HK1068250B - One dose vaccination with <i> mycoplasma hyopneumoniae </i> - Google Patents

Description

Single dose mycoplasma hyopneumoniae vaccine

Technical Field

The present invention relates to a method of treating or preventing a disease or condition in an animal caused by infection with Mycoplasma hyopneumoniae by administering to the animal at about 3 to 10 days of age a single dose of an effective amount of a Mycoplasma hyopneumoniae (m.hyo) vaccine. The mycoplasma hyopneumoniae vaccine may be a completely or partially inactivated cell or modified live preparation, a subunit vaccine, or a nucleic acid or DNA vaccine. The mycoplasma hyopneumoniae vaccines administered according to the present invention may be prepared synthetically or recombinantly.

Background

Mycoplasma hyopneumoniae (m.hyo) is a bacterial pathogen that can cause pneumonia in swine endemic animals. Enzootic pneumonia is a chronic disease that causes the following symptoms: poor food conversion, developmental disorders, and susceptibility to secondary lung infections. Mycoplasma hyopneumoniae is readily transmitted by respiratory secretions and by sow-piglet transmission, and is prevalent in pig farms. About 99% of the herds in the united states are infected, and the swine industry costs about 3 billion dollars each year.

Most of the known vaccines against mycoplasma hyopneumoniae are based on adjuvanted whole cell inactivated preparations of mycoplasma hyopneumoniae. In addition, vaccines based on immunogenic polypeptides or proteins can be synthesized or prepared by cloning and recombinant expression of the mycoplasma hyopneumoniae gene. Mycoplasma hyopneumoniae genes capable of expressing the polypeptides or proteins in vivo are also useful as vaccines.

Examples of whole cell inactivated mycoplasma hyopneumoniae vaccines include RESPISURE and STELLAMUNE, both available from pfeiri, usa.

In addition, some recombinantly produced immunogenic polypeptides and proteins of mycoplasma hyopneumoniae that are useful as subunit vaccines have been described in the art. International patent application publication No. WO96/28472 describes 6 Mycoplasma hyopneumoniae protein antigens having molecular weights of 46-48, 52-54, 60-64, 72-75, 90-94 and 110-114kDa, respectively, and discloses partial protein sequences of the antigens having molecular weights of 52-54, 60-64 and 72-75kDa as well as full-length nucleotide sequences and amino acid sequences of the antigens having molecular weights of 46-48 kDa.

The gene encoding Mycoplasma hyopneumoniae protein P46, P46, has also been cloned by Futo et al (1995; J.Bacteriol 177: 1915-1917). Studies by this group showed that the gene products expressed in vitro could be used to diagnose the antibody immune response to Mycoplasma hyopneumoniae infection without cross-reacting with other Mycoplasma species (Futo et al, 1995; J.Clin.Microbiol.33: 680-683). The sequence and diagnostic use of the p46 gene described by Futo et al is further disclosed in European patent 0475185A 1.

Wise and Kim (1987, J.Bacteriol., 169: 5546-5555) reported 4 Mycoplasma hyopneumoniae membrane-integrated proteins designated P70, P65(P65, supra), P50 and P44, the latter 3 being modified by covalent lipid ligation to induce a strong humoral immune response. The protective effect of this immune response was not studied. The gene encoding the P65 protein has also been cloned, its sequence and use in vaccines and diagnostics has been described in us patent 5,788,962.

International patent application WO91/15593 discloses 5 proteins of Mycoplasma hyopneumoniae with apparent molecular weights of 150, 90, 85, 70 and 43kDa, respectively. Wherein the full-length sequence of the coding gene encoding the 85kDa protein (protein C) is provided, as well as partial nucleotide sequences encoding the other 4 proteins.

U.S. Pat. No. 4, 5,252,328 to Faulds discloses the amino-terminal sequence of an immunoreactive Mycoplasma hyopneumoniae protein having molecular weights of 36, 41, 44, 48, 64, 68, 74.5, 79, 88.5, 96 and 121 kDa. Other proteins identified by electrophoretic mobility without disclosing the protein sequence have apparent molecular weights of 22.5, 34 and 52 kDa. Although US5,252,328 mentions that these proteins can be used in vaccine formulations, no vaccine experimental results are reported.

