HK1130202B - Heat treated bacterins, and emulsion vaccines prepared from such heat treated bacterins - Google Patents

Description

Heat-treated bacterins and emulsion vaccines prepared therefrom

Technical Field

The present invention relates generally to the field of vaccines and methods of stabilizing emulsion vaccines. In particular, the present invention relates to heat-treated bacterins (bacterin), methods of producing heat-treated bacterins, and emulsion vaccines prepared from the heat-treated bacterins.

Background

Vaccination is increasingly used to control infectious diseases in animals. Adjuvants are often used in vaccines because they enhance the humoral and/or cellular immune response to the antigen. Vaccines are typically made as emulsions because emulsions can act as adjuvants and have the property of retaining the antigen as a depot (depot) at the site of injection. Emulsifiers are often used in emulsion vaccines. In addition to the use of emulsifiers, emulsion vaccine stability can also be achieved by mechanically reducing the droplet size of the emulsion.

U.S. Pat. No. 5,084,269 relates to an adjuvant formulation containing lecithin in combination with mineral oil which causes less irritation to the host animal while causing greater systemic immunityAnd (5) epidemic disease. Compositions according to U.S. Pat. No. 5,084,269 have been under the trade name(a trademark of the company pfeiri) is commercially available.

Typically, bacterial antigens are unstable when heated and even brief exposure to higher temperatures can reduce antigen activity. For example, current anthrax vaccines lose all biological activity over a period of 48 hours at 37 ℃ (S.Sing, N.Ahuja, V.Chauhan, E.Rajasekaran, W.S.Mohsin, R.Bhat, and R.Bhatnagar; Bioche.Biophys.Res.Commun.2002Sep.6; 295 (5): 1058-62).

Disclosure of Invention

The present invention relates to heat-treated bacterins, methods of producing heat-treated bacterins, and emulsion vaccines prepared from the heat-treated bacterins. The method includes heating the bacterins to a temperature of about 35 ℃ to about 80 ℃ to form heat-treated bacterins.

Detailed Description

Definition of

Acceptable antigenic activity-the term "acceptable antigenic activity" refers to the ability to elicit a protective immune response in a vaccinated animal after stimulation by a homologous living microorganism or by a stylized utility test (coded locus test) with a homologous living microorganism.

Bacterin-the term "bacterin" refers to a suspension of inactivated bacteria, which can be used as a component of a vaccine.

Emulsifiers-the term "emulsifier" refers to a substance used to make emulsions more stable.

Emulsion-the term "emulsion" refers to a composition of two immiscible liquids in which small droplets of one liquid are suspended in a continuous phase of the other liquid.

Heat-treated bacterins-the term "heat-treated bacterins" refers to bacterins that have been heat-treated, have a lipase activity that is 50% or less of the lipase activity prior to heat treatment, and have an acceptable antigenic activity.

Invert emulsion the term "invert emulsion" refers to a water-in-oil emulsion.

Lipase-the term "lipase" refers to enzymes, esterases, lipases and phospholipases capable of causing emulsion breaking (breaking down) of emulsifiers in emulsion vaccines.

Positive emulsion (normal emulsion) -the term "positive emulsion" refers to an oil-in-water emulsion.

Oil-in-water emulsion-the term "oil-in-water emulsion" refers to an emulsion in which small oil droplets are suspended in a continuous aqueous phase.

Room temperature-the term "room temperature" refers to a temperature of 18-25 ℃.

Water-in-oil emulsion-the term "water-in-oil emulsion" refers to an emulsion in which water droplets are suspended in a continuous oil phase.

