Swine virus vaccines that are liquid stable

Abstract

The present invention is drawn to liquid stable swine vaccines that comprise a live porcine virus. The invention is also drawn to the manufacture of such vaccines and methods of vaccinating animal subjects with these vaccines.

Claims

1. A liquid stable vaccine that comprises a live swine virus, a 5-40% (w/v) sugar alcohol, and 0.15 to 0.75 M of an amino acid selected from the group consisting of arginine, glutamic acid, and glycine; wherein the liquid stable vaccine has a pH of 6.0 to 8.0; and wherein the live swine virus is a Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).

2. The liquid stable vaccine of claim 1 wherein the sugar alcohol is sorbitol.

3. The liquid stable vaccine of claim 2 wherein the amino acid is arginine.

4. The liquid stable vaccine of claim 3 that further comprises a live attenuated PED virus.

5. The liquid stable vaccine of claim 4 that further comprises a live attenuated Porcine Parvo Virus (PPV).

6. The liquid stable vaccine of claim 5 that further comprises a live attenuated Swine Influenza Virus (SIV) or a recombinant attenuated SIV vector that encodes a heterologous antigen.

7. The liquid stable vaccine of claim 6 that further comprises a live Porcine Pseudorabies Virus (PPRV), or a recombinant PPRV vector that encodes a heterologous antigen.

8. The liquid stable vaccine of claim 6 that further comprises a live attenuated Transmissible Gastroenteritis Virus (TGE virus).

9. The liquid stable vaccine of claim 3 that further comprises a bacterium selected from the group consisting of a live attenuated Pasteurella multocida, a live attenuated Salmonella ssp., a live attenuated Mannheimia haemolytica, a live attenuated Lawsonia intracellularis, a live attenuated Clostridium perfringens, a killed Pasteurella multocida, a killed Salmonella ssp., a killed Actinobacillus pleuropneumonias, a killed Lawsonia intracellularis, a killed Mycoplasma hyopneumoniae, a killed Bordetella bronchiseptica, a killed bacterin or extracted pilus antigen from any of the following types of Escherichia coli: K99, K88, 987P, or F41, an inactivated toxoid of Clostridium perfringens, and any combination thereof.

10. The liquid stable vaccine of claim 3, further comprising a live swine virus selected from the group consisting of a Transmissible Gastroenteritis Virus (TGE), Porcine Parvo Virus (PPV), Porcine Pseudorabies Virus (PPRV), Swine Influenza Virus (SIV), Porcine Epidemic Diarrheal Virus (PED), and any combination thereof.

11. The liquid stable vaccine of claim 10 that further comprises a killed swine virus.

12. The liquid stable vaccine of claim 1 further comprising a sugar additive that is a non-alcohol sugar, wherein the total amount of the sugar alcohol and the non-alcohol sugar in the liquid stable vaccine is 15-40% (w/v).

13. The liquid stable vaccine of claim 12 wherein the non-alcohol sugar is selected from the group consisting of sucrose and trehalose.

14. The liquid stable vaccine of claim 1 that further comprises a buffer.

15. The liquid stable vaccine of claim 14 wherein the buffer comprises 2.5 to 50 mM potassium phosphate.

16. The liquid stable vaccine of claim 1, wherein the amino acid is arginine.

17. A method of vaccinating a pig against PRRSV comprising administering to the pig the liquid stable vaccine of claim 1.

18. The method of claim 17, wherein said administering is performed by a method selected from the group consisting of intranasal spray, intramuscular injection, intradermal injection, subcutaneous injection, and feeding.

19. A liquid stable vaccine that comprises a live Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), at least 10 to 20% (w/v) sugar alcohol, and 0.25 to 0.45 M arginine; wherein the liquid stable vaccine has a pH of 6.0 to 8.0.

20. The liquid stable vaccine of claim 19, wherein the sugar alcohol is sorbitol.

21. The liquid stable vaccine of claim 20, that further comprises 5-20% (w/v) sucrose.

22. The liquid stable vaccine of claim 20, that comprises 15% (w/v) sorbitol, and 0.3 M arginine.

23. A method of making a liquid stable vaccine that comprises combining a therapeutically effective amount of a live attenuated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) with a 5-40% (w/v) sugar alcohol, and 0.15 to 0.75 M arginine; wherein the liquid stable vaccine has a pH of 6.0 to 8.0.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Because the liquid stable swine virus vaccines of the present invention comprise live viruses, e.g., live attenuated viruses, heretofore particular care would have been needed during the formulation of the vaccine to maintain the titer of the attenuated viruses at a level that is safely below that which can lead to a significant adverse event. Indeed, most live attenuated swine virus vaccines are lyophilized, and lyophilization can lead to substantial declines in the efficacy of the attenuated live virus vaccines both due to the lyophilization process itself, as well as over time during long-term storage.