International patent application WO95/09870 discloses a biochemical method for the purification of mycoplasma hyopneumoniae adhesin, a membrane-integrating protein of mycoplasma, responsible for adhesion to the cilia of the host's upper respiratory epithelial cells. WO95/09870 also suggests methods for testing these proteins and uses, for example in vaccines and diagnostics.

The study paper by King et al (1997; Vaccine 15: 25-35) discloses an adhesin Mhpl with a molecular weight of 124kDa, a strain variant of P97.

A94 kDa variant of P97 was determined by Wilton et al (1998, Microbiology 144: 1931-. In addition, studies have shown that the P97 Gene is part of an operon which also encodes a second protein, designated P102, which has a predicted molecular weight of about 102kDa (Hsu et al, 1998, Gene 214: 13-23). Minion and Hsu in International patent application WO99/26664 suggested that P102 could be used in vaccines, but no vaccine experiments were reported.

None of the known mycoplasma hyopneumoniae vaccines have been reported to be effective when administered as a single dose treatment to pigs at about 3-10 days of age. Such a vaccine would eliminate the need for multiple doses and thus greatly reduce the cost and labor associated with mass vaccination of swine herds worldwide. There is therefore a need for a mycoplasma hyopneumoniae vaccine that effectively protects and prevents diseases or conditions caused by mycoplasma hyopneumoniae infection by administering a single dose vaccination to pigs of about 3 to 10 days of age.

Summary of The Invention

The present invention provides a method of treating or preventing a disease or condition in an animal caused by infection with mycoplasma hyopneumoniae, said method comprising administering to an animal of about 3 to 10 days of age an effective amount of a single dose of a mycoplasma hyopneumoniae vaccine.

The methods of the invention may eliminate the need for additional doses to generate or maintain immunity against mycoplasma hyopneumoniae. The single vaccination method of the invention can provide protection against virulent mycoplasma hyopneumoniae for seronegative and seropositive pigs. The methods of the invention are effective in treating or preventing symptoms caused by mycoplasma hyopneumoniae infection, such as preventing or alleviating lesions in the porcine lung.

The methods of the invention comprise administering to a pig an effective amount of a mycoplasma hyopneumoniae vaccine comprising a complete or partial cellular preparation (e.g. a bacteri or modified live preparation), a subunit vaccine, e.g. a subunit vaccine comprising one or more mycoplasma hyopneumoniae-derived polypeptides or proteins or immunogenic fragments of said polypeptides or proteins, or one or more mycoplasma hyopneumoniae genes encoding said polypeptides, proteins or immunogenic fragments, which genes or nucleic acids are capable of being expressed in vivo. Mycoplasma hyopneumoniae polypeptides, proteins, and immunogenic fragments and genes or nucleic acids thereof provided in Mycoplasma hyopneumoniae vaccines can be synthesized or recombinantly produced using techniques well known in the art.

The mycoplasma hyopneumoniae vaccine administered according to the present invention may comprise additional components, such as an adjuvant. A variety of adjuvants that can be used include those described herein and well known in the art.

Detailed Description

The invention includes a method of treating or preventing a disease or condition in an animal caused by infection with mycoplasma hyopneumoniae, said method comprising administering to an animal of about 3 to 10 days of age an effective amount of a mycoplasma hyopneumoniae vaccine in a single dose.

The single dose vaccination method of the invention may eliminate the need to administer additional doses to pigs that are intended to generate and/or maintain immunity against mycoplasma hyopneumoniae.

For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following sections which describe or illustrate certain features, embodiments, or applications of the invention.

In certain embodiments, the vaccine used in the methods of the invention comprises a partially or whole cell mycoplasma hyopneumoniae inactivated preparation (bacterin) or modified live vaccine and a pharmaceutically acceptable carrier, or a partially or whole cell mycoplasma hyopneumoniae inactivated preparation (bacterin) or modified live vaccine and an adjuvant.

