Equine herpesvirus vaccine Number:6,803,041 from the United States Patent and Trademark Office (PTO) owispatent A vaccine for protecting a horse against diseases associated with EHV-1 and/or EHV-4 is provided. The vaccine commonly includes inactivated EHV-1 (e.g., chemically inactivated EHV-1 KyA virus) and an adjuvant. The adjuvant can include a cross-linked olefinically unsaturated carboxylic acid polymer which may have bioadhesive properties. The vaccine may also include antigens against other equine pathogens such as inactivated EHV-4 and inactivated A1 and/or A2 strains of equine influenza virus. Methods for protecting horses against diseases associated with EHV-1 and/or EHV-4 and methods of producing the equine herpesvirus vaccine are also provided.<H herpesvirus, vaccine, Equine, herpesvi, 6803041.html, Patent, pto, 6803041

Title: Equine herpesvirus vaccine

Abstract: A vaccine for protecting a horse against diseases associated with EHV-1 and/or EHV-4 is provided. The vaccine commonly includes inactivated EHV-1 (e.g., chemically inactivated EHV-1 KyA virus) and an adjuvant. The adjuvant can include a cross-linked olefinically unsaturated carboxylic acid polymer which may have bioadhesive properties. The vaccine may also include antigens against other equine pathogens such as inactivated EHV-4 and inactivated A1 and/or A2 strains of equine influenza virus. Methods for protecting horses against diseases associated with EHV-1 and/or EHV-4 and methods of producing the equine herpesvirus vaccine are also provided.

Patent Number: 6,803,041 Issued on 10/12/2004 to Mellencamp

Inventors: Mellencamp; Mark W. (St. Joseph, MO)
Assignee: Boehringer Ingelheim Vetmedica, Inc. (St. Joseph, MO)

Appl. No.: 09/812,720

Filed: March 20, 2001

Current U.S. Class: 424/229.1 ; 424/202.1; 424/204.1; 424/205.1; 424/206.1; 424/278.1; 424/280.1; 435/173.3; 435/235.1; 435/236; 435/237; 435/238

Field of Search: 424/229.1,202.1,204.1,205.1,206.1,278.1,280.1,201.1,211.1 435/173.3,235.1,236,237,238

References Cited [Referenced By]

U.S. Patent Documents


3920811	November 1975	Lund
4083958	April 1978	Bryans
4110433	August 1978	Purdy, III
4225582	September 1980	Crandell
4500513	February 1985	Brown et al.
4509949	April 1985	Huang et al.
4615697	October 1986	Robinson
4758641	July 1988	Hsu
5084271	January 1992	Studdert
5221722	June 1993	Sehm
5292653	March 1994	Kit et al.
5462734	October 1995	Letchworth, III et al.
5470718	November 1995	O'Callaghan
5084271	August 1997	Studdert
5707629	January 1998	O'Callaghan
5795578	August 1998	O'Callaghan
5843451	December 1998	Compans et al.
5853715	December 1998	Macek et al.
5922327	July 1999	Crabb et al.
6025181	February 2000	Onions et al.
6083511	July 2000	Onions et al.
6171592	January 2001	Studdert et al.
6193983	February 2001	Crabb et al.
6207166	March 2001	Audonnet et al.
6225111	May 2001	Cochran et al.
6531136	March 2003	Studdert et al.
6544526	April 2003	Crabb et al.

Foreign Patent Documents


0532833	Mar., 1993	EP
0978286	Feb., 2000	EP
0 978 286	Feb., 2000	EP
WO 97/22701	Jun., 1997	WO

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Primary Examiner: Foley; Shanon
Attorney, Agent or Firm: Foley & Lardner LLP

Claims

What is claimed is:

1. A vaccine for protecting a horse against diseases associated with EHV-1, EHV-4 or a combination thereof comprising: chemically inactivated EHV-1 KyA virus; and an adjuvant which includes cross-linked olefinically unsaturated carboxylic acid polymer.

2. The vaccine of claim 1 wherein the EHV-1 KyA virus is chemically inactivated by treatment with a chemical inactivating agent which includes a compound selected from the group consisting of ethylenimine, binary ethylenimine, acetylethylenimine and mixtures thereof.

3. The vaccine of claim 2 wherein the EHV-1 KyA virus is chemically inactivated by treatment with binary ethylenimine.

4. The vaccine of claim 1 further comprising inactivated EHV-4.

5. The vaccine of claim 1 further comprising inactivated equine influenza virus.

6. The vaccine of 5 wherein the inactivated equine influenza virus includes inactivated EIV virus subtype A1.

7. The vaccine of 5 wherein the inactivated equine influenza virus includes inactivated EIV virus subtype A2.

8. The vaccine of 5 comprising inactivated EIV virus subtype A1 and inactivated EIV virus subtype A2.

9. The vaccine of claim 1 wherein said vaccine is capable of protecting horses against EHV-1 and EHV-4.

10. The vaccine of claim 1 wherein the cross-linked olefinically unsaturated carboxylic acid polymer includes cross-linked acrylic acid polymer.