(2) The present invention has overcome this problem by providing liquid stable swine vaccines that remain efficacious, even during storage, without needing to increase the initial titer of the live attenuated viral antigen above a reliably safe level. As an additional benefit, the present invention provides a means for lowering the cost of manufacture of the vaccines provided by significantly reducing the amount of live attenuated swine viruses necessary to make such a safe and efficacious vaccine. In addition, the live attenuated swine virus vaccines of the present invention are more convenient to use than their lyophilized counterparts. Accordingly, the present invention provides safe and efficacious live attenuated swine virus vaccines that can be stored as liquids at refrigerated temperatures and still remain stable for 5 to 7 months, 6 to 9 months, 9-12 months, 12 to 18 months, 18 to 24 months and/or even longer. Unlike its lyophilized counterpart, the liquid stable vaccines of the present invention do not have to be used as soon as it is rehydrated, because they are always hydrated. Since swine vaccines often come in 100 dose vials, as the largest presentation, those with larger farms will use hundreds of glass vials and discard the hazardous waste leftover from these vaccines in the trash. With the liquid stable swine vaccine, larger farms can buy larger packages (e.g., made of plastic) of vaccine, which they can use over weeks or months, providing the vaccine is handled properly and not contaminated, and then burn the residual plastic container when the vaccine is used up, thus decontaminating the container. This opens a whole new exclusive market of convenience to larger farms.

(3) Moreover surprisingly, the liquid stable live swine virus vaccines of the present invention can include swine viruses of any type. Thus, the liquid stable live virus vaccines of the present invention can include both enveloped and non-enveloped swine viruses. In addition, the liquid stable live virus vaccines of the present invention can include live attenuated swine viruses having single-stranded RNA genomes, single-stranded DNA genomes, or double-stranded DNA genomes.

(4) The use of singular terms for convenience in the description is in no way intended to be so limiting. Thus, for example, reference to a sugar additive includes reference to one or more of such sugar additives, unless otherwise specified. The use of plural terms is also not intended to be limiting, unless otherwise specified. Similarly, a chemical compound that can be referred to as an acid or its corresponding base, unless otherwise specified, when denoted herein as either is intended to mean either form of the compound. Thus, the use of the term glutamic acid is meant to include glutamate and vice versa.

(5) As used herein, a vaccine is a composition that is suitable for application to an animal (including, in certain embodiments, humans) which upon administration to the animal induces an immune response strong enough to minimally aid in the protection from a clinical disease arising from an infection with a wild-type micro-organism, i.e., strong enough for aiding in the prevention of the clinical disease, and/or preventing, ameliorating, or curing the clinical disease.

(6) Unless expressly indicated otherwise, the use of the term vaccine includes multivalent vaccines.

(7) As used herein, a multivalent vaccine is a vaccine that comprises two or more different antigens. In a particular embodiment of this type, the multivalent vaccine stimulates the immune system of the recipient against two or more different pathogens.

(8) As used herein, a liquid stable vaccine is a vaccine maintained as a liquid (including a liquid multivalent vaccine) that remains efficacious for at least six months when stored at or below 7 C. (e.g., in a standard refrigerator, and/or at 0 C.-7 C.). In particular embodiments a liquid stable vaccine remains efficacious when stored at or below 7 C. for at least 6 months. In even more particular embodiments a liquid stable vaccine remains efficacious when stored at or below 7 C. for at least 9 months. In still more particular embodiments a liquid stable vaccine remains efficacious when stored at or below 7 C. for at least 1 year. In yet more particular embodiments a liquid stable vaccine remains efficacious when stored at or below 7 C. for at least 1.5 years. In still more particular embodiments a liquid stable vaccine remains efficacious when stored at or below 7 C. for at least 2.0 to 3 years.

(9) The terms pig or swine or porcine are all used interchangeably and include all domesticated porcine species, unless otherwise indicated.

(10) As used herein, the terms protect, protecting, provide protection to, providing protection to, and aids in the protection do not require complete protection from any indication of infection. For example, aids in the protection can mean that the protection is sufficient such that, after challenge, symptoms of the underlying infection are at least reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced and/or eliminated. It is understood that reduced, as used in this context, means relative to the state of the infection, including the molecular state of the infection, not just the physiological state of the infection.

