Mycoplasma hyopneumoniae bacterin vaccine

(12) PATENT DESCRIPTION (19) PL (11) 211174 (13) B1 (21) Application number: 363220 (51) Int.Cl.

(22) Filing date: 12/11/2001 A61K 39/02 (2006.01)

A61K 39/116 (2006.01) (86) Date and number of the international application:

2001-12-11, PCT / US01 / 047865 (87) International application publication date and number:

2002-06-27, WO02 / 49666

Mycoplasma hyopneumoniae vaccine composition, vaccine and use

<td></td><td>(73) The right holder of the patent:</td>

<td>(30) Priority:</td><td>WYETH, Madison, US</td>

<td>12/19/2000, US, 60 / 256,637</td><td>(72) Inventor (s):</td>

<td>(43) Application was announced:</td><td>HSIEN-JUE (STEVE) CHU, Fort Dodge, US</td>

<td>November 15, 2004 BUP 23/04</td><td>WUMIN LI, Fort Dodge, US ZHICHANG XU, Fort Dodge, US</td>

<td>(45) The grant of the patent was announced:</td><td></td>

<td>April 30, 2012 WUP 04/12</td><td>(74) Representative:</td>

<td></td><td>item. stalemate. Magdalena Tagowska</td>

PL 211 174 B1

Description of the invention

The invention relates to a Mycoplasma hyopneumoniae vaccine composition, vaccine and use. The present invention relates to improved methods for inducing protective immunity against Mycoplasma hyopneumoniae, specifically employing an inactivated Mycoplasma hyopneumoniae bacterial vaccine in a single dose effective amount to immunize a recipient animal against Mycoplasma hyopneumoniae infection.

Mycoplasma hyopneumoniae is an etiological agent of porcine mycoplasma pneumonia. This disease is an important cause of economic losses in commercial pig farming because it reduces weight gain and results in poor feed efficiency. The disease causes a chronic cough, a dull coat, delayed growth and abnormal appearance lasting for several weeks. The characteristic organic lesions observed in infected animals are purple to gray areas of tissue densities, particularly in the ventral apical and cardiac lobes. Although the disease causes a low mortality, the affected pigs are often prone to secondary infections with opportunistic pathogens leading to stress or death. Economic losses alone were estimated at $ 200 to $ 250 million a year.

Mycoplasma hyopneumoniae is a slow-growing, demanding bacterium that does not have a cell wall. It is often difficult to isolate from the respiratory tract due to Mycoplasma hyorhinis, a common secondary factor also located in the respiratory tract. The disease is spread by aerosol produced when coughing and by direct contact with an infected or convalescent vector pig. Mixing of infected and uninfected animals resulted in early and frequent reinfection. Infection often begins with infection of the piglets during piglets by the carrier sow. Due to herd management techniques, contamination may not become apparent until the piglets are older. Additional infection is usually observed after weaning when pigs are grouped. Overt disease is normally seen in pigs of six weeks or more of age. In infected animals, the rate of growth and the speed of food processing are significantly reduced. Treatment with antibiotics is expensive and requires prolonged use. Re-infection is also a problem. Vaccines are currently the most effective way to avoid infections and their consequences.

Fort Dodge Animal Health (FDAH) supplies the market with a Mycoplasma hyopneumoniae bacterial vaccine under the name Suvaxyn® Respifend® MH for use as a vaccine to protect healthy pigs against clinical symptoms caused by Mycoplasma hyopneumoniae. The vaccine contains Carbopol as an adjuvant and is recommended as a two-dose vaccine for pigs of at least one week of age, with the second dose being given two to three weeks after the first vaccination. However, a two-dose vaccine has the obvious disadvantage of requiring re-treatment of animals in order to provide complete protection against disease.

Therefore, the present inventors have set themselves the goal of providing an effective vaccine against Mycoplasma hyopneumoniae, which induces protective immunity and prevents the disease caused by this organism, with a single dose of the vaccine being administered.

Another object of the present invention is to provide a vaccine composition suitable for use in pigs against infection and disease caused by Mycoplasma hyopneumoniae, which can be used in combination with other bacterial and / or toxoid vaccines.

Furthermore, it is also an object of the present invention to provide a method of preventing or suppressing a disease in which the causing organism is Mycoplasma hyopneumoniae by using an adjuvant formulation that enhances the immunogenicity of the bacterial vaccine so as to induce protective immunity after a single dose of vaccine.

Other objects and features of the invention will be apparent from the detailed description provided below.

The present invention relates to a vaccine composition for vaccinating an animal against Mycoplasma hyopneumoniae infection containing an immunizing amount of a bacterial vaccine against Mycoplasma hyopneumoniae; an adjuvant mixture comprising an acrylic acid polymer and a mixture of a metabolizable oil and an ethylene propylene oxide block copolymer;

And a pharmaceutically acceptable carrier, which vaccine composition, after a single administration, elicits protective immunity against Mycoplasma hyopneumoniae.

Preferably, the adjuvant mixture in the composition of the invention consists of an acrylic acid polymer and a mixture of a metabolizable oil, which includes one or more terpene hydrocarbons, and an ethylene oxide / propylene oxide block copolymer, in the ratio of acrylic acid polymer to metabolisable oil / oxide block copolymer ethylene and propylene oxide from about 1:25 to 1:50.

Also preferably, the adjuvant mixture in the composition of the invention is typically present at a final concentration of about 1-25% (v / v), more preferably about 2-15% (v / v), and most preferably 5-12% (v / v). (v / v).

In a preferred aspect, the vaccine composition of the invention is characterized in that the acrylic acid polymer is present at a final concentration of about 1% v / v. and the mixture of terpene hydrocarbons / block copolymer of ethylene oxide and propylene oxide is present at a final concentration of from about 5% to 10% v / v.

Preferably, the metabolizable oil used in the composition of the invention is squalane or squalene and the acrylic acid polymer is carbomer (Carbopol).