Description of the invention

The present invention relates to bacterins having reduced lipase activity, vaccines prepared from the bacterins, and methods of reducing lipase activity of a bacterin. In addition to the antigenic component, certain bacterins have lipase activity. When bacterins with lipase activity are introduced into an emulsion, the lipase can break the emulsifier used to construct the emulsion. Emulsion vaccines comprising bacterins with high lipase activity tend to become unstable emulsions, and those comprising bacterins with low lipase levels tend to be stable. Examples of bacterins that may produce bacterins with lipase activity after inactivation include: acinetobacter calcoaceticus (Acinetobacter calcoaceticus), Acetobacter pasteurianus (Acetobacter pasania), Aeromonas hydrophila (Aeromonas hydrophyllum), Alicyclobacillus acidocaldarius (Alicyclobacillus acidocaldarius), Archaeoglobus fulgidus (Arhaegenbus fulgidus), Bacillus pumilus (Bacillus pumilus), Bacillus stearothermophilus (Bacillus stearothermophilus), Bacillus subtilis (Bacillus thermocatenulis), Bacillus thermocatenulatus, Burkholderia cepacia (Burkholderia papacia), Burkholderia glumae (Burkholderia glumae), Campylobacter coli (Campylobacter coli), Campylobacter jejuni (Campylobacter coli), Campylobacter coli (Campylobacter coli), Escherichia coli (Salmonella typhimurium), Escherichia coli (Salmonella typhae), Escherichia coli (Salmonella typhaeformis), Escherichia coli (Salmonella typhimurium), Escherichia coli (Salmonella typhaerothecellus), Escherichia coli (Salmonella typhae), Escherichia coli (Salmonella typhimurium), Escherichia coli (Salmonella typha), Escherichia coli (Salmonella typhae), Escherichia coli (Salmonella typhimurium), Escherichia coli (Salmonella typhi), Escherichia coli (Hippo), Escherichia coli (Salmonella typhi, Pseudomonas aeruginosa), Escherichia coli (Salmonella typhi, Pseudomonas aeruginosa, Mycoplasma filamentous subspecies LC (Mycoplasma mycoides subsp. mycoides LC), Clostridium perfringens (Clostridium perfringens), entomopathogenic nematode symbiotic bacteria (Photorhabdus luminescens), Propionibacterium acnes (Propionibacterium acidimeris), Proteus vulgaris (Proteus vulgares), Pseudomonas wisensis, Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas fluorescens C9(Pseudomonas fluorescens C9), Pseudomonas fluorescens SIKW1(Pseudomonas fluorescens SIKW1), Pseudomonas fragilis (Pseudomonas fragrans) Pseudomonas fragrans, Pseudomonas shallowensis (Pseudomonas aeruginosa), Pseudomonas oleophyla (Pseudomonas aeruginosa), Pseudomonas pseudolytica B-1 (Pseudomonas aeruginosa), Pseudomonas aeruginosa B (Staphylococcus epidermidis), Pseudomonas aeruginosa (Pseudomonas sp), Pseudomonas aeruginosa B-1 (Pseudomonas sp), Pseudomonas aeruginosa (Pseudomonas putida), Pseudomonas putida) (Pseudomonas putida C11), Pseudomonas aeruginosa (Pseudomonas aeruginosa B (Pseudomonas sp), Pseudomonas aeruginosa-1 (Pseudomonas aeruginosa), Pseudomonas aeruginosa (Pseudomonas aeruginosa) Staphylococcus suis (Staphylococcus hyicus), Streptomyces albus (Streptomyces albus), Streptomyces cinnamoneumoniae (Streptomyces cinnamonensis), Streptomyces exfoliatus (Streptomyces scabies), Streptomyces scabies (Streptomyces scabies), Acidophilic thermoacidophilus (Sulfolobus acidodarius), Syechocystis sp., Vibrio cholerae (Vibrio cholerae), Borrelia burgdorferi (Borrelia burgdorferi), Treponema denticola (Treponema pallidum), Treponema minicola (Treponema harderum), Treponema collarum (Treponema gephilum), Treponema curvatum (Treponema pallidum), Treponema pallidum (Treponema pallidum), Leponema pallidum (Leponema pallidum), Leponema pallidum (Leponema pallidum), Leponema pallidum (Leponema pallidum), and Leponema pallidum (Leponema pallidum) and Leponema pallidum (Leponema pallidum pathogen (Leponema pallidum), Leponema pallidum) and Leponema pallidum (.

