm protective antigen specifications for all antigens in the vaccine.

(231) TABLE-US-00028 TABLE 22 The final formulated vaccines contained the following ingredients per 1 mL dose: Ingredients 1 mL Dose Eastern Equine Encephalomyelitis 10.sup.7.5-9.2 TCID.sub.50/mL Western Equine Encephalomyelitis 10.sup.8.2-9.2 PFU/mL Venezuelan Equine 10.sup.7.7-9.2 TCID.sub.50/mL Encephalomyelitis West Nile Virus (North American 10.sup.7.3-9.2 TCID.sub.50/mL Dominant prepared from NAEE159) EHV-1 10.sup.7.0-9.0 TCID.sub.50/mL Equine Influenza A2/Ohio/2003 10.sup.7.3-9.5 TCID.sub.50/mL Equine Influenza A2/Kentucky/95 10.sup.7.3-9.5 TCID.sub.50/mL Equine Influenza A2/NewMarket/ 10.sup.7.3-9.5 TCID.sub.50/mL 2/93 Tetanus Toxoid 5-10 CPU Non-metabolizable Oil Adjuvant 100-200 ?L Diluent - DMEM containing q.s. Gentamicin 30 ?g/mL of diluent volume Formaldehyde 0.1% of diluent volume

(232) Experimental serial 916 was formulated for host animal vaccination studies. Experimental serial 916 is a multi-component vaccine containing VEWT-WNV-EHV-1 and 3 strains of equine influenza type A2 virus. Experimental serial 916 is batched at 10.sup.7.6-9.2 TCID.sub.50/mL of West Nile Virus antigen North American Equine E159(NAEE159). It is a 1 mL dose vaccine in the horse.

(233) This vaccine was also tested in guinea pigs at the time of host animal vaccinations to confirm the WNV efficacy and laboratory animal potency. Four replicate guinea pig sera dilution experiments were performed for experimental serial 916 to validate a guinea pig assay criterion for this one-year duration of immunity (DOI) vaccine.

Experimental Serial 507 (Comparative Efficacy Serial)

(234) Data from Experimental Serial 507 is included in this report to demonstrate that serials formulated with a North American Dominant isolate of WNV antigen show superior efficacy, measured as guinea pig titers, of the relevant North American Dominant isolates of WNV as compared to the earlier NY99 isolate.

Guinea Pig Serological Evaluation

(235) Sera were tested for WNV antibody as follows: 1) West Nile Virus Indicator Strain: North American Equine E159(NAEE159) (ATCC Accession No. PTA-9409) & North American Donkey E159 (NADE159) 2) Growth medium for Vero Cells is DMEM+5% FBS, 2 mM L-glutamine and 30 ?g/mL gentamycin 3) Diluent for Test Serum is DMEM plus 30 ?g/mL gentamycin 4) Diluent for indicator virus working solution is DMEM+10% normal guinea pig serum (specific to WNV assay) 5) Guinea pig test sera is diluted 1:12 7) Use 4 mL overlay instead of 3 mL (specific to WNV assay) 8) Titers are calculated using 50% plaque reduction 9) Nine of ten vaccinated guinea pigs must have an antibody titer of ?1:12 to demonstrate efficacy, and negative guinea pigs must be <1:4 (same criteria as VEE assay in SAM).