The composition may advantageously contain other vaccine components, in particular inactivated bacterial vaccines or purified toxoids from one or more pathogens such as Haemonphilus parasuis, Pasteurella multiocida, Streptococcum suis, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Salmonella choleraesuis and leptospira.

The invention also relates to the use of Mycoplasma hyopneumoniae in combination with an adjuvant mixture comprising an acrylic acid polymer and a mixture of a metabolizable oil and an ethylene oxide / propylene oxide block copolymer; and with a pharmaceutically acceptable carrier, for the preparation of a vaccine composition for protecting an animal against disease caused by Mycoplasma hyopneumonia, wherein the vaccine composition after a single administration induces protective immunity against Mycoplasma hyopneumoniae.

Preferably the immunizing amount of bacteria according to the use of the invention is about 1x10⁸ up to 3x10¹¹ MHDCE / ml, and more preferably about 1x10⁹ up to 3x10⁹ MHDCE / ml.

Furthermore, it is preferred that the vaccine composition obtainable according to the use of the invention is for intramuscular, subcutaneous, intraperitoneal, aerosol, orally or intranasal administration.

Preferably, the adjuvant mixture in the composition obtained according to the invention consists of an acrylic acid polymer and a metabolizable oil mixture which comprises one or more terpene hydrocarbon and a block copolymer of ethylene oxide and propylene oxide present at a final concentration of about 1-25% v / v / v / v. vol.

In a preferred embodiment of the use of the invention, the acrylic acid polymer in the adjuvant mixture is carbomer (Carbopol) and the oil metabolizable in the adjuvant mixture is a terpene hydrocarbon selected from the group consisting of squalene and squalane.

In a preferred embodiment, the vaccine composition according to the use of the method further comprises at least one additional bacterial vaccine selected from the group consisting of Haemophilus parasuis bacteria; Pasteurella multiocida; Streptococcum suis; Actinobacillus pleuropneumoniae; Bordetella bronchiseptica; Salmonella choleraesuis; and leptospira.

The invention further relates to a vaccine comprising inactivated Mycoplasma hyopneumoniae, a metabolizable oil, a block copolymer of ethylene oxide and propylene oxide, and an acrylic acid polymer in the form of an oil-in-water emulsion.

Detailed Description of the Invention

As used herein, "bacterial vaccine (bacterin) is a harvested culture of bacteria that has been inactivated and which, in combination with certain adjuvants, can induce protective immunity when administered to animals to protect against disease or infection.

"An adjuvant is a composition containing at least one substance that enhances the immunogenicity and efficacy of a bacterial vaccine against Mycoplasma hyopneumoniae in the vaccine composition.

As used herein and in the claims, MHDCE (Mycoplasma hyopneumoniae DNA cell equvalents) means the DNA cell equivalents of Mycoplasma hyopneumoniae.

The "immunizing amount" means the amount of the bacterial vaccine that will provide immunity against

Mycoplasma hyopneumoniae. “The amount of immunization will depend on species, breeding, age, size, health, and whether the animal has been previously vaccinated against the same organism.

The present invention provides a vaccine against Mycoplasma pneumoniae which is suitable for single dose vaccination. The vaccine of the present invention comprises an adjuvant mixture which enhances the immunogenicity of the bacterial vaccine and thus ensures that a single administration will elicit protective immunity.

The vaccine can be obtained from freshly harvested cultures by methods that are standard in the art (see, for example, US Patent 5,338,543 or US Patent 5,565,205, and Example 2 below). That is, the organism can be reproduced in a culture medium such as complete PPLO (for Pleuropneumonia) [Difco Laboratories]. Body growth is monitored by standard techniques such as determination of color change units (CCUs). color changing units) and cells are harvested when a sufficiently high titer is obtained. The starting broths may also be concentrated or lyophilized by conventional methods prior to incorporating the vaccine into the formulation. Other methods, such as those described in Thomas et al., Agri-Practice, Vol. 7 No. 5, pp. 26-30, may be used.

The vaccine of the present invention comprises an inactivated bacterial vaccine against Mycoplasma hyopneumoniae in combination with an adjuvant mixture and one or more pharmaceutically acceptable carriers. Suitable carriers for use include aqueous media, for example, saline, phosphate buffered saline, minimal basal medium (MEM), or MEM with HEPES buffer.

The adjuvant mixture for use in the vaccine compositions of the present invention enhances the immune response and comprises a mixture of an acrylic acid polymer with a mixture of a metabolizable oil, e.g. an unsaturated terpene hydrocarbon or its hydrogenation product, preferably squalane (2,3,10,15,19,23). -hexamethyltetracosan) or squalene, and a block copolymer of ethylene oxide and propylene. Such acrylic acid polymer may be a homopolymer or a copolymer. The acrylic acid polymer is preferably a carbomer. Carbomers are commercially available under the trademark Carbopol. Acrylic acid polymers are described, for example, in US Pat. Nos. 2,909,462 and 3,790,665. Block copolymers of ethylene oxide and propylene are surfactants, preferably liquid surfactants, which aid in suspending solid and liquid ingredients. Surfactants are commercially available as polymers under the trade name Pluronic®. A preferred surfactant is poloxamer 401 which is commercially available under the trade name Pluronic® L121.

The immunogenically stimulating adjuvant mixture is usually present in a v / v amount in the vaccine composition of the invention. about 1% to 25%, preferably about 2% to 15%), more preferably about 5% to 12% v / v. The amounts of the adjuvant mixture used and the ratio of the two adjuvant components may vary depending on the addition of other bacterial vaccines or the purified toxoid. The adjuvant mixture generally comprises a metabolizable oil, an acrylic acid polymer, and a block copolymer of ethylene oxide and propylene oxide formulated as an emulsion in an aqueous medium.

In this adjuvant mixture, the metabolizable oil and acrylic acid polymer may be present in an amount ranging from about 10 to 150 ml / L and from about 0.5 to 10 g / L, respectively. In a preferred embodiment of the adjuvant mixture, the constituent of the mixture of metabolizable oil and ethylene oxide / propylene oxide block copolymer is a mixture of squalane and Pluronic® L121 (poloxamer 401), which may be present in an amount of about 50 to 100 ml / 1 and the carboxymethyl polymer is Carbopol 934P (Carbamer 934P) which may be present in an amount of about 2 ml / L. Typically, the ratio of acrylic acid polymer to metabolisable oil / ethylene oxide propylene block copolymer mixture in the adjuvant mixture is about 1:25 to 1:50.