Lipases which can break the emulsifiers used to construct the emulsion, thereby causing the emulsion to destabilize and break, can include one or more demulsifying enzymes, such as esterases, lipases, and phospholipases. These enzymes, esterases, lipases and phospholipases are collectively referred to as lipases. The lipase activity of bacterins can be measured using a synthetic substrate (substrate) called O-pivaloyloxymethylumbelliferone (C-POM). The rate of hydrolysis by lipase is a measure of the activity of the lipase. The reaction rate of hydrolysis by lipase in this reaction is monitored by the increase in fluorescence density of the lipase activity product. The reaction rate depends on the exact hydrolysis test conditions chosen, and therefore comparisons between lipase activity levels when measured by hydrolysis rate should be made using data obtained under the same test conditions. Literature methods are disclosed in several papers, including Kurioka s, and Matsuda m. (1976) ana. biochem.75: 281-289; de Silva NS and Quinn PA, (1987) j.clin.microbiol.25: 729-; and grace, a. and Ortiz, a. (1998) chem.phys.of lipids.91: 109-118.

In emulsion vaccines, breaking of the emulsion causes the components to separate in phase. This is undesirable because when there is phase separation, individual doses taken from the container may not contain the same amount of vaccine components. In addition, the loss of emulsion results in a loss of adjuvant activity of the emulsifier and results in a reduction of the antigenic effect of the vaccine.

Live attenuated viruses and bacterins are often included in vaccines. Such vaccines are useful because a single vaccine can be used to immunize against different diseases with one vaccine. If lipase activity is present in the bacterin, it results in the release of the emulsifier from the emulsion. This free emulsifier is capable of destroying and inactivating live vaccine viruses, resulting in a loss of viral infectivity.

Bacterins useful in vaccines can be formed by the following method: the target bacteria are cultured and subsequently inactivated to produce a vaccine containing a plurality of bacterial components, including cell wall components. Bacteria can be inactivated by a variety of methods, including exposing them to compounds such as thimerosal, formalin, formaldehyde, diethylamine, diethyleneimine (BEI), beta-propiolactone (BPL), and glutaraldehyde. Combinations of these compounds may also be used. In addition, the bacteria may be inactivated by germicidal radiation.

It has now been found that the lipase activity of bacterins having lipase activity can be reduced by heat treatment. Specifically, the lipase activity of a bacterin can be reduced by heating the bacterin to a temperature of about 35 to about 80 ℃ to form a heat-treated bacterin having acceptable antigenic activity. The heat treatment is carried out for a time sufficient to provide a heat-treated bacterin having a lipase activity which is 50% or less of the lipase activity of the bacterin prior to the heat treatment. In order to obtain good emulsion vaccine stability, lipase activity does not need to be reduced to zero. We have found that vaccines with good shelf life can be prepared from heat-treated bacterins whose lipase activity is 50% or less of the level of lipase activity prior to heat treatment.

When the hydrolysis rate of the test substrate is used as a measure of the activity of the bacterin lipase, the hydrolysis rate of the test substrate before heat treatment is compared to the hydrolysis rate after heat treatment. The heat treatment is carried out to reduce the rate of hydrolysis to 50% or less of that observed for fresh bacterins.

The exact method of measuring the lipase activity level is not critical, as long as the activity before heat treatment and the activity after heat treatment are measured using the same method. For example, if one substrate is used to measure the rate of hydrolysis of a test substrate, a different substrate may produce a different rate. However, if the same substrate is used for both the initial activity assay and the post-treatment activity assay, the relative rates still show the effect of heat treatment.