(11) The term prophylactically-effective amount refers to the amount of a composition that when administered to swine significantly reduces the likelihood and/or extent of an infection/infestation due to a given pathogen.

(12) Metaphylaxis is the timely mass medication of an entire group of animals to eliminate or minimize an expected outbreak of disease, e.g. in one or more animals at high risk of infection/infestation.

(13) The term chemoprophylaxis refers to the administration of a medication/treatment, e.g., one or more prophylactic compositions, for the purpose of preventing or reducing viral, bacterial, and/or parasitic infection/infestation; and/or preventing or reducing disease and/or symptoms related to that infection/infestation.

(14) The term prophylactic composition refers to any agent used singularly or in combination with other agents that significantly reduces the likelihood and/or extent of an infection/infestation due to a given pathogen in swine. In one such embodiment the swine are at high risk of developing swine enteric disease following commingling, changes in weather, changes in nutrition, and/or other stressors that can initiate a symptom and/or a disease related to the presence of the viral, bacterial, or parasitic pathogens commonly associated with swine, targeted by the agent or combination of agents.

(15) As used herein, the term therapeutically effective amount is an amount of a given antigen, e.g., a live attenuated swine virus, which is sufficient to provide protection to and/or aid in the protection from the pathogen that the antigen is being administered to protect against, when provided in a single administration and/or when intended, provided as an initial administration with one or more subsequent booster administration(s).

(16) As used herein, an efficacious vaccine comprises a therapeutically effective amount of a given antigen. An efficacious vaccine retains sufficient titer for a given antigen to be compliant with the regulatory requirements for that antigen for the jurisdiction where the vaccine is administered, e.g., the administration of a vaccine in the United States is governed by the United States Department of Agriculture (USDA).

(17) As used herein, the term pharmaceutically acceptable is used adjectivally to mean that the modified noun is appropriate for use in a pharmaceutical product. When it is used, for example, to describe an excipient in a pharmaceutical vaccine, it characterizes the excipient as being compatible with the other ingredients of the composition and not disadvantageously deleterious to the intended recipient.

(18) The term carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Pharmaceutical acceptable carriers can be sterile liquids, such as water and/or oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous sugar, e.g., dextrose and/or glycerol, solutions can be employed as carriers, particularly for injectable solutions. In addition, the carrier can be and/or comprise a hydrocolloid and/or polymer solution e.g., to thicken the porcine vaccines that are to be sprayed on to the pigs.

(19) As used herein, an adjuvant is a substance that is able to favor or amplify the cascade of immunological events, ultimately leading to a better immunological response, i.e., the integrated bodily response to an antigen. An adjuvant is in general not required for the immunological response to occur, but favors or amplifies this response.

(20) As used herein, systemic administration is administration into the circulatory system of the body (comprising the cardiovascular and lymphatic system), thus affecting the body as a whole rather than a specific locus such as the gastro-intestinal tract (via e.g., oral administration) and the respiratory system (via e.g., intranasal administration). Systemic administration can be performed e.g., by administering into muscle tissue (intramuscular), into the dermis (intradermal, transdermal, or supradermal), underneath the skin (subcutaneous), underneath the mucosa (submucosal), in the veins (intravenous) etc.

(21) Parenteral administration includes subcutaneous injections, submucosal injections, intravenous injections, intramuscular injections, intradermal injections, and infusion.

(22) As used herein a sugar additive is a 5 to 12 carbon sugar (e.g., sucrose, maltose, trehalose, dextrose, lactose, glucose, fructose, galactose) or sugar alcohol/polyol (e.g., sorbitol, mannitol, arabitol, inositol, maltitol). Unless otherwise specifically stated to the contrary, the percent (%) of the sugar additive is provided as a weight (w) of the sugar additive to the volume (v) of the vaccine, (w/v) in the vaccine.

(23) As used herein a non-reducing sugar is a sugar additive which in basic aqueous medium does not generate any compounds containing an aldehyde group. Examples of non-reducing sugars of the present invention include sucrose and trehalose.

(24) As used herein, the terms non-sugar alcohol and non-alcohol sugar are used interchangeably. A sugar additive that is a non-sugar alcohol (or non-alcohol sugar) as used herein, can be any sugar additive that is not a sugar alcohol, e.g., a non-reducing sugar.