A preferred acrylic acid polymers are those sold by B.F Goodrich as Carbopol 934 P NF and 941 NF which are polyallyl sucrose cross-linked acrylic acid polymers and which have the chemical formula (CH2CHOOOH) n. These polymers form aqueous gels which are suitably formulated with aqueous carriers. Preferred block copolymers of ethylene oxide and propylene oxide are nonionic surfactants supplied by BASF as Pluronic® L121, L61, L, 81 or L101.

The vaccine according to the invention may be administered by intramuscular, subcutaneous, intranasal, intraperitoneal or oral routes, preferably intramuscularly or subcutaneously.

PL 211 174 B1

The vaccine of the invention generally comprises inactivated Mycoplasma hyopneumoniae, a metabolizable oil, a block copolymer of ethylene oxide and propylene oxide, the acrylic acid polymer is in the form of an oil-in-water emulsion. The vaccine preferably contains the acrylic acid polymer in a concentration ranging from 0.5 to 10 g / l. The vaccine preferably contains the metabolizable oil in a concentration ranging from 2 to 6 ml / L. The vaccine preferably contains a block copolymer of ethylene oxide and propylene oxide at a concentration ranging from 1 to 3 ml / l.

For single dose administration, the vaccine should preferably contain an amount of Mycoplasma pneunomoniae bacterial vaccine corresponding to about 1 × 10⁹ up to 3x10⁹ MHDCE / ml, preferably about 1x10⁸ up to 3x10¹¹ MHDCE / ml. About one to five ml, preferably 2 ml, may be administered per animal, intramuscularly, subcutaneously, or intraperitoneally. One to ten ml, preferably 2 to 5 ml, may be administered orally or nasally.

The following examples are intended to further illustrate the invention without limiting its scope.

Example 1

Bacterial vaccine against Mycoplasma hyopneumoniae

Preparation of a vaccine composition

Description of starting broths. Mycoplasma hyopneumoniae can be obtained from any number of readily available sources. In one embodiment, Mycoplasma hyopneumoniae strain P-5722-3 can be used. The culture was obtained from C. Armstrong, of Purdue University, West Lafayette, Indiana, USA. As recommended, the Mycoplasma hyopneumoniae culture was passaged in the Mycoplasma hyopneumoniae broth seven times to establish the stock culture.

Cell culture. Mycoplasma hyopneumoniae was cultured in a medium containing Bacto PPLO, yeast extract, glucose, L-cysteine ​​hydrochloride, ampicillin, thallium acetate, phenol red, antifoam, normal sterile pig serum and water for 18-144 hours. For inactivation, double ethyleneimine (BEI) was added directly to the cultivation in the fermentation vessel. The pH was adjusted to 7.4 and the cells were then harvested according to traditional procedures to provide a bacterial vaccine against Mycoplasma hyopneumoniae.

Preparation of a vaccine composition

The composition of preservatives and the proportions used. The collected bacterial vaccine is protected by the addition of thiomersal and tetra-sodium salt of ethylenediaminetetraacetic acid (EDTA) in an amount not greater than 0.01% and 0.07%, respectively. Ampicillin USP is present in the growth medium at a concentration of 0.250 grams / liter. The residual concentration of ampicillin in the final product will vary depending on the volume of fluid collected, with the residual concentration of ampicillin in the final product not exceeding 30 µg / ml.

Product standardization. Mycoplasma concentrate is quantified by a DNA fluorimetric assay.

A metabolizable oil mixture that includes one or more terpene hydrocarbons and a block copolymer of ethylene oxide and propylene oxide, e.g. Squalane / Pluronic L121 mixture, was prepared by dissolving 10 g of sodium chloride, 0.25 g of potassium chloride, 2.72 dibasic sodium phosphate , 0.25 ml of monobasic potassium phosphate, 20 ml of Pluronic L121 (BASF Corporation), 40 ml of Squalane (Kodak), 3.2 ml of Tween 80 in 900 ml of purified water, made up to 1000 ml. Once mixed, the ingredients can be autoclaved. The mixture was then homogenized until a stable emulsion was formed. Formalin may be added to a final concentration of 0.2% or thiomersal may be added to a final concentration of 1: 10,000.

Combine units to form a series. Satisfactory Mycoplasma hyopneumoniae concentrates are aseptically combined with adjuvants, preservative and solvent in a sterile container equipped with an agitator and mixed for not less than 30 minutes.

Quantities for 1,000,000 doses (2 ml each):

<td colspan="3">% vol / vol</td>

<td>Mycoplasma concentrate (&gt; 1.0x10¹⁰ MHDCE / ml)</td><td>400,000 ml</td><td> 20,0</td>

<td>Squalane / Pluronic L121 mixture</td><td>100,000 ml</td><td> 5,0</td>

<td>Carbopol (2% conc w / v)</td><td>200,000 ml</td><td> 10,0</td>

<td>Thiomersal concentration 1% conc. conc. weight / vol. in water and EDTA (tetrasodium salt) 7% conc. weight / vol.</td><td>18,000 ml</td><td> 0,9</td>

<td>Sterile saline</td><td>1,282,000 ml</td><td> 64,1</td>

The pH of the series is adjusted to 7.0 0.2.

MHDCE = Mycoplasma hyopneumoniae DNA cell equivalents

Method and technique of filling and sealing final containers. The product may generally be filtered through a sterile 200-500 micron filter element and placed under the conditions specified in 9 CFR 114.6 in sterile containers in the room dedicated to the filling operation. The glass or plastic containers were closed with a rubber stopper and crimped and sealed with aluminum closures. Each 2.0 ml dose contains no less than 2x10⁹ Mycoplasma hyopneumoniae DNA cell equivalents.