In other embodiments, the vaccine used in the methods of the invention comprises an immunogenic protein or polypeptide or fragment thereof and a pharmaceutically acceptable carrier, or an immunogenic protein or polypeptide or fragment thereof and an adjuvant.

Definitions and abbreviations

The term "treating or preventing" when referring to a mycoplasma hyopneumoniae infection refers to inhibiting replication of mycoplasma hyopneumoniae bacteria, inhibiting spread of mycoplasma hyopneumoniae, or preventing colonization of mycoplasma hyopneumoniae in its host, as well as alleviating a symptom of a disease or disorder caused by mycoplasma hyopneumoniae infection. Treatment is considered to be therapeutically effective if the bacterial load is reduced, the pulmonary infection is reduced and/or the food intake and/or growth is increased.

The term "mycoplasma hyopneumoniae vaccine" refers to a vaccine that can be used to prevent or treat a condition or disease caused by mycoplasma hyopneumoniae infection, and may include any vaccine that is effective in treating or preventing mycoplasma hyopneumoniae infection in pigs. Mycoplasma hyopneumoniae vaccines that can be used in the invention include, for example, complete or partial Mycoplasma hyopneumoniae cell preparations, inactivated or modified live vaccines, subunit vaccines that contain one or more Mycoplasma hyopneumoniae-derived polypeptides or proteins or immunogenic fragments of such polypeptides or proteins, or one or more Mycoplasma hyopneumoniae genes or nucleic acids encoding one or more Mycoplasma hyopneumoniae-derived polypeptides or proteins or immunogenic fragments thereof, and which genes or nucleic acids are capable of being expressed in swine. Mycoplasma hyopneumoniae polypeptides, proteins, immunogenic fragments of such polypeptides and proteins, or Mycoplasma hyopneumoniae genes or nucleic acids can be synthesized or recombinantly produced using techniques well known in the art. Preferably, the mycoplasma hyopneumoniae vaccine used in the method of the invention is a bacterin.

The term "animal" refers to all non-human animals, including mammals.

The term "pig" refers to piglets (piglets), swine, pigs, porcine, sows (sow), gilts (gilts), barrows, boars (boars) and members of the porcine family.

Preferably, the methods of the invention are administered to a non-human mammal, most preferably, a pig.

The term "vaccine" refers to an inactivated whole or partial mycoplasma hyopneumoniae cell preparation suitable for vaccine use.

The term "effective amount" refers to an amount sufficient to elicit an immune response in a subject to which it is administered. The immune response may include, but is not limited to, induction of an innate, cellular, and/or humoral immunity.

Inactivated (partial or whole cell) and improved live vaccines

Methods for preparing conventional inactivated or modified live vaccines for use in the methods of the invention are well known in the art.

The single dose vaccination methods of the present invention provide for administration of mycoplasma hyopneumoniae bacterins from a variety of publicly available sources. For example, Mycoplasma hyopneumoniae bacterins can be prepared from Mycoplasma hyopneumoniae isolates. Many isolates of mycoplasma hyopneumoniae are well known to those of ordinary skill in the art and can be obtained, for example: american type culture Collection, 10801 university of Boulevard, Manassas, VA 20110-2209. Including for example: ATTC numbers 25095, 25617, 25934, 27714 and 27715.

Mycoplasma hyopneumoniae isolates can also be obtained directly from lung injury tissue of naturally or experimentally infected pigs using known techniques.

Mycoplasma hyopneumoniae isolates can be inactivated by a variety of known methods, for example, by treating the bacterial isolates with Binary Ethyleneimine (BEI) as disclosed in U.S. Pat. No. 5,565,205, or by inactivation with, for example, formalin, heat, BPL, radiation, or glutaraldehyde.

Mycoplasma hyopneumoniae bacterins suitable for use in the methods of the invention are also available from a variety of commercial sources. Such sources include, but are not limited to: RESPIFEND (Fort Dodge, american household), HYORESP (merria), M + PAC (pionbauy), PROSYSTEM M (Intervet), INGLEVAC M (Boehringer), RESPISURE (fevere), and STELLAMUNE myoplasma (fevere).