11. A vaccine for protecting a horse against diseases associated with EHV-1 EHV-4 or a combination thereof comprising: EHV-1 KyA virus inactivated by treatment with a chemical inactivating agent which includes ethylenimine, binary ethylenimine, acetylethylenimine or a mixture thereof; and a bioadhesive adjuvant which includes a cross-linked olefinically unsaturated carboxylic acid polymer.

12. The vaccine of claim 11 wherein the chemical inactivating agent includes binary ethylenimine.

13. The vaccine of claim 11 further comprising inactivated EHV-4.

14. The vaccine of claim 11 futher comprising inactivated EIV virus subtype A1 and inactivated EIV virus subtype A2.

15. A vaccine for protecting a horse against diseases associated with EHV-1, EHV-4 or a combination thereof comprising: EHV-1 KyA virus inactivated by treatment with a chemical inactivating agent which includes ethylenimine, binary ethylenimine, acetylethyleflimine or a mixture thereof; and. a bioadhesive adjuvant which includes a cross-linked acrylic acid polymer.

16. The vaccine of claim 15 further comprising inactivated EHV-4.

17. The vaccine of claim 15 further comprising inactivated equine influenza virus.

18. The vaccine of claim 17 wherein the inactivated equine influenza virus includes inactivated EIV virus subtype A1.

19. The vaccine of claim 17 wherein the inactivated equine influenza virus includes inactivated EIV virus subtype A2.

20. The vaccine of claim 17 comprising inactivated EIV virus subtype A1 and inactivated EIV virus subtype A2.

21. The vaccine of claim 15 further comprising gentamicin.

22. The vaccine of claim 15 comprising EHV-1 KyA virus inactivated by treatment with a chemical inactivating agent which includes binary ethylenimine.

23. The vaccine of claim 15 wherein the cross-linked acrylic acid polymer has a viscosity of no more than about 20,000 cPs at 20 rpm as a 1.0 wt. % aqueous solution at pH 7.5.

24. A method for protecting a horse against diseases associated with EHV-1, EHV-4 or a combination thereof comprising: administering to said horse a vaccine comprising chemically inactivated EHV-1 KyA virus and an adjuvant which includes cross-linked olefinically unsaturated carboxylic acid polymer.

25. The method of claim 24 wherein administering the vaccine to said horse comprises: parenterally administering the vaccine; and intranasally administering the vaccine.

26. The method of claim 25 wherein administering the vaccine to said horse comprises: parenterally administering the vaccine at least once in a first step; and intranasally administering the vaccine in a subsequent step.

27. The method of claim 24 wherein the vaccine further comprises inactivated EHV-4.

28. The method of claim 24 wherein the vaccine further comprises inactivated equine influenza virus.

29. The method of claim 28 wherein the vaccine comprises inactivated EIV virus subtype A1 and inactivated EIV virus subtype A2.

30. A kit comprising in combination, (1) a dispenser capable of administering a vaccine to a horse; and (2) a composition to protect against diseases associated with EHV-1, EHV-4 or a combination thereof, wherein the composition comprises: chemically inactivated EHV-1 KyA virus; and an adjuvant which includes cross-linked olefinically unsaturated carboxylic acid polymer.

31. The kit of claim 30 wherein the dispenser is capable of dispensing its contents as droplets; and the composition is capable of protecting against diseases associated with EHV-1, EHV-4 or a combination thereof when administered intranasally.

32. The kit of claim 30 wherein the chemically inactivated EHV-1 KyA virus is chemically inactivated by treatment with binary ethylenimine.

33. The kit of claim 30 wherein the composition further comprises inactivated EIV virus subtype A1 and inactivated EIV virus subtype A2.

Description

BACKGROUND

Respiratory diseases are a major cause of economic loss to the equine industry. Equine herpesviruses (EHV), equine influenza viruses (EIV), and the bacterium, Streptococcus equi are pathogens most often associated with infectious respiratory disease in horses. World wide, equine herpesviruses are major pathogens associated with morbidity in horses as a result of respiratory infection. Both equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) can cause respiratory disease. EHV-1 is also associated with abortions and neurological disease. Because of the high degree of mobility and the international nature of the equine industry, efficacious vaccines are needed to reduce the disease and control the spread of these pathogens.

A number of EHV vaccines are available commercially. None, however, generally is capable of conferring long lasting protection and most require frequent booster immunizations to achieve a significant level of protection against EHV infection. The most commonly recommended route of administration is via intramuscular injection, despite the respiratory system being a primary site of the infection in many instances. In addition, some of the commercial vaccines have been reported to cause undesirable side effects. A number of attempts at developing a recombinant vaccine for EHV have been reported. This approach, however, has not yet resulted in the introduction of a commercial recombinant vaccine which has achieved widespread acceptance.

Literature reports have consistently documented a high degree of variability in the capability of vaccines based on EHV-1 strains to provide cross protection against infection by EHV-4 strains. While vaccines based on EHV-4 strains have shown a greater propensity to provide some protection against both EHV-1 and EHV-4 strains, cross protection based on EHV-4 strains has also been reported to show variability.