Efficiency testing.

Product samples from the total volume and from the final container may be tested for effectiveness as follows:

Efficacy testing method: female ICR mice, six to seven weeks of age, were used from one shipment from Harlan Sprague Dawley or from another accepted recipient. A minimum of 20 mice are required for immunization with each unknown and bacterial reference vaccine. A minimum of five mice were kept as unvaccinated controls. Test vaccines and reference bacterial vaccines were thoroughly mixed. Sterile disposable syringes fitted with 5/8 inch long needles were used to vaccinate mice subcutaneously in the groin region using 1/10 of the target animal dose (0.2 ml). Each group of mice was placed as a separate unit and food and water was allowed free for 14 days. Each mouse was then put to sleep. The anesthetized mouse was placed on its back. With one hand held, the head was kept pointing downwards and the front one paw was extended in front of the rest of the body. Using a scalpel, an approximately nac inch of skin was cut between the front of the extended leg and the thorax, dissecting the brachial artery. Using a 3.0 ml syringe without needle, blood that leaked through the incision was collected. The blood was placed in a labeled tube and allowed to clot. The clot tubes were centrifuged at 1000 xg to separate the serum from the clot. Individual sera were stored at -20 ° C or lower until used for testing.

Serological Testing: The ELISA procedure was used to measure the antibody response of the mice to the reference and / or unknown bacterial vaccines. The ELISA procedure was performed using Immulon II flat bottom disposable microtiter plates from Dynatech or equivalent and an ELISA plate reader.

Reagents for the test:

Phosphate Buffer Saline - Tween (PBST) (pH adjusted to 7.2-7.4 with 5N NaOH solution or 5N HCl).

Quantity per liter

<td>Ingredients</td><td>Liter</td>

<td>NaCl</td><td>8.50 grams</td>

<td>NaH2PO4</td><td>0.22 grams</td>

<td>Na2HPO4</td><td>1.19 grams</td>

<td>Tween-20</td><td>0.50 ml</td>

<td>Deionized water made up for</td><td>1000.00 ml</td>

Buffered saline with glycine (GBS) (pH adjusted to 9.5 to 9.7 with 5N NaOH solution or 5N HCl).

<td>Ingredients</td><td>Quantity per liter</td>

<td>Glycine</td><td>0.75 grams</td>

<td>NaCl</td><td>8.50 grams</td>

<td>Deionized water made up for</td><td>1000.00 ml</td>

Positive control serum: The positive control serum is harvested serum from mice vaccinated with the Mycoplasma hyopneumoniae bacterial vaccine.

Conjugate and substrate:

Affinity purified peroxidase labeled anti-mouse IgG conjugate was obtained from Kirkegaard and Perry Laboratories, Inc. (Catalog No. 074-1802). Procedure to be specified

The optimal dilution of the conjugate is detailed below. Peroxidase substrate solutions (ABTS) were obtained from Kirkegaard and Perry, Inc.

Conjugate titration: Immulon II flat bottom microtiter plate was coated with 100 µg per well of 20 µg per ml of Mycoplasma hyopneumoniae cell antigen diluted in 10 mM GBS. The plate was incubated for not less than one hour at 37 ° C ± 2 ° C and transferred to 2-7 ° C, at which temperature it was incubated for not less than 18 hours but not more than one week. Before use, the plate was washed three times with PBST, with a one minute soak between each wash, and taped when dry. A 1:40 dilution of positive control serum was prepared in PBST and the diluted positive control serum (100 µΐ / well) was added to one half of the wells of the plate. PBST was added to the other half of the wells. The plate was incubated for one hour at room temperature and then washed three times. Conjugate serum was gradually diluted in PBST two fold starting with a 1:10 dilution and ending with a 1:10 240 dilution. 100 μl of each conjugate dilution was added to four wells of positive serum and four wells of PBST, and allowed to react for half an hour at temperature. room. The plates were washed four times and 100 µl of peroxidase substrate solution (abts) was added per well. The plate was read at the double wavelength setting T λ = 450. The dilution of the conjugate was chosen to give a reading of 0.850 to 1.050 for the positive control serum when the PBST control value was subtracted from the serum for the positive control.

Test antigen:

Mycoplasma hyopneumoniae antigen is a whole cell preparation and is provided by Fort Dodge Animal Health.

The ELISA assay is performed as follows: Immulon II flat bottom microtiter plates from Dynatech are used. One vial of lyophilized whole cell Mycoplasma hyopneumoniae antigen was reconstituted with ten ml of glycine buffered saline (GBS). The concentration of the reconstituted mycoplasma protein was 20 µg / ml. Then 100 µl (2 µg) of the diluted antigen was added to the wells of the plate. The plate was incubated at 37 ° C 2 ° C for not less than one hour, then transferred and incubated at 2-7 ° C for a minimum of 18 hours and a maximum of one week. The plates were washed three times with PBST, soaked one minute between each wash, and then taped dry. Sera were diluted 1:40 in PBST. The positive control serum will be included in quadruplicate on each plate. The sample volume per well is 100 µl. Serial test serum samples and reference serum samples will be tested in duplicate on the same plate. Plates were incubated for one hour at room temperature and washed three times with PBST. 100 µl of a peroxidase labeled anti-mouse IgG conjugate (Kirkegaard and Perry), diluted in PBST, was added to all wells and the wells were incubated for 30 minutes at room temperature. Plates were washed four times with PBST. 100 µl of Peroxidase Substrate Solution (ABTS) was added to all wells and the plates were incubated until the positive serum control reached an OD405 (450) 0.850 to 1.050 when the instrument was calibrated against the PBST control wells. The plates were read and compared to the blank of the PBST wells. For the test to be valid, the sera of mice vaccinated with the reference bacterial vaccine must reach a minimum mean value of 0.500 and the sera of unvaccinated control mice must not exceed a maximum mean value of 0.100. But the difference between the mean value for the serum of mice vaccinated with the reference bacterial vaccine and that of unvaccinated control mice must be greater than or equal to 0.400.