Stylized tests for antigen activity were present for vaccines comprising one or more of leptospira canicola, leptospira icterohaemic, leptospira typhosa, leptospira pomona (9CFR 113.101, § 113.102, § 113.103, § 113.104 and§ 113.105). For these species, acceptable antigenic activity is defined as the ability to induce protective immunity in vaccinated hamsters as follows: when hamsters were challenged with homologous live bacteria, at least 75% of the vaccinated hamsters survived in the model, while at least 80% of the unvaccinated hamsters were not viable. In the case where the antigen is Leptospira hardjors, acceptable antigenic activity is defined as the ability of the vaccine to elicit a geometric mean titer of serum agglutination for Leptospira hardjors of > 40 in calves that have been vaccinated with a vaccine comprising the bacterial antigen Leptospira hardjors. For other bacterins, acceptable antigenic activity is defined as the ability to elicit a protective immune response in a vaccinated animal, after stimulation by a homologous living microorganism, or by a stylized utility test with a homologous living microorganism.

The heat treatment may be performed over a range of temperatures and may last for different lengths of time. Typically, the heating may be carried out at a temperature of about 35 to about 80 ℃ for about 20 minutes to about 24 hours. When the vaccine is heated to a higher temperature, such as about 75 to about 80 ℃, the heating time is at the shorter end of the time frame. When the heating is performed at a lower temperature, the heating is performed for a longer period of time. Another combination of temperature and time is heating at about 60 to about 70 c for about 9 to about 10 hours. Another combination of temperature and time is heating at about 65 to about 70 c for about 5 to about 8 hours. Another combination of temperature and time is heating at about 65 to about 70 c for about 1 hour. Another combination of temperature and time is heating at about 55 to about 65 c for about 5 to about 8 hours.

After heat treatment, the bacterins have lower lipase activity than freshly prepared bacterins, but can also be formulated in the same manner as freshly prepared bacterins. Thus, heat-treated bacterins can be incorporated into vaccines by conventional methods of vaccine production. These methods are also well known in the art.

Emulsion vaccines can be formed by mixing the desired bacterin with an oil phase and an emulsifier or emulsifiers. The mixture may then be vigorously stirred to form an emulsion. Suitable stirring methods include homogenization and subsequent microfluidization (microfluidization). Preservatives and excipients may also be added to the mixture prior to emulsification.

Vaccines may also include both bacterin and viral antigens. In preparing a vaccine comprising a bacterin and a viral antigen, the bacterin, any viral antigen to be added, an emulsifier or emulsifiers, and optionally preservatives and excipients are mixed with an oil phase and emulsified. After the emulsion is formed, the pH of the formulation can be adjusted to the appropriate pH using NaOH solution or HCl solution. For vaccine applications, it is generally desirable that the pH be near neutral to avoid irritation of the injection site. Typically a pH of about 7.0 to about 7.3.

Oil phases suitable for forming emulsion vaccines include non-metabolizable oils and metabolizable oils. Non-metabolizable oils include mineral oils (e.g., paraffin oils) and light mineral oils. Metabolizable oils include vegetable oils, fish oils, and synthetic fatty acid glycerides.

Examples of emulsifiers that can be used in the preparation of the emulsion vaccines of the present invention are phospholipids, sorbitan esters, polyethoxy sorbitan esters and mannitol derivatives, which are commonly used vaccine emulsifiers. Phospholipid emulsifiers include lecithin, phosphatidylethanolamine, phosphatidylinositol (phosphatidylinositol), phosphatidylserine, and lecithin (e.g.,). Sorbitan ester emulsifiers include sorbitan monolaurate (e.g.,20 and20) sorbitan monooleate (e.g.,80 and80) sorbitan monopalmitate (e.g.,40 and40) and sorbitan monostearate (for example,60 and60). Polyethoxylated sorbitan esters include polyethoxylated sorbitan monolaurate (e.g.,20 and21) polyethoxy sorbitan monooleate (for example,80) polyethoxy sorbitan monopalmitate (for example,40) and polyethoxy sorbitan monostearate (e.g.,60). The mannitol derivative emulsifier comprises mannitol octadecanoic acid ether.Andis a trademark of ICI Americas.Is a trademark of pfeiri. Vaccines are typically formulated as oil-in-water orthoemulsions, although water-in-oil invert emulsions may also be prepared.