(25) As used herein, unless otherwise specifically stated to the contrary, the percent (%) of a solid additive, e.g., sugar additive or gelatin, in a vaccine is based on a 1% solution being 1 g of solid/100 ml of vaccine volume (w/v).

(26) As used herein, unless otherwise specifically stated to the contrary, the percent (%) of a liquid additive, e.g., ethanol, in a vaccine is based on a 1% solution being 1 ml of liquid additive/100 ml of vaccine volume (v/v).

(27) As used herein, the term, approximately, is used interchangeably with the term about and generally signifies that a value is within twenty-five percent of the indicated value, unless otherwise indicated, i.e., a concentration of about 2 mM EDTA can be 1.5 mM to 2.5 mM EDTA.

(28) As used herein, unless otherwise specifically stated to the contrary, the pH value provided is the pH value determined/measured at 25 C.

(29) Because the liquid stable vaccines of the present invention ideally range in pH from pH 6.0 to pH 8.0, the liquid stable vaccines of the present invention can comprise a buffer. Buffers for use in the liquid stable vaccines of the present invention include but are not limited to: potassium phosphate, sodium phosphate, Tris, Tris-Histidine, BIS-Tris, BIS-Tris-Propane, sodium or potassium pyrophosphate, imidazole, PIPES, ACES, MOPS, MOPSO, BES, TES, tricine, glycylglycine, and HEPES. The buffers can be brought to the desired pH with the use of any suitable counter ion.

(30) The hydrolysate of whole casein that can be used in the liquid stable vaccines of the present invention can be obtained by a number of procedures including e.g., as an acid hydrolysate or an enzymatic hydrolysate. Such hydrolysates contain in the form of mixed amino acids and peptides having all of the amino acids originally present in casein. One pancreatic hydrolysate of whole casein that can be used in the liquid stable vaccines of the present invention is sold as CASEIN HYDROLYSATE ENZYMATIC by MP Biomedicals. Comparable products are sold under the name of NZ-AMINE, NZ-AMINE A, NZ-AMINE AS, and NZ-AMINE B, and Tryptone by Sigma-Aldrich.

(31) Examples of hydrocolloids that can be comprised by the vaccines of the present invention include: gelatin, starch polymers, and gums, such as xanthum gum, carragenin, and gum Arabic (acacia gum).

(32) Multivalent Vaccines: The present invention provides liquid stable multivalent vaccines. A liquid stable multivalent swine vaccine of the present invention can include two or more antigens including one or more of the following live attenuated swine viruses: PRRS, PEDV, PRV, TGEV, PPRV, SW, and/or a recombinant SIV encoding one or more heterologous antigens. As noted above, a liquid stable multivalent swine vaccine of the present invention can also include one or more of the following live attenuated viruses: PRRS, PEDV, PRV, TGEV, PPRV, SW, and/or a recombinant SIV encoding one or more heterologous antigens, along with one or more killed swine viruses.

(33) In addition, a liquid stable vaccine of the present invention can be subsequently combined with one or more live attenuated or killed bacterial vaccine comprising an antigen such as Pasteurella multocida, Salmonella ssp., Escherichia coli of multiple pillus types, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Mycoplasma hyopneumoniae, Lawsonia intracellularis, Erysipelas spp., Clostridium perfringens and Clostridium difficile prior to administration. Examples of monovalent and multivalent vaccines that can be used in the liquid stable vaccines of the present invention are provided in Tables 1A-1C below:

(34) TABLE-US-00001 TABLE 1A Live Virus Vaccines Single Combo Porcine reproduction and Porcine rotavirus/E. coli/ respiratory syndrome C. perfringens Type C virus (PRRS) Porcine Rotavirus (PRV) TGE/PRV/C. perfringens type C/ multiple serotypes E. coli multiple pilus types Transmissible gastroenteritis virus (TGE)

(35) TABLE-US-00002 TABLE 1B Live Bacteria Single Combo Salmonella cholerasuis M. haemolytica/P. multocida Mannheimia haemolytica Lawsonia/PCV/M. hyopneumoniae Pasteurella multocida Lawsonia intracellularis