The mean values ​​of the serial vaccines, reference vaccines and controls, were calculated and estimated as follows: in order to be considered satisfactory, the tested bacterial vaccines must show a mean optical density value equal to or greater than the reference. Or, using a one-end Student T-test, the test bacterial vaccine must not be significantly (p &lt;0.05 confidence level) lower than the reference bacterial vaccine. Any calculated T value equal to or greater than 1.686 will indicate a significant difference between the reference vaccine and the test bacterial vaccine, and will result in the test bacterial vaccine being rejected. Any computed T value less than 1.686 will indicate a satisfactory series. Any test bacterial vaccine determined by the test as unsatisfactory for any reason not related to product efficacy is rejected and the preliminary test is considered invalid.

PL 211 174 B1

Example 2

Test vaccine:

The vaccine to be tested was prepared according to the procedures detailed in Example 1 using a 5% metabolizable oil mixture that includes one or more terpene hydrocarbon and an ethylene oxide propylene block copolymer (Squalane / Pluronic L121 mixture) and 0.2% acrylic acid polymer (Carbopol ) as an adjuvant, and 2x10⁹ M. hyopneumoniae DNA (MHDCE) cell equivalents per dose.

Example 3

This study was designed to demonstrate the four-month duration of immunity (DOI) induced in pigs at three weeks of age in a single dose vaccination with the vaccines of Example 2.

To this end, two separate animal trials were conducted. All pigs in both trials were serum negative (antibody titres &lt;10) at the time of vaccination, indicating that the animals were susceptible to Mycoplasma hyopneumoniae infection. All pigs in the control groups remained serum negative prior to the challenge test. This indicates that the immune response in vaccinated pigs was due to the vaccine and not due to environmental exposure.

In a first trial, the vaccine of example 2 was tested together with a commercially available product, Ingelvac M. hyo® manufactured by Boehringer Ingelheim (BI), in a high-virulent M. hyopneumoniae (0.4 × 10) challenge test.⁶ organisms). Twenty-two (22) pigs 18-21 days old were vaccinated intramuscularly with the vaccine of Example 2 (IM) and eight (8) pigs were vaccinated with Ingelvac M. hyo® [lot 271 032]. Twenty-two (22) pigs were the controls of the challenge test and 10 pigs were the non-challenge controls. Pigs in vaccinated and challenged control groups were challenged with virulent M. hyopneumoniae four months after vaccination. The pigs vaccinated with the vaccine of Example 2 had a mean lung injury of 15.9% and the challenge control pigs had a mean lung injury of 19.6%. Mean lung lesions in the group vaccinated with the vaccine of Example 2 were less than the controls although pigs were challenged with a higher dose than recommended by Iowa State University (ISU), however, the difference was not significant (p = 0.19). Likewise, when commercially available, Ingelvac M. hyo® was evaluated in the same group of animals at the same challenge dose, a similar level of lung lesions was also observed (14.6%). There was also no significant difference between the Ingelvac M. hyo® vaccinated group and the control group (p = 0.27), and between the Ingelvac M. hyo® vaccinated group and the group vaccinated with the vaccine of Example 2.

In a second trial, the vaccine of Example 2 was tested by challenging virulent M. hyopneumoniae at the level suggested by ISU (1.0 x 10⁶ organisms) four months after vaccination. Twenty-three (23) pigs, 21 days of age, were vaccinated with one dose of the vaccine, 25 pigs as challenge controls and 7 pigs as non-challenge controls. Pigs in the vaccinated and challenge control groups were challenged with virulent M. hyopneumoniae four months after vaccination. The control group had mean lung lesions of 10.4%) and the vaccinated group had mean lung lesions of 5.5%. There was a significant difference between the vaccinated group and the control group (p = 0.031). This indicates that the vaccine of example 2 is effective in stimulating a protective immunity that can last at least four months after a single dose of vaccination in pigs at three weeks of age.

When the data related to the vaccine of Example 2 in the two trials were combined and analyzed, the mean lung lesions in the vaccinated group were significantly lower than in the control group.

In summary, the vaccine of Example 2 induces protective immunity against a challenge with virulent M. hyopneumoniae in the fourth month following a single vaccination of pigs at three weeks of age.

Experimental data

Two separate trials were conducted in this study. In trial one, sixty-seven (67) pigs were assigned to four groups using the Microsoft Excel randomization program. Twenty-four (24) pigs 18-21 days of age were vaccinated intramuscularly (IM) with one dose of the vaccine of Example 2. Twenty-four (24) pigs served as challenge controls and 10 pigs as unchallenged controls. Nine pigs were vaccinated by IM with a commercial product, Ingelvac M. hyo®, [lot 271 032, manufactured by Boehringer Ingelheim (BI)], according to the attached instructions. Five pigs (two pigs vaccinated with the vaccine of Example 2, one with the BI vaccine, and two control pigs) died during the protective period of vaccination due to reasons not related to vaccination. The remaining pigs in the vaccinated and challenge control groups were challenged with 14 ml of virulent M. hyopneumoniae (1.4 x 10⁶ organisms) four months after vaccination. Pigs in all four groups were sacrificed 30 days post-challenge and lung lesions were determined for each pig in the trial.

In a second trial, twenty-five (25) pigs of 21 days of age were vaccinated IM with one dose of the vaccine of Example 2. Twenty-five (25) pigs served as challenge controls and 10 pigs as unchallenge controls. Two pigs died due to reasons unrelated to vaccination and one pig was accidentally sold during the holding of vaccination. The remaining pigs in the vaccinated and challenge control groups were challenged with 10 ml of virulent M. hyopneumoniae (1.0 x 10⁶ organisms) four months after vaccination. Two pigs died during the post-challenge observation due to reasons unrelated to vaccination / challenge. The remaining pigs in all three groups were sacrificed 30 days post-challenge and lung lesions were determined for each pig in the trial.

Grafting:

Each pig in the vaccinated groups received one 2 ml dose of the IM test vaccine in the side of the neck.