Various adjuvants such as Quil a, cholesterol, aluminum phosphate and aluminum hydroxide and preservatives such as thimerosal may be used in the vaccine. Quil A is a purified mixture of quillaja saponin (quillaja saponin) extracted from the bark of Quillaia Saponaria Molina (south America Quillaia tree). Quil a acts directly on the immune system to activate the systemic (genized) state of sensitivity. By this action, it induces both humoral and cell-mediated responses. The lipophilic chain allows the interaction of the antigen and the adjuvant to be delivered into the cytosol to proceed by endogenous means. Quil A is commonly used with cholesterol because less undesirable side effects are eliminated when cholesterol is added in the proper ratio. Cholesterol forms an insoluble complex with Quil a, which forms a helical structure when it binds to Quil a, thereby exposing the sugar units of the molecule, which helps stimulate an immune response.

It is common to add viral antigens to vaccines containing bacterins. One advantage of this approach is that one vaccine can be used to immunize against several diseases without the need to use multiple doses of several different vaccines to achieve the same effect. Both inactivated and live attenuated viruses may be used in vaccines. Viruses that may be used include avian herpes virus, bovine herpes virus, canine herpes virus, equine herpes virus, feline viral rhinotracheitis virus, marek's disease virus, ovine herpes virus, porcine herpes virus, pseudorabies virus, avian paramyxovirus, bovine respiratory syncytial virus, canine distemper virus, canine parainfluenza virus, bovine parainfluenza virus 3, ovine parainfluenza virus 3, bovine pestivirus, border disease virus, Bovine Viral Diarrhea (BVD) virus, classical swine fever virus, avian leukemia virus, bovine immunodeficiency virus, bovine leukemia virus, equine infectious anemia virus, feline immunodeficiency virus, feline leukemia virus, ovine progressive (progressive) pneumonia virus, ovine lung adenocarcinoma virus, canine coronavirus, bovine coronavirus, feline infectious peritonitis virus, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, porcine reproductive rhinomyelitis virus, bovine herpes virus, porcine respiratory syncytial virus, porcine respiratory syncytial virus, canine distemper, Porcine parvovirus, transmissible gastroenteritis virus, turkey coronavirus, bovine transient fever virus, rabies virus, vesicular stomatitis virus, avian influenza virus, equine influenza virus, porcine influenza virus, canine influenza virus, eastern equine encephalitis virus (EEE), Venezuelan equine encephalitis virus, and western equine encephalitis virus.

If the bacterin has lipase activity, it can cause the release of the emulsifier from the emulsion. Such free emulsifiers may disrupt the envelope of live virus and inactivate live vaccine virus, resulting in loss of viral infectivity. Thus, the heat treatment of the vaccine serves to stabilize the emulsion and maintain its adjuvant effect, while maintaining the viral infectivity of the virus.

The following examples are provided for purposes of further illustration and are not intended to limit the scope of the claimed invention.

Step (ii) of

Step 1 turbidity measurement

Turbidity was determined by light scattering methods in Nephelometric Units (NU). The intensity of light scattered by the sample under the given conditions is compared with the intensity of light scattered by the standard reference suspension. The higher the intensity of the scattered light, the greater the turbidity of the sample. A light source is directed at the sample and light scattering is detected at 90 deg. to the direction of the light source. The apparatus was calibrated by detecting light scattering from a formalin (formazin) suspension.

Calibration of turbidimeter apparatus

The ultrafiltrate was prepared by filtering the distilled water through a membrane filter having a pore size of 0.2 μm. 1.00g of hydrazine sulfate ((NH)₂)·H₂SO₄) The first solution was prepared by dissolving in ultrafiltration water and diluting to 100ml with ultrafiltration water in a volumetric flask. A second solution was prepared by dissolving 10.00g of hexamethylenetetramine in ultrafiltration water and diluting to 100ml with ultrafiltration water in a volumetric flask. 5.0ml of the first solution was mixed with 5.0ml of the second solution, thereby preparing a formalin suspension. The mixture was allowed to stand at about 24 ℃ for 24 hours. The mixture was diluted to 100ml with ultrafiltration water to form a stock turbiditysuspension concentrate (stock turbiditysis suspension) having a turbidity of 400 NU. 10.00ml of the turbidity suspension stock solution was diluted to 100ml with ultrafiltration water to prepare a 40NU formalin turbidity suspension. Other calibration solutions were prepared by diluting the mother liquor.