(36) TABLE-US-00003 TABLE 1C Killed Vaccines Single Combo Clostridium Perfringens Clostridium perfringens Type C/ Type A toxoid D toxoids Clostridium perfringens Porcine Parvovirus, Erysipelas, Type C toxoid Leptospira canicola, pomona, hardjo-icterhaemorrhagia, Grippotyphosa Clostridium perfringens Swine influenza virus (multiple Type D toxoid serotypes) Mycoplasma hyopneumoniae C. perfringens type C toxoid/ E. coli Porcine Circovirus E. coli (multiple pilus types) Porcine Parvovirus E. coli (multiple pilus types)/ C. perfringens type C Swine Influenza virus B. bronchiseptica/P. multocida Escherichia coli multiple pilus types including K99, K88, 987P, Type 1 Lawsonia intracellularis Bordetella bronchiseptica Actinobacillus pleuropneumoniae

(37) Adjuvants: The vaccines of the present invention can either contain an adjuvant or alternatively not contain an adjuvant, often depending on the antigen(s) that the vaccine contains. In particular embodiments, the adjuvant comprises an aluminum salt. The use of aluminum salts in conjunction with live viral vaccines has been described. In particular embodiments the aluminum salt is chosen from the group consisting of aluminum phosphate, aluminum potassium phosphate, and aluminum hydroxide. One aluminum phosphate adjuvant is REHYDROPHOS (General Chemical, Parsippany, N.J.). Examples of aluminum hydroxide adjuvants include: REHYDROGEL, REHYDROGEL HPA, or REHYDROGEL LV (General Chemical, Parsippany, N.J.). Other well-known adjuvants include hydrocarbon oils, polymers, saponins and/or an adjuvant made up of gel particles of sodium acrylate in water, e.g., MONTANIDE PET GEL A (Seppic, Paris France). One low molecular weight copolymer adjuvant can form cross-linkage in solution to become a high molecular weight gel, e.g., POLYGEN (MVP Laboratories, Omaha) When added, the amount of adjuvant is usually between about 1% and 20% (v/v) in the vaccine. In particular embodiments the amount of adjuvant is between about 2% to 10% (v/v). In more particular embodiments the amount of adjuvant is between about 3% to 6% (v/v).

(38) Vaccine Administration: The liquid stable virus vaccines of the present invention may be administered by any conventional means, for example, by systemic administration, including by parenteral administration such as, without limitation, subcutaneous or intramuscular administration. The liquid stable virus vaccines of the present invention also may be administered by mucosal administration, such as by intranasal, oral, and/or ocular administration. Alternatively, the vaccines may be administered via a skin patch, in a delayed release implant, scarification, or topical administration. It is contemplated that a liquid stable virus vaccine of the present invention also may be administered via the drinking water and/or food of the recipient swine.

(39) The vaccines (including multivalent vaccines) of the present invention also may be administered as part of a combination therapy, i.e., a therapy that includes, in addition to the vaccine itself, administering one or more additional active agents, therapies, etc. In that instance, it should be recognized the amount of vaccine that constitutes a therapeutically effective amount may be more or less than the amount of vaccine that would constitute a therapeutically effective amount if the vaccine were to be administered alone. Other therapies may include those known in the art, such as, e.g., analgesics, fever-reducing medications, expectorants, anti-inflammation medications, antihistamines, and/or administration of fluids.

(40) The immunogenicity level may be determined experimentally by vaccine dose titration and challenge study techniques generally known in the art. Such techniques typically include vaccinating a number of animal subjects with the vaccine at different dosages and then challenging the animal subjects with the virulent virus to determine the minimum protective dose.

(41) Factors affecting the preferred dosage regimen may include, for example, the breed (e.g., of a swine), age, weight, diet, activity, lung size, and condition of the subject; the route of administration; the efficacy, safety, and duration-of-immunity profiles of the particular vaccine used; whether a delivery system is used; and whether the vaccine is administered as part of a drug and/or vaccine combination. Thus, the dosage actually employed can vary for specific animals, and, therefore, can deviate from the typical dosages set forth above. Determining such dosage adjustments is generally within the skill of those in the art of vaccine development using conventional means.

(42) Similarly, the volume with which such a dose can be administered typically lies between 0.1 mL (intradermal applications) and 2.0 mL. A typical range for the administration volume is between 0.2 and 1.0 mL, and about 0.2 to 0.5 mL for intradermal administration.

(43) It is contemplated that the vaccine may be administered to the vaccine recipient at a single time or alternatively, two or more times over days, weeks, months, or years. In some embodiments, the vaccine is administered at least two times. In certain such embodiments, for example, the vaccine is administered twice, with the second dose (e.g., a booster) being administered at least 2 weeks after the first dose. In particular embodiments, the vaccine is administered twice, with the second dose being administered no longer than 8 weeks after the first dose. In other embodiments, the second dose is administered from 1 week to 2 years after the first dose, from 1.5 weeks to 8 weeks after the first dose, or from 2 to 4 weeks after the first dose. In other embodiments, the second dose is administered about 3 weeks after the first dose.