There is accordingly a continuing need to develop additional vaccines capable of protecting horses against diseases associated with EHV-1 and/or EHV-4. It would also be advantageous to develop vaccine that is effective against EHV-1 and/or EHV-4 which could be administered via intranasally as well as via parenteral methods (e.g., intramuscularly, subcutaneously or intravenously).

SUMMARY

The present invention relates to immunogenic compositions which include an inactivated form of EHV-1. In particular, the application provides a vaccine for protecting horses against diseases associated with EHV-1 and/or EHV-4. The vaccine includes inactivated EHV-1 (e.g., chemically inactivated EHV-1 KyA virus) and typically also includes an adjuvant. The vaccine may also include other components, such as preservative(s), stabilizer(s) and antigens against other equine pathogens. Typically, the antigens against other equine pathogens are also present in an inactivated form, such as inactivated forms of EHV-4 and inactivated strains of equine influenza virus ("EIV"). For example, the vaccine may be a combination vaccine which includes inactivated forms of A1 and/or A2 strains of equine influenza virus in addition to the inactivated EHV-1. Examples of suitable antigens against EIV include inactivated EIV A1 virus strain A/EQ1/Newmarket/77, inactivated EIV A2 virus strain Newmarket/2/93, and inactivated EIV A2 virus strain Kentucky/95.

The terms "vaccine" and "immunogenic composition" are defined herein in a broad sense to refer to any type of biological agent in an administratable form capable of stimulating an immune response in an animal inoculated with the vaccine. For purposes of this invention, the vaccine (immunogenic composition) typically includes the viral agent in an inactivated form. Vaccines in general may be based on either the virus itself or an immunogenic (antigenic) component of the virus. Herein, the term "protection" when used in reference to a vaccine refers to the amelioration (either partial or complete) of any of the symptoms associated with the disease or condition in question. Thus, protection of horses from EHV by the present vaccines generally results in a diminishing of virus shedding and/or one or more of the clinical symptoms associated with infection by EHV-1 and/or EHV-4 (e.g., pyrexia, nasal discharge, conjunctivitis, coughing, dyspnea, depression, and antibiotic treatment required for secondary bacterial infection).

In one embodiment, the present immunogenic compositions include a chemically inactivated form of EHV-1. Vaccines which include chemically inactivated EHV-1 KyA virus are particularly desirable. A variety of chemical inactivating agents known to those skilled in the art may be employed to inactivate the virus. Ethylenimine and related derivatives, such as binary ethylenimine ("BEI") and acetylethylenimine, are examples of suitable chemical inactivating agents for use in inactivating the EHV-1 virus. Other chemical inactivating agents, e.g., beta-propiolactone or aldehydes (such as formaldehyde and glutaraldehyde), can also be used to inactivate the virus.

The present vaccines generally include an adjuvant which desirably may have bioadhesive properties, particularly where the virus is designed to be capable of intranasal administration. Examples of suitable adjuvants include cross-linked olefinically unsaturated carboxylic acid polymers, such as cross-linked acrylic acid polymers. As used herein the term "cross-linked acrylic acid polymer" refers to polymer and copolymers formed from a monomer mixture which includes acrylic acid as the preodominant monomer in the mixture. Examples of suitable cross-linked acrylic acid polymers include those commercially available under the tradenames Carbopol.RTM. 934P and Carbopol.RTM. 971 (available from B. F. Goodrich Co., Cleveland, Ohio). One particularly suitable adjuvant for use in the present vaccines is a cross-linked acrylic acid polymer having a Brookfield viscosity of no more than about 20,000 cPs (as measured at 20 rpm as a 1.0 wt. % aqueous solution at pH 7.5). Where a bioadhesive adjuvant is desired, it may be advantageous to utilize an adjuvant which has a bioadhesive property of at least about 50 dynes/cm2 as measured between two pieces of freshly excised rabbit stomach tissue (as determined by the procedure described in U.S. Pat. No. 4,615,697).

Methods for protecting horses against diseases associated with EHV-1 and/or EHV-4 which include administering a vaccine containing inactivated EHV-1 to the horses. The vaccine can be administered using a variety of methods including intranasal and/or parenteral (e.g., intramuscular) administration. In one embodiment of the method, the inactivated EHV-1 containing vaccine is first administered intramuscularly one or more times (e.g., at intervals of 2-4 weeks), followed by administration of the vaccine at least once intranasally (e.g., 2-4 weeks after the last parenteral administration of vaccine). The vaccine is advisedly administered to horses that are 6 months or older. Ideally, all horses in a given herd are vaccinated annually in order to protect against the spread of respiratory symptoms of the disease.

A method of producing an equine herpesvirus vaccine is also provided. The method typically includes inoculating simian cells with EHV-1 virus, e.g., with EHV-1 KyA virus. The inoculated simian cells are incubated, generally at least until CPE is observed (commonly after 24 to 120 hours at 36.degree. C.), and then the EHV-1 virus is harvested from the incubated cells (e.g., by decanting and filtering the culture fluids). The harvested virus-containing fluids can be treated with a chemical inactivating agent, such as binary ethylenimine, to form inactivated EHV-1 virus. Typically, the inactivated virus is further processed, e.g., by concentration and blending with other components, to produce a commercial formulation. For example, the fluids containing the inactivated virus may be concentrated and blended with an adjuvant and/or antigen(s) to one or more other equine pathogens.