Turbidity detection

The sample to be tested is diluted with ultrafiltration water so that the turbidity falls within the calibration range of the turbidimeter. The turbidity was measured and the raw turbidity was calculated using the following formula:

wherein: m is the turbidity (NU) of the diluted sample

D is volume of dilution water (mL)

O is the original volume of the sample (mL)

Step 2 Lipase Activity

Lipase activity was measured using O-pivaloyloxymethylumbelliferone as fluorogenic substrate. Lipase-catalyzed hydrolysis of this non-fluorescent substrate produces hydroxymethyl ethers that are unstable under aqueous conditions. Decomposition of the unstable methylol ether produces formaldehyde and the fluorescent product umbelliferone. Monitoring the fluorescence intensity of the generated umbelliferone as a function of time provides a sensitive dynamic measure of lipase enzyme activity.

Preparing a solution of O-pivaloyloxymethyl umbelliferone (molecular Probe product No. P35901) in neat DMSO at a mother liquor concentration of 5 mM; the unused solution was stored at-20 ℃ protected from light. A5 mM solution of O-pivaloyloxymethyl umbelliferone was diluted to 750. mu.M with 58mM TRIS-HCl buffer (pH8.0) and the resulting solution was preheated to 37 ℃. The leptospira sample or control buffer/matrix was centrifuged at room temperature and 6500 times gravity for 10 minutes to form pellet (pellet) and supernatant. In low volume 96-well plate (Corning 3393, black polystyrene non-stick surface, half area) analysis of the hole in 15 u L100 mM TRIS-HCl buffer (pH8.0) and 15 u L at room temperature from Leptospira sample or control buffer/matrix supernatant mixed for reaction; preincubation at 37 ℃ for 10 min; the reaction was then initiated by the addition of 20 μ L of 750 μ M O-pivaloyloxymethyl umbelliferone or control buffer/matrix. The resulting reaction mixture contained 53mM TRIS-HCl buffer (pH8.0) and 0 or 300. mu.M O-pivaloyloxymethylumbelliferone. Fluorescence intensity was measured at 30-45 second intervals over a period of 1 hour (Spectramax Gemini XS, 37 ℃ C.,. lambda.)_ex＝360nm，λ_em460nm, PMT sensitivity set 'medium', read 6 times per well). The reaction rate was determined from the slope of the resulting progress curve.

Examples

Example 1 reduction of Lipase Activity by Heat treatment

Preparing a pool (pool) of leptospira inactivated with thimerosal to form a bacterin, the leptospira comprising the following species: leptospira canicola, leptospira icterohaemorrhagiae, leptospira influenzae, leptospira hardjo, and leptospira pomona. Samples of 6 bacterins were stored overnight (approximately 12 hours) at 4 ℃, 37 ℃, 45 ℃, 56 ℃, 65 ℃ and 80 ℃. Samples stored at 4 ℃ served as untreated controls. The samples stored at 37 deg.C, 45 deg.C, 56 deg.C, 65 deg.C and 80 deg.C for 12 hours were heat-treated samples. After storage, the hydrolysis rates of the test substrates in the presence of the various bacterins were measured according to the method of step 2. The hydrolysis rate of the sample divided by the hydrolysis rate of the sample stored at 4 ℃ multiplied by 100 is the percentage of the original lipase activity retained by each bacterin after storage. The following table shows the storage temperature and the percentage of original lipase activity retained after storage.