(44) In the above embodiments, the first and subsequent dosages may vary, such as in amount and/or form. Often, however, the dosages are the same in amount and form. When only a single dose is administered, the amount of vaccine in that dose alone generally comprises a therapeutically effective amount of the vaccine. When, however, more than one dose is administered, the amounts of vaccine in those doses together may constitute a therapeutically effective amount. In addition, a vaccine may be initially administered, and then a booster may be administered from 2 to 12 weeks later, as discussed above. However, subsequent administrations of the vaccine may be made on an annual (1-year) or bi-annual (2-year) basis, regardless as to whether a booster was administered or not.

(45) The vaccines of the present invention can also contain an anti-bacterial such as an antibiotic. Examples of such antibiotics can include: 10-1000 g/mL gentamicin, 0.5-5.0 g/mL amphotericin B, 10-100 g/mL tetracycline, 10-100 units/mL nystatin (mycostatin), 10-100 units/mL penicillin, 10-100 g streptomycin, 10-100 g polymyxin B, and 10-100 g neomycin.

(46) The present invention may be better understood by reference to the following non-limiting Example, which is provided as exemplary of the invention. The following Example is presented in order to more fully illustrate embodiments of the invention. It should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLE

Stability of Liquid Swine Virus Vaccines

(47) Materials and Methods

(48) Bulk antigen preparation: Frozen bulk PRRS virus antigen was obtained and kept at <50 C. until the time to blend the vaccine. The PRRS bulk antigen has a titer around 9 logs log.sub.10(EID.sub.50).

(49) Materials: US Pharmacopeial (USP) or higher grade sucrose and sorbitol are purchased from Fisher Scientific. Molecular biology grade L-arginine monohydrochloride with purity higher than 98% are purchased from Sigma. NZ Amine (bloom 250) solutions are prepared from the best available commercial reagents. The following solutions have been prepared and sterilized by autoclaving or 0.2 um filtration: 80% (w/v) sucrose, 70% (w/v) sorbitol, 2.3 M L-arginine monohydrochloride (pH 7.2), 5% (w/v) methionine hydrochloride solution, 45% (w/v) polyvinylpyrrolidone K-60, 0.5 M ethylenediaminetetraacetic acid (EDTA), Pleuronic F-68, 3 M glutamic acid, monosodium salt, 1.0 M potassium phosphate buffer (pH 7.2), tryptose phosphate broth (TPB) solution and 250 mg/mL gentamicin. Stock Solutions are summarized in Table 2A.

(50) Stabilizer solution and vaccine blend pH adjustment: The pH of the final vaccine blend can be critical to the stability of the virus in liquid. The pH is measured using a very sensitive pH probe and meter. The meter displays the pH to 3 significant figures to the right of the decimal. There is a separate temperature probe with meter and both must be in the solution and stable. This pH meter is capable of a 5 point calibration curve with 3 points being an absolute minimum. During the vaccine blend preparation, the pH of the stabilizer solution is adjusted to the target pH prior to adding the virus antigen, and the pH is measured again after mixing the stabilizer solutions and virus antigens.

(51) Preparation of the stabilizer solutions: Once the initial pH adjustment has been made, all formulations were filter sterilized using a 0.2 M filter (PES is preferred filter matrix simply due to improved filter capacity). Currently filtration is performed using vacuum. A secondary benefit of vacuum filtering is the additional de-gassing of the formulation. After the formulation has been filter sterilized, it is sparged with argon gas to increase the depletion of O.sub.2 which will hopefully yield lower reactivity of the formulation over time. Once the sparging is complete an argon overlay is placed prior to storage and a tight a seal is made. After the formulation is prepared, the pH is confirmed/adjusted to pH 7.2 at the desired temperature (e.g., 4, 15, or 25 C.), i.e., for many experiments performed herein the desired temperature was 4 C. If the formulation and previous procedures have been performed correctly the pH should be close to target pH. With an overnight incubation the pH may drift slightly due to the completion of chemical reactions associated with the earlier pH adjustment and further de-gassing.

(52) Thawing virus bulk antigen: The frozen antigens are slowly thawed at room temperature (15-30 C.) or at refrigerated temperature (2-8 C.). The thawed antigen should be kept at 2-8 C. for no more than 8 hours prior to usage.