The present application is also directed to a kit which includes in combination, (1) a dispenser capable of administering a vaccine to a horse; and (2) a chemically inactivated EHV-1 containing vaccine capable of protecting against diseases associated with EHV-1 and/or EHV-4. The kit may include a dispenser which is capable of dispensing its contents as droplets, e.g., as aerosol, atomized spray and/or liquid droplets, and a form of the vaccine which is capable of protecting against diseases associated with EHV-1 and/or EHV-4 when administered at least in part intranasally.

Throughout this application, the text refers to various embodiments of the present compositions and/or related methods. The various embodiments described are meant to provide a variety of illustrative examples and should not be construed as descriptions of alternative species. Rather, it should be noted that the descriptions of various embodiments provided herein may be of overlapping scope. The embodiments discussed herein are merely illustrative and are not meant to limit the scope of the present invention.

DETAILED DESCRIPTION

The present immunogenic compositions include an inactivated form of EHV-1. The vaccines are designed for protecting horses against diseases associated with EHV-1 and/or EHV-4. The vaccines typically include a chemically inactivated form of EHV-1 and those which include chemically inactivated EHV-1 KyA virus are particularly desirable. A variety of chemical inactivating agents known to those skilled in the art may be employed to inactivate the virus. Ethylenimine and related derivatives, such as binary ethylenimine ("BEI") and acetylethylenimine, are examples of suitable chemical inactivating agents for use in inactivating the EHV-1 virus. Other chemical inactivating agents, e.g., beta-propiolactone, aldehydes (such as formaldehyde) and/or detergents (e.g., Tween.RTM. detergent, Triton.RTM. X, or alkyl trimethylammonium salts) can also be used to inactivate the virus. The inactivation can be performed using standard methods known to those of skill in the art. Samples can be taken at periodic time intervals and assayed for residual live virus. Monitoring of cytopathic effect on an appropriate cell line and/or fluorescent staining with an appropriate specific monoclonal antibody can be used to detected the presence of residual live virus.

Inactivation with BEI can be accomplished by combining a stock BEI solution (e.g., a solution formed by adding 0.1-0.2 M 2-bromo-ethylamine hydrobromide to 0.1-0.2 N aqueous NaOH) with viral fluids to a final concentration of about 1-2 mM BEI. Inactivation is commonly performed by holding the BEI-virus mixture at 35-40.degree. C. (e.g., 37.degree. C.) with constant mixing for 36-72 hours. Virus inactivation can be halted by the addition of sodium thiosulfate solution to a final concentration in excess of the BEI concentration (e.g., 2-3 mM sodium thiosulfate with 1-2 mM BEI solutions) followed by mixing for several hours.

The present immunogenic compositions usually include an adjuvant and, if desired, one or more emulsifiers such as Tween.RTM. detergent incorporated with the inactivated EHV-1. Suitable adjuvants include, for example, vitamin E acetate solubilisate, aluminium hydroxide, aluminium phosphate or aluminium oxide, (mineral) oil emulsions, non-ionic detergents, squalene and saponins. Other adjuvants which may be used include an oil based adjuvants such as Freund's complete adjuvant (FCA), and Freund's incomplete adjuvant (FIA). It has been found that cross-linked olefinically unsaturated carboxylic acid polymers, such as Carbopol.RTM. 971 polymer, are particularly suitable adjuvants for use in the present inactivated EHV-1 immunogenic compositions.

One example of such an adjuvant is an olefinically unsaturated carboxylic acid polymer produced by reaction of a monomer mixture which includes one or more olefinically unsaturated carboxylic acid monomers (such as acrylic acid and/or methacrylic acid) and a cross-linking agent. Typically, at least about 90 wt. % of the monomer mixture is olefinically unsaturated carboxylic monomer. The resulting polymer product desirably contains no more than about 0.5 wt. % and, preferably, no more than about 0.2 wt. % unreacted olefinically unsaturated carboxylic monomer. The polymerization reaction can be carried out by reaction of the monomer mixture in the presence of solvent which includes aliphatic ketone, alkyl ester or a mixture thereof. Suitable aliphatic ketones include those having 3 to 6 carbon atoms, such as acetone and cyclohexanone (as used herein the term "aliphatic ketone" includes cycloaliphatic ketones). Examples of suitable alkyl esters include those having 3 to 6 carbon atoms, such as ethyl acetate, ethyl formate, isopropyl acetate, n-propyl acetate, butyl acetate or a mixture thereof.

Suitable olefinically unsaturated carboxylic acid polymer adjuvants desirably have a Brookfield viscosity of no more than about 40,000 cPs (at 20 rpm as a 0.5 wt. % aqueous solution at pH 7.5). Particularly suitable examples include olefinically unsaturated carboxylic acid polymers with a viscosity of no more than about 15,000 cPs and more desirably about 4,000-11,000 cPs (at 20 rpm as a 0.5 wt. % aqueous solution at pH 7.5).