Storage temperature (12 hours)

4℃

37℃

45℃

56℃

65℃

80℃

Percentage of original Lipase Activity

100％

55.4％

32.5％

15.7％

10.8％

8.4％

Example 2 preparation of a test vaccine formulation

Cultures of leptospira canicola, leptospira icterohaemorrhagiae, leptospira influenzae, leptospira hardjo, and leptospira pomona were grown. The turbidity (NU) of each culture was measured in a turbidimeter unit. The bacteria were inactivated with thimerosal to form a bacterin. Each of the bacterins was heat-treated at 65 ℃ for 8 hours to reduce lipase activity. Combining the bacterins, followed by combination withAdjuvants, preservatives and dilution buffers were mixed so that each 5ml dose of vaccine contained the components listed in the table below.

Antigen concentration

Components	Component concentrations/dosages
		Leptospira canicola	1200NU/5ml dose
Leptospira for hemorrhagic jaundice	1200NU/5ml dose
		Leptospira influenzae	1200NU/5ml dose
Leptospira Johnsonii (Leptospira)	2400NU/5ml dose
		Leptospira pomona	1200NU/5ml dose

The formulation was homogenized using a Silverson homogenizer and microfluidised using a microfluidiser from Microfluidics (microfluidize). After homogenization and microfluidization, the pH of the formulation is adjusted to pH 7.0-7.3.

Example 3 efficacy tests in hamster and cattle

The vaccine of example 2 was administered to hamsters and cattle to test efficacy using standard laboratory and host animal models. The test hamsters were then stimulated with a dose of leptospira canicola, leptospira icterohaemic, leptospira typhosa, or leptospira pomona to test the efficacy of the vaccine. The number of survivors was measured as a demonstration of efficacy. Bovine microscopic agglutination titers were measured against leptospira hardjo to verify the efficacy of the vaccine components in cattle. The following table shows vaccines prepared from heat-treated leptospira bacterins that are capable of producing an antigenic response that passes the utility standard.

EXAMPLE 4 physiochemical testing of vaccines

Vaccines were prepared from heat-treated leptospira bacterins according to the method of example 2. Similar vaccines were prepared from non-heat treated leptospira bacterins according to the method of example 2. Both vaccine formulations were stored at 4 ℃ for 60 days. Each vaccine was analyzed for particle size using a laser diffractometer at day 0 when freshly prepared and again at day 60.

The lower panel shows the particle size distribution of each vaccine after day 0 and 60 days of storage.

Granulometry on day 0 of freshly prepared vaccine containing non-heat treated leptospira bacterins

Granulometry at day 60 for vaccines containing non-heat treated leptospira bacterins

Particle size analysis on day 0 for freshly prepared vaccines containing heat-treated leptospira bacterins

Particle size analysis at day 60 for vaccines containing heat-treated leptospira bacterins

Vaccines prepared from heat treated leptospira bacterins showed particle size retention, indicating that the emulsion was stable. Vaccines prepared from non-heat treated leptospira bacterins showed a particle size increase indicating emulsion breaking.

Example 5 virucidal assay

Vaccines were prepared according to the method of example 2 from both non-heat treated leptospira bacterins and heat treated leptospira bacterins. The vaccines were tested for virucidal activity against BHV-1 virus, PI3 virus and BRSV virus after 5-6 months of aging. Virucidal activity testing was performed by rehydrating a monovalent Virucidal Assay Control (VAC) with the adjuvant diluent (adjuvanted diluent) to be tested. Two monovalent virucidal assay controls were rehydrated at each dose volume. After titration and cell seeding to pass TCID₅₀(50% tissue culture infectious dose) determination of viable virus titres before, two rehydrated monovalent VAC were combined (pooled) and incubated at 20-25 ℃ for 2 hours. Viral titer loss in excess of 0.7TCID₅₀The/ml is not acceptable.

The results of the virucidal assay showed a loss of viral titer as shown in the table below.

Vaccines prepared with non-heat treated leptospira bacterins showed higher levels of virucidal activity. Vaccines prepared with heat treated leptospira bacterins are non-virucidal.