Preferred sources of mycoplasma hyopneumoniae bacterin for use in the methods of the invention are: RESPISURE and STELLAMUNE myccoplasma.

A particularly preferred source of Mycoplasma hyopneumoniae bacterin for use in the methods of the invention is RESPISURE-1 (Peyer Co.), comprising strain P-5722-3(NL1042), available from the university of Purpura in the United states.

Preferably, strain P-5722-3 is inactivated with BEI and adjuvanted with a commercially available adjuvant, preferably AMPHIGEN (Hydronics, USA). The preferred dosage is 2.0 ml. Common preservatives include thimerosal/EDTA. A carrier, preferably PBS, may be added. Methods for preparing improved live vaccines, such as by subculturing to reduce the toxicity of the strains, are well known in the art.

Subunit vaccine

The methods of the invention may be practiced using immunogenic proteins, polypeptides and immunogenic fragments of said proteins and polypeptides comprising purified Mycoplasma hyopneumoniae. The proteins and polypeptides may be prepared using techniques well known in the art. Alternatively, protein purity or homogeneity can be determined by methods well known to those skilled in the art, for example, by subjecting the sample to polyacrylamide gel electrophoresis followed by observation of individual polypeptide bands on the stained gel. Higher resolution assays can be performed using HPLC or other similar methods well known in the art.

In a particular embodiment, the vaccine for use in the present invention comprises at least one mycoplasma hyopneumoniae protein, such as, but not limited to, P46, P65, P97, P102, P70, P50 and P44.

In another embodiment, the vaccine for use in the method of the invention comprises a mycoplasma hyopneumoniae bacterin (inactivated whole or partial cells or modified live) or a mycoplasma hyopneumoniae protein or polypeptide or immunogenic fragment thereof and at least one further immunogen (inactivated whole or partial cells or modified live) or an immunogenic or antigenic protein, polypeptide or immunogenic fragment thereof, preferably a viral, bacterial or parasitic polypeptide. Such additional pathogens and proteins, polypeptides or immunogenic fragments thereof include, but are not limited to, Swine Influenza Virus (SIV), porcine reproductive and respiratory syndrome virus (PRRS or mystery swine disease), post-weaning diarrhea (PWD), and Porcine Proliferative Enteritis (PPE). The composition is useful as a combination vaccine.

In another specific embodiment, the immunogenic fragment of a protein or polypeptide comprises at least 10, at least 20, at least 30, at least 40, at least 50, or at least 100 contiguous amino acids of the immunogenic protein or polypeptide (including but not limited to P46, P65, P97, P102, P70, P50, and P44) used in the methods of the invention.

In addition, the mycoplasma hyopneumoniae protein used in the vaccine is substantially purified or homogeneous. The proteins or polypeptides employed in the methods of the invention are typically purified from host cells expressing recombinant nucleotides encoding the proteins. Purification of the protein can be carried out by a variety of methods well known in the art. See, e.g., "Methods in Enzymology", academic press, inc., san diego, "protein purification: principles and methods ", 1982, Springer-Verlag, New York.

Purified Mycoplasma hyopneumoniae polypeptides and proteins, and immunogenic fragments thereof, can also be prepared using known synthetic methods.

Vaccine formulations

Suitable formulations for the vaccines of the present invention include injectable liquid solutions or suspensions; or in solid form suitable for dissolution or suspension in a liquid prior to injection. It can also be made into emulsifier. The active immunogenic ingredients are often mixed with a pharmaceutically acceptable adjuvant that can be matched to the active ingredient.

The polypeptides may be formulated into neutral or salt vaccine forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and salts with inorganic acids such as hydrochloric or phosphoric acid, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts with free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or triiron hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, and procaine.