(53) Preparation of vaccine blend: To make a 100 mL of vaccine blend, the volume of each stock solution required to reach the final concentration for each component as listed in Table 2B was calculated and then added to a sterilized container first, and the stabilizers and excipients were mixed using a stiffing bar. After the stabilizer solutions and all excipients were thoroughly mixed, the virus antigens at the indicated volume were added into the container and thoroughly mixed. The generating of bubbles and foams were avoided during this mixing step. When the virus is added in a very short time frame (a few minutes) then the virus may be added with no further issues. When the virus is not immediately added, an argon overlay is put in place to displace residual O.sub.2. Once the virus has been added a fresh argon overlay is put into place prior to mixing. The argon gas is added to the bottle using a low flow rate. After the vaccine blending is complete, the vaccine blend is kept at 2-8 C. until dispensing into small aliquots.

(54) Filling the vaccines: The vaccine blend was dispensed into glass ampule vials at 1.8 mL per vial. Each vial is then back-filled and overlaid with argon gas. The ampule vials were flame sealed, labelled and then transferred into boxes, and stored in the incubator at the designated temperature.

(55) Stability testing at elevated temperature and real-time conditions: Liquid PRRS vaccines in ampule vials were stored at 27 C. and 4 C. respectively in the corresponding incubators. At the designated time point, 2 or more vials from each formulation were retrieved and the titer of each antigen was measured by tissue cell culture based virus titration assay.

(56) Analytical Methods

(57) PRRSv titration assay: The infectivity of test samples containing PRRSv is determined by titration on susceptible tissue culture cells, preferably African Green Monkey kidney cell lines such as MARC145 or MA104. Three to four days prior to the assay, cells are seeded into suitable culture plates or dishes, such as 96-well tissue culture plates.

(58) In the following, the assay is described for standard 96-well tissue culture plates: Actively growing cells are seeded at 510.sup.4 cells/well in assay media (for instance Dulbecco's Minimum Essential Media, supplemented with 10 mM HEPES, 2 mM L-Glutamine, 60 g/mL Gentamycin and 7% fetal bovine serum), and incubated at 37 C. under 5% CO.sub.2 atmosphere in a humidified incubator until use in the assay. On the day of the assay, appropriate serial dilutions of the test samples are prepared in assay media in dilution tubes, for instance glass tubes, or polypropylene tubes. For example, 10-fold dilutions may be prepared by adding 0.2 mL of undiluted test sample to 1.8 mL of assay media, briefly mixing by means of a vortex mixer, transferring 0.2 mL of the mixture into a separate test tube containing 1.8 mL of assay media, and so on.

(59) For each test sample, one 96-well plate is removed from incubation, and the media is removed by inverting the plate and aseptically dumping the media into a suitable receptacle. The sample is tested by adding 0.1 mL of each dilution to each of 10 wells on the plate. Dilutions are added from the highest dilution to the lowest dilution to be tested. Incubation is then continued for an additional 7 days.

(60) Following incubation, the plates may be read either by visually observing cytopathic effects (CPE) using a light microscope at 100-1000 magnification, or macroscopically after staining with Crystal Violet (CV). In the CV method, a stock solution is prepared by dissolving 5 g of CV powder per 100 mL of 95% ethanol, followed by the addition of an equal volume of formaldehyde (37% stock solution), and the addition of deionized or better quality water to a final volume of 1,000 mL per 100 mL of ethanol. Media are removed from the 96-well plates by dumping, and CV stock solution is carefully added at 0.15 mL/well without disturbing remaining monolayers. The plate is left at ambient temperature for 15 minutes, after which the CV solution is removed by dumping. The wells are then carefully rinsed under gently running tap water. Finally, the water is removed by dumping.

(61) Each well is observed for the absence of intact monolayers and presence of characteristic CPE indicative of PRRSv infection for both the CPE method and CV method. Wells are scored as positive or negative for signs of PRRSv infection. Virus titers are calculated according to the method of Spearman and Krber, and are expressed as Log.sub.10 TCID.sub.50/mL.

(62) Results and Conclusions

(63) Table 2B below, lists 10 liquid stable formulations for swine virus vaccines of the present invention. Formulation 11 is a tryptone, lactose formulation that was designed in view of the freeze-drying methodology that was employed heretofore to stabilize live swine virus vaccines. In accelerated testing performed at 27 C., the standard freeze drying formulation (Formulation 11) started to fail immediately, see Table 3 below. In the corresponding real time stability study at 2 C.-7 C., the titer of all of the formulations were comparable to formulation 2 (see, Table 2B below). The titer of all of the 10 variations of this formulation appear to remain relatively stable at 2 C.-7 C. for at least 6 months, the final time point measured. In direct contrast, the titer of formulation 11 is measurably decreasing at this 6 month time point, having lost close to 1.5 logs of in its titer, see, Table 4 below.