Taunt and section:

The starting virulent M. hyopneumoniae challenge, frozen (-70 ° C) lung homogenate was obtained by Dr. Eileen Thacker of Iowa State University (ISU). The starting challenge suspension was confirmed to be pure and contains approximately 10⁷ M. hyopneumoniae organisms per ml. The recommended dose for challenge is 10 ml of the 1: 100 diluted stock suspension (i.e., 1.0 x 10⁶ organisms).

Pigs in the vaccinated and challenge controls in the first trial were challenged with 14 ml of a 1: 100 dilution of the stock (i.e., 1.4 x 10⁶ organisms). The pigs in the second trial were challenged with 10 ml of a 1: 100 dilution of stock (i.e., 1.0 x 10⁶ organisms) as recommended.

On the day of challenge, the homogenate was thawed rapidly under warm water and diluted as recommended by ISU using sterile M. hyopneumoniae growth medium. Pigs were sedated with the Xylazine-Ketamine-Telazol ™ mixture consisting of 50 mg / ml xylazine, 50 mg / ml ketamine, and 100 mg / ml telazole. The anesthetic mixture was administered IM in an amount of 0.022-0.044 ml / kg (0.01-0.02 ml / lb) body weight. Each pig was intratracheally administered a single 14 ml dose (first trial) or a 10 ml dose (second trial) of the challenge material. To ensure proper needle placement, air was drawn into the syringe prior to administration of the challenge dose. Unvaccinated unchallenged control pigs were housed in separate rooms and not challenged.

by days post challenge (DPC), all pigs were sacrificed. The lungs were removed and general lung lesions were quantified by a person ignorant of the results of the test groups.

Collection and testing of samples:

Blood samples were collected from all pigs on the day of vaccination (0 DPV), one month post vaccination (1 MPV), 4 months post vaccination / 0 days post challenge (MPV / 0 DPC) and 30 days post challenge (DPC) for antibody testing. serum against M. hyopneumoniae as detected by a competitive ELISA kit (made by DAKO Co.). Serum samples were stored at -20 ° C prior to testing.

Data analysis:

Lung lesion scores were compared between vaccinated and unvaccinated groups using analysis of variance (ANOVA). Lung lesion scores were converted by arcsin to improve residue distribution.

Results and discussion

Serology: All pigs in both trials were tested for serum antibodies to M. hyopneumoniae using a commercial ELISA kit using a 1:10 serum dilution for all tests. All pigs at the time of vaccination were serum negative (antibody titres &lt;10) indicating that the animals were susceptible to infection with M. hyopneumoniae. All pigs in the control groups remained serum negative before challenge. This indicates that the immune response in vaccinated pigs was due to the administration of the vaccine and not to environmental exposure. All pigs in the vaccinated groups and the majority of the challenge pigs in the control groups (18 of 22 pigs in trial 1 and 15 out of 25 in trial 2) seroconverted against M. hyopneumoniae after challenge, while all unchallenged animals remained serum negative. This suggests

That the challenge was specific for M. hyopneumoniae. The serological status of the test animals is summarized in Table 1.

Test of immunogenicity in the first sample:

A first trial was carried out to determine if the vaccine of Example 2 could stimulate a strong immunity that could protect against higher levels of challenge than recommended by ISU four months after vaccination. This trial also compared the vaccine of Example 2 with the commercially available Ingelvac M. hyo® for the ability to stimulate protective immunity four months after vaccination.

Twenty-two pigs vaccinated with the FDAH Suvaxyn MH-One and eight pigs with the licensed product, Ingelvac M. hypo® lot 271 032, were challenged with 1.4 x 10⁶ organisms per pig (1.0 x 10⁶ organisms per pig was recommended by the ISU, see chapter 5.5). Twenty-two pigs served as challenge controls and 10 pigs were used as unchallenged controls. The percentages of lung lesions are summarized in Table 2. Pigs vaccinated with the vaccine of Example 2 had a mean lung lesion of 15.9% and the challenge control pigs had a mean lung lesion of 19.6%. Lung lesions in the group vaccinated with the vaccine of Example 2 were less than the control even when pigs were challenged with a higher dose of M. hyopneumoniae. However, the difference was not significant (p = 0.19). Similarly, when the commercial product, Ingelvac M. hyo® was evaluated in the same group of animals at the same challenge dose, a similar level of lung lesions was also obtained (14.6%)). There were no significant differences between the Ingelvac M vaccinated group. hypo® and the control group (p = 0.27), and between the M. hyo® vaccinated group with Ingelvac and the group vaccinated with the vaccine of Example 2 (p = 0.88).

Although an insignificant numerical reduction in lesions was recorded in the Example 2 vaccine group, the data obtained in this trial suggest that higher challenge doses (1.4 x 10⁶ organisms) used in this trial were probably too high, even with pig immunity stimulated by the commercial Ingelvac product. This level of challenge is likely not appropriate for estimating vaccination / challenge studies using group sizes of 2025 animals, but with larger groups it is possible to prove such significance.

Secondary immunogenicity test:

Pigs in the vaccinated and challenge control groups in the second trial group were tested at the challenge dose (1.0 x 10⁶ organisms) as recommended by the ISU four months after vaccination to demonstrate the four-month duration of protection (DOI). The percentage of lung lesions is summarized in Table 3. Control groups had a mean lung lesion of 10.4%. The vaccinated groups had a mean lung lesion of 5.5%. There is a significant difference between vaccinated and control groups (p = 0.031). This indicates that the vaccine of example 2 is effective in stimulating a protective immunity that can last at least four months after a single dose of vaccination in pigs at three weeks of age.

Evaluation of the combined results of both trials:

While the two trials were significantly different, the effect of the trial on the group was not significant. The size of the group influence was similar in both trials. Thus, the group impact could be estimated without considering the sample. As the analysis of the full model showed that the interactions between the groups and the sample were not significant, this supports the notion that the group influence was the same in both trials and justifies combining the data from both trials in one analysis. Thus, when the vaccine data of Example 2 from the two trials were combined and the arcsin converted variable for lung lesions was analyzed treating group and trial as independent variables (reduced model). The group is statistically significant (p = 0.013).