Results and Discussion

Serological Evaluation for West Nile Virus

(236) TABLE-US-00029 TABLE 23 Number of Plaques/Sera Dilution Experimental Serial 916 Replicate I West Nile Guinea Pig Plaque Reduction Neutralization Results Using North American Equine E159 (NAEE159) as Indicator Virus The study was initiated and the guinea pigs were bled 25 days later. 916 (I) 2 3 4 6 8 12 16 32 64 GP1 0 2 2 4 5.5 2.5 17 14 11 GP2 6.5 9.5 11 14 9 11.5 13 20 22.5 GP3 7 5 6 9.5 10 9.5 11 10 16.5 GP4 7 5 4 4 1 6 15.5 9.5 12.5 GP5 6 4.5 9 7 17.5 13.5 12 16 17 GP6 1 1 2.5 7.5 3 8 19.5 19.5 20.5 GP7 9.5 8 8 15.5 17.5 21.5 36 17.5 20.5 GP8 5 3.5 7.5 7.5 14.5 11 26.5 26.5 27.5 GP9 0 2 7 6 7 12 9 10.5 11.5 GP10 4.5 4.5 5.5 11 22 8.5 23 29 30.5 # 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 Passed Neg. 68.5 64.5 72.5 93.5 68 82 89.5 78.5 71 Control 1 Neg. 72 61 87 69.5 70.5 85.5 77.5 69.5 88 Control 2 Virus Control Values: 99, 70, 68, 88, 77, 64 Virus Control Average Plaques: 79 Virus Control 50% Reduction: 39.5

(237) TABLE-US-00030 TABLE 24 Experimental Serial 916 Replicate II West Nile Guinea Pig Plaque Reduction Neutralization Results Using North American Equine E159 (NAEE159) as Indicator Virus Guinea Pigs were bled 35 days after the initiation of the study Number of Plaques/Sera Dilution 916 (II) 2 3 4 6 8 12 16 32 64 GP1 1 3 6 6 14.5 6.5 13.5 18 17.5 GP2 1.5 3.5 6.5 6.5 8.5 9.5 10.5 12.5 19 GP3 12 5.5 22 21 41.5 34 41 41 43.5 GP4 4 8.5 17.5 16.5 21 36.5 28 41.5 42.5 GP5 3.5 4.5 9.5 15 26.5 25.5 13.5 23 40 GP6 1 3 7 13.5 17.5 14 24.5 26 23 GP7 8 5.5 14.5 11.5 21.5 15.5 34 26.5 28 GP8 1 1.5 2.5 3.5 4 11 9 20 17.5 GP9 13.5 18 25.5 29.5 29.5 35.5 28 37.5 43 GP10 9 8 10.5 21.5 21 20.5 27 31.5 27.5 # 10/10 10/10 10/10 10/10 9/10 10/10 9/10 8/10 6/10 Passed Neg. 69.5 67 71.5 69.5 72.5 73 83 68 78 Control 1 Neg. 68 70.5 69 72.5 82.5 66 75 76 74 Control 2 Virus Control Values: 99, 70, 68, 88, 77, 64 Virus Control Average Plaques: 79 Virus Control 50% Reduction: 39.5

(238) TABLE-US-00031 TABLE 25 Experimental Serial 916 Replicate III West Nile Guinea Pig Plaque Reduction Neutralization Results Using North American Equine E159 (NAEE159) as Indicator Virus Guinea Pigs were bled 35 days after initiation Number of Plaques/Sera Dilution 916 (III) 2 3 4 6 8 12 16 32 64 GP1 0.5 1.5 0 0 0 1 4 6 3.5 GP2 0 0 1 0.5 0 0 0 0 0 GP3 7.5 9 9 16 17 13.5 12 18.5 23 GP4 2.5 0 0 3 2 2 3.5 1 2.5 GP5 13.5 15.5 18 18 19.5 24 16.5 21.5 33.5 GP6 6.5 15.5 31.5 10 29.5 26.5 28.5 31.5 32 GP7 14.5 12 17.5 20.5 19.5 29.5 21.5 16 22.5 GP8 21 24.5 36 28 34.5 30.5 27.5 29 26 GP9 0.5 0.5 3 4 6.5 5 10 9.5 18 GP10 2.5 7.5 11 8 12.5 9 20 10.5 17 # 10/10 10/10 8/10 10/10 9/10 10/10 10/10 9/10 8/10 Passed Neg. 73 61.5 79.5 53.5 58.5 78 57 70 63 Control 1 Neg. 55 54.5 64 52.5 58 68.5 66 67.5 79 Control 2 Virus Control Values: 51, 58, 61, 66, 78 Virus Control Average Plaques: 62 Virus Control 50% Reduction: 31