Claims (13)

1. An emulsion vaccine comprising a) an emulsion comprising an oil and one or more emulsifiers; and b) a heat-treated leptospira bacterin comprising a suspension of inactivated leptospira bacteria, said bacterin having a lipase activity of 50% or less compared to the lipase activity of the bacterin prior to heat treatment and having an acceptable antigenic activity; wherein said heat-treated leptospira bacterin is prepared by a process comprising heating said bacterin to a temperature of 60 to 70 ℃ for 5 to 10 hours.

2. The emulsion vaccine of claim 1, wherein the heat-treated leptospira bacterin is prepared by a process comprising heating the bacterin to a temperature of 65 ℃ for 8 hours.

3. The emulsion vaccine of claim 1 or 2, wherein the inactivated leptospira bacteria is 1-5 species of leptospira species selected from the group consisting of leptospira canicola, leptospira influenzae, leptospira harderi, leptospira icterohaemorrhagiae, and leptospira pomona.

4. The emulsion vaccine of claim 1 or 2, further comprising a live virus.

5. The emulsion vaccine of claim 4, wherein the live virus is 1-3 viruses selected from the group consisting of bovine rhinotracheitis (IBR) virus, parainfluenza 3(PI3) virus, and Bovine Respiratory Syncytial Virus (BRSV).

6. The emulsion vaccine of claim 1 or 2, further comprising an inactivated virus.

7. The emulsion vaccine of claim 6 wherein the inactivated virus is 1 or 2 viruses selected from the group consisting of Bovine Viral Diarrhea (BVD) type 1 virus and Bovine Viral Diarrhea (BVD) type 2 virus.

8. The emulsion vaccine of claim 1, further comprising a lecithin formulation, Quil a, and cholesterol.

9. The emulsion vaccine of claim 1 or 2,

a) the inactivated leptospira bacteria are 1-5 species of leptospira species selected from the group consisting of leptospira canicola, leptospira influenzae, leptospira hardicola, leptospira icterohaemorrhagiae, and leptospira pomona;

b) the vaccine further comprises a live virus selected from bovine rhinotracheitis (IBR) virus, parainfluenza 3(PI3) virus, and Bovine Respiratory Syncytial Virus (BRSV); and

c) the vaccine further comprises 1 or 2 inactivated viruses selected from the group consisting of Bovine Viral Diarrhea (BVD) type 1 virus and Bovine Viral Diarrhea (BVD) type 2 virus.

10. The emulsion vaccine of claim 9, further comprising a lecithin formulation, QuilA, and cholesterol.

11. A method of preparing a heat-treated leptospira bacterin of the emulsion vaccine of claim 1, comprising the steps of:

a) measuring lipase activity of the bacterin;

b) heating the vaccine to a temperature of 60 to 70 ℃ for 5 to 10 hours;

c) measuring lipase activity of the bacterin after heat treatment;

d) comparing the lipase activity of the bacterin before heating with the lipase activity of the bacterin after heating; and

e) the following heat-treated bacterins were selected: wherein the lipase activity after the heat treatment is 50% or less of the lipase activity of the vaccine before the heat treatment.

12. The method of claim 11, wherein the bacterin is heated to a temperature of 65 ℃ for 8 hours.

13. An emulsion vaccine comprising heat-treated leptospira canicola, leptospira influenzae, leptospira hardjo, leptospira icterohaemorrha, and leptospira pomona, obtained by: cultures of Leptospira canicola, Leptospira icterohaemorrhagiae, Leptospira influenzae, Leptospira hardtii and Leptospira pomona were grown, the turbidity (NU) of each culture was measured in a turbidimeter unit,inactivating the bacteria with thimerosal to form bacterins, heat treating each bacterin at 65 deg.C for 8 hours, combining the bacterins, and subsequently combining with the bacterinsAdjuvants, preservatives and dilution buffers were mixed to give a concentration of 1200NU/5ml dose for all leptospira bacterins except for leptospira hardjo and a concentration of 2400NU/5ml dose for leptospira hardjo, wherein the emulsion vaccine was homogenized using a Silverson homogenizer and microfluidized using a microfluidizer from Microfluidics, and then the pH was adjusted to 7.0-7.3.