One example of a suitable adjuvant includes a cross-linked acrylic acid polymer formed from a monomer mixture which includes acrylic acid and a cross-linking agent. The cross-linking agent may include a polyalkenyl polyether cross-linking agent, such as a divinyl glycol. Examples of suitable divinyl alcohols include allyl sucrose, allyl pentaerythritol, polyalkylene diol diallyl ether having a molecular weight of no more than 1000, trimethylolpropane diallyl ether, and mixtures thereof. Examples of other useful cross-linking agents are divinylbenzene, N,N-diallylacrylamide, 3,4-dihydroxy-1,5-hexadiene, 2,5-dimethyl-1,5-hexadiene and the like.

Where the vaccine is to be administered intranasally, it may be advantageous to use an adjuvant is bioadhesive with respect to mucous membranes. Bioadhesive polymers generally have the property of being able to adhere to a mucous membrane in the eyes, nose, mouth, gastrointestinal tract, vaginal cavity and rectal canal. Bioadhesive may be broadly defined as a material that adheres to a live or freshly killed biological surface such as mucus membrane or skin tissue. Bioadhesion as that phrase is used herein to define a useful bioadhesive is assayed by a procedure that measures the force required to separate two layers of freshly excised rabbit stomach tissue that are adhered together by an adhesive. Using this procedure, a bioadhesive may be defined as a material that requires a force of at least about 50 dynes/cm.sup.2 to separate two adhered, freshly excised pieces of rabbit stomach tissue, following the procedure described in U.S. Pat. No. 4,615,697, the disclosure of which is herein incorporated by reference. The upper limits for forces required to separate the freshly excised rabbit tissue are not precisely known, but are believed to be at least about 2000 dynes/cm.sup.2.

Suitable examples of adjuvants include cross-linked olefinically unsaturated carboxylic acid polymers with bioadhesive properties (e.g., Carbopol.RTM. 971 polymer, a cross-linked acrylic acid polymer available from B. F. Goodrich Co., Cleveland, Ohio). Polyacrylic acids of this type are generally crosslinked carboxy-functional polymers that contain specified amounts of carboxyl functionality and crosslinking agent. Such polymers can be a bioadhesive such that the polymers exhibit an adhesion between two pieces of freshly excised rabbit stomach tissue of at least 50 dynes/cm.sup.2 (when measured in the manner described in U.S. Pat. No. 4,615,697).

It is generally advantageous to formulate the present compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to the treated; each unit containing a predetermined quantity of the active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of inactivated EHV-1 (as well as inactivated EHV-4 and/or inactivated EIV) are dictated by and depend on among other factors (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved; (b) the limitations inherent in the art of compounding such active material for the treatment of disease; and (c) the manner of intended administration of the dosage unit form.

The principal active ingredient is typically compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as disclosed herein. A unit dosage form can, for example, contain the EHV-1 antigen in amounts ranging from 1 to about 5 relative potency units ("RPUs"). This amount of the antigen is generally present in from about 1 to about 25/ml of carrier. In the case of compositions containing supplementary active ingredients (e.g., inactivated EIV and/or inactivated EHV-4), the dosages are determined by reference to the usual dose and manner of administration of the supplementary active ingredients.

The present vaccines typically include inactivated EHV-1 formulated with a pharmaceutically acceptable carrier. The pharmaceutical forms suitable for injectable use commonly include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The formulation should desirably be sterile and fluid to the extent that easy syringability exists. The dosage form should be stable under the conditions of manufacture and storage and typically is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. One possible carrier is a physiological salt solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal (sodium ethylmercuri-thiosalicylate), deomycin, gentamicin and the like. In many cases it may be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions, if desired, can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum, monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the inactivated virus in the desired amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions can be prepared by incorporating the various active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

It may also be advantageous to add a stabilizer to the present compositions to improve the stability of inactivated virus. Suitable stabilizers include, for example, glycerol/EDTA, carbohydrates (such as sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose), proteins (such as albumin or casein) and protein degradation products (e.g., partially hydrolyzed gelatin). If desired, the formulation may be buffered by methods known in the art, using reagents such as alkali metal phosphates, e.g., sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate and/or potassium dihydrogen phosphate. Other solvents, such as ethanol or propylene glycol, can be used to increase solubility of ingredients in the vaccine formulation and/or the stability of the solution. Further additives which can be used in the present formulation include conventional antioxidants and conventional chelating agents, such as ethylenediamine tetraacetic acid (EDTA).

The compositions and methods of the present invention may be illustrated by the following examples, which are presented to illustrate the present invention and to assist in teaching one of ordinary skill how to make and use the same. These examples are not intended in any way to narrow or otherwise limit the scope of the present invention.

EXAMPLE 1

Production of the Fluids Containing Inactivated EHV-1 Strain

To produce the Equine Rhinopneumonitis Vaccine, killed virus, a master seed culture of an EHV-1 was first produced. From this master seed, a culture of EHV-1 was grown and then inactivated. The inactivated virus culture was then mixed with an adjuvant in order to produce the Equine Rhinopneumonitis Vaccine. The following method was used to produce the Equine Rhinopneumonitis Vaccine.

In order to produce the EHV-1 master seed virus culture ("EHV-1 MSV"), equine herpesvirus type 1 strain KyA (EHV-1 KyA) was passaged four times on Vero A139 cells and four times on EVero cells. The fourth passage was used as a master seed virus designated EHV-1 KyA, MSV Lot 001-dil.