Table 1: Summary of serological status against M. hyopneumoniae of control and vaccinated pigs

First attempt

Positive / negative (1:10) in

<td>Group</td><td>The number of pigs</td><td>ODPV</td><td>-1 DPC</td><td>30DPC</td>

<td>FDAH vaccine</td><td> 22</td><td> 0/22</td><td> 0/22</td><td> 22/22</td>

<td>Vaccine BI</td><td> 8</td><td> 0/8</td><td> 4/8</td><td> 8/8</td>

<td>Provoked control</td><td> 22</td><td> 0/22</td><td> 0/22</td><td> 18/22</td>

<td>Unprovoked control</td><td> 10</td><td> 0/10</td><td> 0/10</td><td> 0/10</td>

PL 211 174 B1

Second attempt

<td colspan="2">Positive / u</td><td colspan="3">emny (1:10) v</td>

<td>Group</td><td>The number of pigs</td><td>ODPV</td><td>-3DPC</td><td>30DPC</td>

<td>FDAH vaccine</td><td> 23</td><td> 0/23</td><td> 6/23</td><td> 23/23</td>

<td>Control</td><td> 25</td><td> 0/25</td><td> 0/25</td><td> 14/25</td>

<td>Unprovoked control</td><td> 7</td><td> 0/7</td><td> 0/7</td><td> 0/7</td>

Table 2: Summary of the percentage of lung lesions in the first trial (higher dose) *

<td>Group</td><td>The number of pigs</td><td>Average percentage of lung lesions</td><td>the value of P</td>

<td>FDAH vaccine</td><td> 22</td><td> 15,90%</td><td> 0,19**</td>

<td>Vaccine BI</td><td> 8</td><td> 14,60%</td><td> 0 27***</td>

<td>Control</td><td> 22</td><td> 19,60%</td><td></td>

<td>Unprovoked control</td><td> 10</td><td> 0%</td><td> 0 88****</td>

* Pigs were challenged 1.4x10⁶ organisms per pig (ISU recommended dose 1.0x10⁶ pigs) ** comparison between the FDAH vaccinated group and the control group *** comparison between the BI vaccinated group and the control group * comparison between the BI vaccinated group and the FDAH vaccinated group

Table 3: Summary of Percentage of Lung Lesions in Sample 2 (Recommended Dose)

<td>Group</td><td>The number of pigs</td><td>Average percentage of lung lesions</td><td>the value of P</td>

<td>FDAH vaccine</td><td> 23</td><td> 5,50%</td><td> 0,031 **</td>

<td>Control</td><td> 25</td><td> 10,40%</td><td></td>

<td>Unprovoked control</td><td> 7</td><td> 0,77%</td><td></td>

* Pigs were challenged with the donor recommended by ISU (1.0x10⁶ pigs) ** comparison between the FDAH vaccinated group and the control group

Example 4

Evaluation of the long-term immunity induced by the vaccine composition of the invention against a virulent challenge six months after a single dose

Test vaccine A was prepared using essentially the same procedures as described in Examples 1 and 2, and using the amounts set out below.

Test vaccine A

Amounts per 1,000,000 doses (2 ml each):% v / v

<td>Mycoplasma concentrate (&gt; 1.0 x 10¹⁰ MHDCE / ml)</td><td>1,200,000 ml</td><td> 60,0</td>

<td>Squalane / Pluronic L121 mixture</td><td>200,000 ml</td><td> 10,0</td>

<td>Carbopol (2% w / v in water)</td><td>200,000 ml</td><td> 10,0</td>

<td>Thiomersal concentration 1% w / v. in water and EDTA (tetrasodium salt) 7 w / v%</td><td>18,000 ml</td><td> 0,9</td>

<td>Sterile saline</td><td>382,000 ml</td><td> 19,1</td>

The batch pH was adjusted to 7.0 0.2.

MHDCE = Mycoplasma hyopneumoniae DNA cell equivalents

Summary

Thirty-three 21-day-old pigs were used in this evaluation. Twenty pigs, three weeks of age, were vaccinated with one dose of A vaccine intramuscularly (IM). Ten pigs served as unvaccinated controls and three pigs as unchallenged environmental controls.

All pigs were serum negative at the time of vaccination (antibody titres &lt;10), indicating that the animals were susceptible to infection with M. hyopneumoniae. All pigs prior to challenge

PL 211 174 B1 in the control groups remained serum negative. This indicates that the immune response in vaccinated pigs was due to the vaccine and to exposure to environmental factors.

Six months after vaccination, 20 vaccinated pigs and 10 unvaccinated control pigs were challenged with virulent M. hyopneumoniae (1.0 x 10⁶ organisms on a pig). Three pigs served as unchallenged controls. Vaccinated pigs had lung lesion numerical mean lesions of 3.6% and challenge control pigs had lung lesion numeric mean lung lesions of 14.6%). Lung lesions in the vaccinated groups were significantly less than in the controls (p = 0.0215).

The data obtained in this evaluation demonstrate that Test Vaccine A induced long-term protective immunity against challenge with virulent M. hyopneumoniae six months after single dose vaccination.

Experimental project

Thirty-three 21-day-old pigs were randomly assigned to three groups (vaccinated group, challenge control group and unchallenged environmental control group) using Microsoft Excel for litter randomization. Twenty pigs, three weeks of age, were vaccinated intramuscularly with one dose of test vaccine A. Ten pigs served as challenge controls and three pigs as unchallenged environmental controls. Pigs in the vaccinated group and challenge controls were challenged with 10 ml per pig of a virulent M. hyopneumoniae colony (1.0 x 10⁶organisms) in the sixth month after vaccination. Three unvaccinated pigs were used as unchallenged controls. Pigs in the challenge and unchallenged control groups were sacrificed 26 days post-challenge and lung lesions were determined for each pig.

Each pig in the vaccinates groups received one 2 ml dose of the IM test vaccine in the side of the neck.