(64) TABLE-US-00004 TABLE 2A STOCK SOLUTIONS Component Concentration pH Sucrose 80% (w/v) N/A Sorbitol 70% (w/v) N/A L-arginine hydrochloride 2.3M pH 7.2 L-methionine hydrochloride 5% (w/v) pH 7.2 Polyvinylpyrrolidone K-60 45% (w/v) N/A EDTA 0.5M pH 7.2 Pleuronic F-68 100% N/A L-glutamic acid, monosodium salt 3M pH 7.2 Potassium phosphate buffer 1M pH 7.2 Gentamicin sulfate solution 250 mg/mL N/A N/A indicates that the pH was not adjusted.

(65) TABLE-US-00005 TABLE 2B FORMULATIONS F-68 K-60 Sucr. Sorb. ARG MET GLU EDTA (L/ PVP (% w/v) (% w/v) (M) (% w/v) (M) (mM) mL) (% w/v) 1 10 15 0.3 2 15 0.3 3 15 0.3 1.0 0.5 0.8 4 15 0.3 0.5 5 20 0.3 1.0 0.5 0.8 6 15 0.3 0.5 0.8 7 15 0.3 2.0 8 15 0.3 0.25 9 15 0.25 10 15 0.46 All 10 formulations were made up in potassium phosphate buffer pH 7.3; sucrose (Sucr.), sorbitol (Sorb.), arginine (ARG), methionine (MET), glutamic acid (GLU), ethylenediaminetetraacetic acid (EDTA), nonionic surfactant Pluronic F-68 (F-68), polyvinylpyrrolidone K-60 (K-60 PVP) were included as indicated.

(66) TABLE-US-00006 TABLE 3 STABILITY TESTS at 27 C. Formultn. 0 Time 2 weeks 4 weeks 6 weeks 1 6.2 4.4 3.6 0.7 2 5.6 4.6 3.6 1.0 3 6.3 4.8 3.5 0.7 4 6.0 5.0 3.4 1.0 5 6.3 4.9 2.8 0.6 6 5.9 5.1 3.6 1.3 7 5.8 4.7 3.6 1.1 8 5.9 3.9 0.5 0.5 9 6.1 5.0 3.1 0.7 10 5.8 4.5 0.6 0.6 11 6.2 1.4 0.5 0.5 Control 6.5 6.7 6.7 6.6 The values provided are the Log TCID50 virus. The times represent the storage times at 27 C. All of the formulations (Formultn.) are described in Table 2B above, except a commercial formulation (Formulation 11). The control is a sample of Formulation 11 that is maintained frozen prior to the assay. Formulation 11 comprised: 3.75% (w/v) Bacto Tryptone; 1.5% (w/v) dextran; 0.1% (w/v) gelatin; 5.0% (w/v) lactose; 0.1% (w/v) sodium glutamate; 0.5% (w/v) albumin Fraction V, and buffered with monobasic and dibasic potassium phosphate.

(67) TABLE-US-00007 TABLE 4 STABILITY TESTS at 2-7 C. Formultn. 0 Time 1 month 2 months 3 months 6 months 1 6.2 6.5 5.8 6.13 5.55 2 5.6 6.5 6.1 6.25 5.95 3 6.3 6.5 6.3 6.38 5.9 4 6.0 6.3 6.2 6.25 5.9 5 6.3 6.8 6.3 6.53 6.2 6 5.9 6.5 6.4 6.40 5.8 7 5.8 6.4 6.2 6.28 5.9 8 5.9 5.7 5.3 5.48 5.15 9 6.1 6.3 6.0 6.13 5.65 10 5.8 6.0 5.9 5.93 5.7 11 6.2 6.3 5.6 5.95 4.75 Control 6.5 6.7 6.7 6.7 6.7 The values provided are the Log TCID50 virus. The times represent the storage times at 2 C.-7 C. The control is a sample of Formulation 11 that is maintained frozen prior to the assay. The formulations (Formultn.) are described in Table 2B above, except the commercial formulation (Formulation 11) which is described in Table 3 above.

(68) The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.