From the master seed virus, a culture of EHV-1 was produced by infecting EVero cells with EHV-1 MSV in a minimum essential medium ("MEM") having 0 to 5% serum. Gentamicin was added to the culture medium in an amount sufficient to inhibit bacterial growth. The EVero cells were typically infected with the EHV-1 MSV with a target multiplicity of infection ("MOI") of 0.001. Such cultures can be grown in glass roller bottles or on microcarrier beads. The culture was incubated at 36.degree. C..+-.2.degree. C. for 24 to 120 hours until cytopathic effect ("CPE") was observed. During incubation, the culture was monitored for EHV induced CPE to ensure a pure EHV strain. If a typical CPE was observed or any macroscopic or microscopic evidence of contamination existed, the culture was discarded. Pure virus culture was aseptically harvested into sterile glass carboys, sterile plastic carboys, or sterile stainless steel tanks and was clarified by filtration through filters of 8 microns or greater. Bulk virus harvest fluids were tested to ensure the absence of mycoplasma prior to inactivation. Harvested fluids which were not immediately inactivated were stored at -40.degree. C. or below.

After being harvested, the virus culture was inactivated in order to produce a killed vaccine. To inactivate the virus, the culture temperature was brought to 36.degree. C..+-.2.degree. C. Next, a 0.2M solution of 2-bromoethyleneamine hydrobromide was cyclized to binary ethylenimine ("BEI") in 0.15M NaOH and added to the culture to give a final concentration of 2 mM BEI. The resulting mixture was stirred continuously for 48 hours at 36.degree. C..+-.2.degree. C.

After treatment with BEI, the culture was tested for its ability to induce CPE typical of EHV to ensure inactivation of the virus using the procedure described in Example 3. This task was accomplished by passing the BEI treated viral fluids over EVero cells and checking the EVero cells for any viral infection. The BEI treated culture fluids were typically stored at 2-7.degree. C. or below until the inactivation assay had been completed. After a satisfactory inactivation test showing no viral infection, excess BEI was neutralized by adding a sufficient amount of a cold (4.degree. C..+-.2.degree. C.) solution of 1.0M sodium thiosulfate to give a final concentration of 6 mM.

Following the inactivation and testing of the EHV-1 culture, the inactivated culture was concentrated, if necessary, by ultrafiltration to a concentration that would allow formulation as a vaccine with a relative potency ("RP") for EHV-1 of at least 1.0 as determined by the EHV potency release assay described in Example 6 herein.

The inactivated virus was formulated as an adjuvanted vaccine by thoroughly blending the inactivated EHV-1 culture with saline and a 0.5% stock solution of the adjuvant Carbopol.RTM. 971 to form a bulk serial. A typical 60 L serial was made by blending 3.5-4.0 L inactivated EHV-1 culture fluid, 12 L Carbopol.RTM. 971 stock solution and 44-44.5 L saline. The bulk serial was maintained at 2-8.degree. C. until being transferred into vials containing either one or ten doses (@2.2 ml per dose). Each dose of the inactivated vaccine contained at least 1.0 RP value of inactivated EHV-1, 2 mg Carbopol.RTM. 971 and a residual amount of gentamicin.

EXAMPLE 2

Production of the Fluids Containing Inactivated Equine Influenza Virus

Equine Influenza Virus--A/EQ1/Newmarket/77, Subtype A1

The MSV Rec ER1K subtype A1 of equine influenza was developed at the Wellcome Foundation Ltd., Beckenham, Kent, U.K. The original equine strain was passaged ten times, alone or in combination with A/Puerto Rico/8/34 virus, in specific pathogen free (SPF) embryonated eggs. Reassortant ER1K was passaged an additional seven times in Vero tissue culture to produce MSV designated A/E/Newmarket/77 (Equi) (H7N7) Rec ER1K. The virus was received by Boehringer Ingelheim Vetmedica, Inc. (BIVI) from Coopers Animal Health, Inc., Est. Lic. No. 107. The virus was passed one time in the EVero cell line [Vero cell line received from Coopers Animal Health has been designated as EVero at BIVI] at BIVI to establish the new master seed virus. The master seed virus is designated as Lot 111795.

Equine Influenza Virus--Newmarket/2/93, Subtype A2

The Newmarket/2/93 subtype A2 of equine influenza was obtained from Dr. J. A. Mummford at the Animal Health Trust, P.O. Box 5 Newmarket, Suffolk CB8 8JH, England. The virus was isolated from a horse with rhinitis. The virus was passaged five times in specific pathogen free (SPF) embryonated chicken eggs and then passaged five times in the Madin-Darby canine kidney (MDCK) cell line. The fifth passage in MDCK cells was designated as MSV. The MSV is designated as EIV NM/2/93, MSV Lot 001-dil.