Provocation and section

A challenge stock of virulent M. hyopneumoniae, frozen (&lt;-70 ° C) lung homogenate, was obtained by Dr. Eileen Thacker of Iowa State University (ISU). The challenge stock was confirmed to be clean and contain approximately 10⁷ M. hyopneumoniae organisms per ml.

Pigs were challenged with 10 ml of a 1: 100 dilution of the stock (i.e., approximately 1.0 x 10 cm)⁶ organisms).

On the day of challenge, the homogenate was thawed rapidly under warm water and diluted as recommended by ISU using sterile M. hyopneumoniae growth medium. Pigs were sedated with the XylazineKetamine-Telazol ™ mixture consisting of 50 mg / ml xylazine, 50 mg / ml ketamine, and 100 mg / ml telazol. The anesthetic mixture was administered IM in an amount of 0.022-0.044 ml / kg (0.01-0.02 ml / lb) body weight. Each pig was given a single 10 ml dose of challenge material (1.0x10⁶ organisms), endotracheally. To ensure proper needle placement, air was drawn into the syringe prior to administration of the challenge dose. Unvaccinated, unchallenged control pigs were housed in separate rooms and not challenged.

on the days post-challenge (DPC), all pigs were sacrificed. The lungs were removed and the overall lung lesions were determined as described in Example 3.

Collection and testing of samples:

Blood samples were collected from all pigs on the day of vaccination (0 DPV), 35 days post vaccination (35 DPV), - 1 DPC (one day prior to challenge) and 26 DPC (days post challenge) to test for antibodies to M. hyopneumoniae in serum detected by a competition ELISA kit (made by DAKO Co.). Serum samples were stored at -20 ° C prior to testing.

Data analysis

Lung lesion scores were compared between vaccinated and control groups using a one-way analysis (ANOVA). Lung lesion scores were converted by arcsin to improve residue distribution. Since the assumption of normality for the assessment of lung lesions was questionable, both the assessment of lung lesions and the converted arcsin of lung lesions were analyzed by the Wilcoxon rank sum test. The significance level was set at p &lt;0.05. Results from the nonparametric Wilcoxon rank sum test were used for reporting.

Results and discussion

Serology:

All pigs were tested for serum antibodies against M. hyopneumoniae using a commercially available ELISA kit using a 1:10 dilution of serum for all assays. Samples with uncertain test results were considered positive in the data analysis. All pigs at the time of vaccination were serum negative (antibody titres &lt;10) indicating that the animals were susceptible to infection with M. hyopneumoniae. All pigs in the control groups remained serum negative before challenge. After vaccination, fifteen of twenty (15/20) vaccinated animals became serous positive for M. hyopneumoniae at least once (samples were collected at 35DPV and 1 DPC). This suggests that the immune response in vaccinated pigs was due to the vaccine and not to environmental exposure. All vaccinated pigs and four out of ten pigs in the challenge control group became serous positive for M. hyopneumoniae after challenge, while all unchallenged animals remained sero negative. The serological status of the test animals is summarized in Table 4.

Scoring of pathological changes in the lungs

Twenty pigs vaccinated with test vaccine A and ten unvaccinated control pigs were challenged with virulent M. hyopneumoniae (1.0 x 10⁶ pigs) in the sixth month after vaccination. Three pigs served as unchallenged controls. Eight out of twenty vaccinated animals (40%) did not develop lung lesions after challenge, while in the control group only one out of 10 pigs (10%) showed no lung lesions. The percentage of lung lesions is summarized in Table 5. The vaccinated pigs had a mean lung lesion of 3.6% and the challenge control pigs had a mean lung lesion of 14.6%). Lung lesions in the vaccinated groups were significantly less than in the controls (p = 0.0215).

Table 4: Porcine serological status against M. hyopneumoniae in the study *

<td></td><td></td><td></td><td colspan="4">Number of positive (1:10) / all</td>

<td>Group</td><td>Treatment</td><td>The number of pigs</td><td>0 DPV **</td><td>35 DPV</td><td>-1 DPC ***</td><td>26 DPC</td>

<td> 1</td><td>Vaccine / challenge</td><td> 20</td><td> 0/20</td><td> 9/20</td><td> 12/20</td><td> 20/20</td>

<td> 2</td><td>Control / provoke</td><td> 10</td><td> 0/10</td><td> 0/10</td><td> 0/10</td><td> 4/10</td>

<td> 3</td><td>Control / no provocation</td><td> 3</td><td> 0/3</td><td> 0/3</td><td> 0/3</td><td> 0/3</td>

* Serum samples were tested for serum antibodies against M. hyopneumoniae using a commercial ELISA kit.

All samples were tested at a 1:10 dilution of the serum. The results were interpreted according to the instructions attached to the kit.

Suspect samples at 1:10 were considered positive.

** DPV = Day Post Vaccination *** DPC = Day Post Challenge

Table 5: Summary of lung lesion assessment results (%) in pigs challenged with M. hyopneumoniae and unchallenged controls

<td>Group</td><td>Treatment</td><td>number pigs</td><td>Average% Lung Damage Score</td><td>Deviation standard</td><td>Below 95% CL * of the mean</td><td>Above 95% CL * of the mean</td><td>Value P **</td>

<td> 1</td><td>Vaccine/ provoking</td><td> 20</td><td> 3,6</td><td> 7,6</td><td> 0,07</td><td> 7,19</td><td> 0,0215</td>

<td> 2</td><td>Control/ provoking</td><td> 10</td><td> 14,6</td><td> 20,0</td><td> 0,33</td><td> 28,94</td><td></td>

<td> 3</td><td>Control / no provocation</td><td> 3</td><td> 1,8</td><td> 1,8</td><td> -2,67</td><td> 6,27</td><td></td>

* CL = confidence level ** P value was obtained from the comparison of groups 1 and 2.

As can be seen from the data presented in Tables 4 and 5, test vaccine A induces protective immunity against challenge with virulent M. hyopneumoniae for six months after a single dose of the vaccine administered to pigs at three weeks of age.