(239) TABLE-US-00032 TABLE 26 Experimental Serial 916 Replicate IV West Nile Guinea Pig Plaque Reduction Neutralization Results Using North American Equine E159 (NAEE159) as Indicator Virus Guinea Pigs were bled 30 days after the initiation of the study Number of Plaques/Sera Dilution 916 (IV) 2 3 4 6 8 12 16 32 64 GP1 11.5 7 5.5 20.5 12 17.5 22.5 20 25 GP2 31 21.5 20.5 36.5 32 30 27 15 28.5 GP3 16 20.5 18 23.5 21.5 16 35.5 16.5 15 GP4 1 2 6.5 7 11 15 19 14 18.5 GP5 4 1 9.5 12 20 17 24.5 19.5 23.5 GP6 0 0 0 1.5 2.5 2.5 0.5 3 3 GP7 5.5 6.5 5.5 10 7 13 5.5 9.5 13 GP8 1 4.5 0.5 3 7.5 4.5 13.5 17.5 11.5 # 8/8 8/8 8/8 7/8 8/8 8/8 7/8 8/8 8/8 Passed Neg. 48 39 46.5 39 48 54 39.5 54 67.5 Control 1 Neg. 40.5 53 44 44.5 54 48.5 59.5 61.5 45.5 Control 2 Virus Control Values: 93, 53, 56, 92, 67, 44 Virus Control Average Plaques: 67.5 Virus Control 50% Reduction: 33.8
EXPERIMENTAL SERIAL 507 (Demonstrating Superior Efficacy of North American Equine E159(NAEE159) Vaccines, and that other North American Dominant Vaccines, such as North American Donkey E159(NADE159 will provide superior efficacy to NY99 vaccines)

(240) Guinea Pig Serological Evaluation for West Nile Virus

(241) TABLE-US-00033 TABLE 27 Experimental Serial 507 West Nile Guinea Pig Plaque Reduction Neutralization Results using WNV NY1999 Isolate as Indicator Virus Number of Plaques/Sera Dilution Guinea Pig Number 4 8 16 32 64 GP1 ?10 ?15 ?15 ?14.5 ?10.5 GP2 4.5 5 7.5 9.5 13 GP3 1 3.5 3.5 6 6.5 GP4 8 11.5 ?13.5 ?14.5 ?14.5 GP5 6 7.5 7.5 8.5 9 GP6 8.5 9.5 9.5 12 14 GP7 7 8.5 9 ?10.5 ?14.5 GP8 5 10.5 10.5 14 14 GP9 5.5 7 7.5 10 10 GP10 5.5 6.5 8.5 ?12.5 14 # Passed 9/10 6/10 5/10 2/10 1/10 Neg. Control 1 ?13.5 ?13.5 ?13 ?13 ?14.5 Neg. Control 2 ?15.5 ?14.5 ?16 ?17 ?16.5 Virus Control Values: ?24, ?15, ?14, ?16, ?18, ?20 Virus Control Average Plaques: ?17.8 Virus Control 50% Reduction: ?8.9

(242) TABLE-US-00034 TABLE 28 Experimental Serial 507 West Nile Guinea Pig Plaque Reduction Neutralization Results Using North American Equine E159 (NAEE159) as Indicator Virus Number of Plaques/Sera Dilution Guinea Pig Number 2 4 8 16 32 64 GP1 17 14 18.5 14.5 19.5 26 GP2 2.5 3.5 2 6 17 15 GP3 5.5 10 4.5 9 5 11.5 GP4 11.5 10 15 15.5 19 19.5 GP5 13 23 23.5 19.5 32.5 13 GP6 5.5 8.5 12 11 13 14 GP7 8.5 12 14 15 16 12 GP8 14.5 14 18 19.5 25.5 27.5 GP9 1.5 3 3 8.5 15.5 7 GP10 12.5 12 7 18.5 14 13 # Passed 9/10 9/10 7/10 7/10 5/10 7/10 Neg. Control 1 24.5 21 22 23.5 22 25 Neg. Control 2 24 33 29 29 24.5 34.5 Virus Control Values: 40, 38, 34, 35, 28, 23 Virus Control Average: 33
Virus Control 50% Reduction: 16.5

Discussion and Conclusions

(243) Guinea pigs were vaccinated and sera tested for West Nile Virus antibody. This assay established that a titer of 1:12 in vaccinated guinea pigs correlates to protection in a horse vaccination/challenge study that provides at least one year duration of immunity for WNV vaccine prepared using a North American Dominant isolate of WNV, such as North American Equine E159(NAEE159).