Equine Influenza--Kentucky/95, Subtype A2

The Kentucky/95 subtype A2 of equine influenza was obtained from the Gluck Equine Research Center, Lexington, Ky. The virus was isolated from a horse with rhinitis. The virus was passaged two times in specific pathogen free (SPF) embryonated chicken eggs. The virus was then passaged three times in Madin-Darby canine kidney (MDCK) cell line and three times in EVero cells. The third passage in EVero cells was designated as MSV. The MSV is designated as EIV K95, MSV Lot 001-dil.

The following procedures were used to produce the three strains of equine influenza components separately, but by similar methods. Each production lot of Newmarket/77 virus was identified as equine influenza virus (EIV) by observing characteristic EIV induced cytopathic effects (CPE) in EVero cells. Each production lot of Newmarket/2/93 and Kentucky/95 virus was identified as EIV by observing characteristic EIV induced CPE in MDCK cells. The Newmarket/77 and Kentucky/95 are cytocidal for monkey kidney cell cultures and produced typical EIV CPE in monolayer cultures. The Newmarket/2/93 is cytocidal for MDCK cell cultures and produced typical EIV CPE in monolayer cultures. Virulence in horses was not evaluated for any of the viruses. The master seed viruses were tested for purity in accordance with 9 C.F.R. 113.27 (c), 113.28, and 113.55. The Newmarket/77 master seed virus was for Swine Vesicular Disease, Akabane, Bovine Ephemeral Fever, Bluetongue, and Velogenic Viscerotrophic Newcastle disease. Bulk virus harvest fluids are tested for mycoplasma prior to inactivation in accordance with the 9 C.F.R. 113.28. The master seed viruses were tested for immunogenicity using the following procedure.

Bulk or final container samples of completed product from each serial were tested for potency by a guinea pig potency test. Each of at least ten guinea pigs, weighing 300-500 grams, was injected intramuscularly. Each guinea pig dose was one half of the dose recommended as a unit dose of the vaccine on the label for a horse. A second dose was injected 14 to 21 days after the first dose. Two additional guinea pigs from the same source were held as controls. Fourteen to 21 days after the second injection, serum samples from each vaccinate and each control were tested for Newmarket/77, Newmarket/2/93 and Kentucky/95 antibodies by hemagglutination inhibition (HAI). The potency of the EIV fractions in the vaccine were determined using the National Veterinary Services Laboratories Testing Protocol, Supplemental Assay Method for Conducting the Hemagglutination Inhibition Assay for Equine Influenza Antibody (MVSAM0124.01, dated Oct. 2, 1998).

If the controls did not remain seronegative at 1:10 for the EIV fraction under test, the test was considered a no test and was repeated. If eight of the ten vaccinates in a valid test did not develop HAI antibody titer of 80 or greater for the Newmarket/77 fraction and a titer of 40 or greater for the Newmarket/2/93 and Kentucky/95 fractions, the sample was considered unsatisfactory. Vaccines containing inactivated EIV are desirably formulated to include at least about 64 HAU/unit dose of inactivated EIV virus subtype A1 and/or at least about 256 HAU/unit dose of inactivated EIV virus subtype A2

The first passage through the fourth passage from master seed virus were utilized for working and production seed. Vaccine was produced from the first passage through the fifth passage from master seed virus. Equine Influenza, strain Newmarket/77, subtype A1 and Equine Influenza, strain Kentucky/95, subtype A2 were propagated in EVero Cell Line Cultures.

The MDCK cell line used for propagation of Newmarket/2/93 was confined between passages 71 and 91. Passage 71 was frozen and was designated as the Master Cell Stock. The MDCK cell cultures were propagated in MEM containing 5-10% fetal bovine serum. Seed and production culture of Newmarket/2/93 were propagated in MEM supplemented with 10 units of trypsin/mL. Cell cultures are grown on microcarrier beads in 1, 3, 10, 30, 50, or 140 liter cell culture vessels, 9 L glass roller bottles, or plastic roller bottles. Master and working seed viruses are stored at -60.degree. C. or below.

MDCK cells cultures were grown in sterile glass or plastic containers. The cultures were grown in 75 cm.sup.2 or 150 cm.sup.2 plastic flasks with 40 to 100 mL of media. Expansion cultures were grown in roller bottle cultures in 100-1000 mL media and then subcultured in roller bottle cultures also. Either a 75 cm.sup.2 or 150 cm.sup.2 surface attachment area was chosen, based upon the attachment and growth of the cell line when first coming out of the liquid nitrogen repository. Due to the stress of liquid nitrogen storage, in some instances it was advantageous to start cell growth in a smaller surface area, and when growth had been achieved, subculture the cells further into a 150 cm.sup.2 T-flask. If the cells came out of liquid nitrogen storage and produced a healthy monolayer of cells sufficient for expansion in a 150 cm.sup.2 T-flask, then the 75 cm.sup.2 T-flask step was omitted and the cells were planted directly into the 150 cm.sup.2 T-flask.

Prior to inoculation with virus, the cell growth medium was decanted from the culture vessel. Minimum essential medium (MEM) without serum was added to the culture vessel and the cells were rinsed for 30 to 45 minutes. The volume of rinse medium was dependent on the culture vessel being used. The culture vessel was inoculated by decanting rinse medium and adding MEM without serum containing 10 Units of purified trypsin/ml MEM and to which seed virus has been added. Cell cultures were infected with a target multiplicity of infection (MOI) of 0.01 for the Newmarket/77, Newmarket/2/93 and Kentucky/95 strains.