(244) Concurrently with guinea pig vaccinations, WNV vaccine prepared using a North American Dominant isolate of WNV, North American Equine E159(NAEE159), was also administered to horses (20 vaccinates and 10 controls) via primary immunization followed by booster immunization 3 weeks later. More than one year post-booster vaccination, horses were subjected to virulent West Nile Virus challenge, and were protected when compared to non-vaccinated controls. Vaccinated horses were protected from viremia, clinical signs, mortality, and encephalitic lesions after virulent heterologous challenge with West Nile Virus.

(245) In addition the data substantiate the superior efficacy of WNV vaccines prepared using North American Dominant WNV, as opposed to previously developed vaccines derived from WNV NY99. The sera from the vaccinated guinea pigs were tested for neutralizing antibody to both a North American Dominant isolate of WNV and to WNV isolate NY99. Titers to the isolate frequently occurring in North America, namely North American Dominant (NAEE159), were consistently higher in vaccinated guinea pigs as compared to titers to the isolate that is no longer reported to be present in nor causing disease in North America, WNV NY99. Hence, the vaccine displayed superior activity in stimulating neutralizing antibodies to North American Dominant WNV, as opposed to NY99 WNV. These data support the conclusion of the superior efficacy of WNV vaccines prepared from North American Dominant WNV isolates as contrasted with earlier less effective vaccines prepared from or based on the NY99 WNV isolate.

Example 10

(246) This Example illustrates the genetic differences between North American WNV strains and North American Dominant WNV strains, as used in the present invention.

Materials and Methods

(247) Relevant areas of the genome of WNV NY99 and the North American Dominant WNV isolates suitable for preparation of a novel, superior vaccine were sequenced and compared to confirm the key genetic differences. Examples of North American Dominant isolates used in vaccine preparation include North American Equine E159(NAEE159) (ATCC Accession No. PTA-9409) and North American Donkey E159(NADE159).

Results and Conclusions

(248) The critical Envelope (E) protein and Non-Structural 5 (NS5) protein was sequenced in these WNV isolates using standard laboratory techniques to determine genetic differences in nucleotide sequence as contrasted with WNV NY99. Notably, the North American Dominant isolates, of which specific examples are North American Equine E159(NAEE159) and North American Donkey E159(NADE159), displayed the changes which characterize North American Dominant WNV isolates and distinguish them from NY99 WNV, namely, the U to C mutation and C to U mutation at positions 1442 and 2466, respectively, of the nucleotide sequence encoding the E protein and the C to U mutation at position 9352 in the sequence encoding the NS5 protein. FIGS. 10-17 show the sequence alignments of various regions of isolates. The alignments in the E region are relative to publically available reference sequences for a NY 99 isolate (deposited in GenBank as AY590210) and a North American Dominant isolate (WN 02 isolate) deposited in GenBank as AY590223. The alignments in the NS5 region are also relative to publically available reference sequences for a NY 99 isolate (deposited in GenBank as AY369442) and a North American Dominant isolate (WN 02 isolate) deposited in GenBank as AY369440). As shown by these alignments, North American Dominant WNV isolates have the same sequence changes relative to the NY 99 isolate as those defined in the definition for a North American Dominant WNV isolate. These sequences are provided herein as SEQ ID NOS. 1-22, the full length genome of a WN99 isolate is provided as SEQ ID NO. 23, and the protein encoded by the full length genome of SEQ ID NO. 23 is provided as SEQ ID NO. 24.