The cell cultures were maintained at 36.degree. C..+-.2.degree. C. until the fluid was harvested. Monolayers were observed following inoculation for characteristic EIV induced changes in cell morphology. CPE included rounding and refractile cells that detached from the monolayer surface. CPE was usually apparent 24-96 hours after inoculation. The culture was examined throughout the incubation period for macroscopic and/or microscopic evidence of contamination or for a typical changes in cell morphology. Any culture exhibiting evidence of microbial contamination or nonspecific cellular degeneration was discarded.

Prior to harvesting, each culture was examined macroscopically and microscopically. Any culture exhibiting evidence of contamination or a typical CPE was discarded. The fluid from the culture vessel(s) was harvested one to four days after inoculation. The virus culture fluid in the culture vessel was aseptically harvested into sterile glass or plastic carboys or a sterile stainless steel tank after clarification by filtration through filters of 8 microns or greater. Pooled fluids were either inactivated immediately or stored at -40.degree. C. or below for inactivation at a later time. Samples were collected for sterility and antigen testing after inactivation.

The volume of culture fluids was determined and the temperature of the fluids was brought to 36.degree. C..+-.2.degree. C. A 0.2M solution of 2-bromoethyleneamine hydrobromide which had been cyclized to binary ethlylenimine (BEI) in 0.15M NaOH was added to the culture fluids to give a final BEI concentration of 2 mM. The inactivated virus fluid was stored either frozen at .ltoreq.-40.degree. C. or at 4-8.degree. C. until completion of inactivation testing.

Each lot of Newmarket/77 and Kentucky/95 was tested for inactivation by passage in Vero A139 cells. Each lot of Newmarket/2/93 was tested for inactivation by passage in MDCK cells. Appropriate cell culture monolayer (150 ml) was inoculated with 1.0 ml of inactivated EIV fluids and maintained at 36.degree. C..+-.2.degree. C. for 14 days with at least two passages. At the end of the maintenance period, the cell monolayers were examined for CPE typical of EIV. For positive virus controls, one culture flask each of EVero cells was inoculated with reference Newmarket/77 and Kentucky/95 virus, respectively, and one culture of MDCK cells was inoculated with a reference Newmarket/2/93 virus (each to a target MOI of 0.01). One flask of EVero cells and one flask of MDCK cells remained uninoculated as negative controls. After incubation and passage, the absence of virus-infected cells in the BEI treated viral fluids constituted a satisfactory test for inactivation. The control cells inoculated with the reference virus showed CPE typical of EIV and the uninoculated flask showed no evidence of EIV CPE.

After a satisfactory inactivation test, residual BEI was neutralized by the addition of a cold (4.degree. C..+-.2.degree. C.) solution of 1.0 M sodium thiosulfate to give a final concentration of 6 mM. The inactivated fluids were stored at 4.degree. C..+-.2.degree. C. or below until bulking of the final product. If the inactivation test was unsatisfactory, the fluids could be retreated with BEI using the procedure described above and retested for inactivation.

If necessary, each lot of inactivated bulk virus fluid was concentrated 2.times. to 50.times. by ultrafiltration to achieve the desired concentration. One or more bulk lots of inactivated bulk virus fluids were usually pooled for concentration. The concentrated bulk virus was held at 4-8.degree. C. until bulking.

The resulting product after inactivation and concentration contained gentamicin in residual amounts from the medium used in the production of the virus harvest fluids. These levels did not exceed the level allowed per dose of product. The vaccine was formulated to contains 2 mg of Carbopol.RTM. 971 per dose of vaccine.

The components for bulking were aseptically added to a glass, plastic, or stainless steel container by siphoning, or by positive pressure (sterile-filtered nitrogen). The serial was blended thoroughly and then maintained at 2-8.degree. C. until ready for filling into final containers. The following provides an illustrative inactivated EIV vaccine composition:

Newmarket/77 3,000 ml Newmarket/2/93 6,000 ml Kentucky/95 6,000 ml Carbopol .RTM. 971 (0.5% stock solution) 12,000 ml Saline 33,000 ml

If desired, the saline or a portion thereof can be substituted by a solution that contains inactivated EHV-1 and, optionally, inactivated EHV-4 to produce a combination EHV/EIV vaccine.

After bulking, the serial was drawn off into sterile plastic or stainless steel containers for transfer to a sterile fill area for filling into final containers, or the bulk serial was sampled and drawn off into sterile plastic carboys. If the serial was drawn off into carboys, it was stored at 4-8.degree. C. If two or more carboys of bulk vaccine were to be filled at a single time, the product was pooled into a sterile stainless steel vessel when required for filling. Each dose of vaccine was formulated to contain sufficient inactivated virus harvest fluids to provide at least 64 hemagglutination units (HAU) of Newmarket/77 and 128 HAU of Newmarket/2/93 and 128 HAU of Kentucky/95.

EXAMPLE 3

Method of Monitoring Inactivation of Viruses

Each lot of EHV-1, EHV-4, EIV Newmarket/77, and EIV