The vaccine preparation of the invention contains an effective immunizing amount of mycoplasma hyopneumoniae immunogen and a pharmaceutically acceptable carrier. The vaccine formulation comprises an immunologically effective amount of one or more antigens and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. An example of such an acceptable carrier is a physiological equilibration medium containing one or more stabilizing agents, such as stabilized hydrolyzed protein, lactose, and the like. Preferably, the carrier is sterile. The form of the preparation should be adapted to the mode of administration in question.

The purified antigen can be used in vaccine formulations using standard methods. For example, the purified protein should be adjusted to a suitable concentration, compounded with any suitable vaccine adjuvant and packaged. Suitable adjuvants include, but are not limited to: mineral gels, such as aluminum hydroxide; surfactants such as lysolecithin; glycosides, such as saponins and saponin derivatives such as Quil A or GPI-0100; cationic surfactants, such as DDA (quaternary hydrocarbon ammonium halide; Pluronic polyols; polyanions and polyatomic ions; polyacrylic acids, nonionic block polymers, such as Pluronic F-127(B.A.S.F., USA), Avridine and Rantidine; peptides; recombinant mutant labile toxins, such as leukotoxin (rmLT) or Cholera Toxin (CT), chemically bound or near molecular transporters (chemical bound or near molecular transporters), mineral oils, such as MonideiSA-50 (Seppic, Paris, France), polycarboxylates, Amphigen (Hydronics, USA), Omaha, NE. USA, Hydrogel, (Superfos bisemulsus, Freulssund, Denmark) oil (Baysil), such as Bayhalol, sodium alginate, and vegetable oil, such as sodium alginate, and sodium alginate, such as sodium alginate, n-acetyl-nor (nor) -muramyl-L-alanyl-D-isoglutamine, N-acetyl muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 '-2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine; cholesterol cytokine and adjuvant composition. Polyatomic ions can act as dispersants, thickeners, anticaking agents, allowing the vaccine to be resuspended as a monodisperse suspension after an extended settling period. The adjuvant composition can be in the form of aqueous solution, capsule (controlled release or sustained release) or microcapsule, etc.

The immunogen may also be incorporated into liposomes or conjugated to polysaccharides and/or other polymers useful in vaccine formulations. When the recombinant antigen is a hapten, i.e. a molecule which is antigenic and therefore selectively reactive with the relevant antibody, but which is not immunogenic and therefore incapable of eliciting an immune response, the hapten may be covalently coupled to a carrier or immunogenic molecule; for example, a macromolecular protein such as serum albumin may confer immunogenicity on the hapten to which it is coupled. The hapten-carrier can be formulated for use as a vaccine.

Gene and nucleic acid vaccines

The invention may employ mycoplasma hyopneumoniae genes or nucleic acids encoding immunogenic proteins, polypeptides, and immunogenic fragments of the proteins and polypeptides. The genes and nucleic acids can be expressed in vivo and can be prepared by methods well known in the art.

In a particular embodiment, the vaccine for use in the present invention comprises at least one gene or nucleic acid encoding a mycoplasma hyopneumoniae protein, such as, but not limited to, P46, P65, P97, P102, P70, P50 and P44.

In another embodiment, the gene or nucleic acid used in the present invention encodes an immunogenic fragment of mycoplasma hyopneumoniae protein or polypeptide comprising at least 10, at least 20, at least 30, at least 40, at least 50, or at least 100 contiguous amino acids of the immunogenic protein or polypeptide employed in the present invention, including, but not limited to, P46, P65, P97, P102, P70, P50, and P44.

In other embodiments of the methods of the invention, the gene or nucleic acid is administered using methods well known in the art, for example, using a gene gun.

In other embodiments of the methods of the invention, the gene or nucleic acid used is a DNA vaccine. In addition, the nucleic acid or gene may be used in combination with liposomes or other transfection-promoting factors well known in the art.

Methods for preparing and delivering DNA vaccines are well known in the art. See, e.g., Krishnan, b.r, "current state of the art in veterinary medicine" Advanced Drug delivery reviews, Elsevier Science (2000).

Expression system

A variety of host expression vector systems may be used to express the antigenic protein sequences of the present invention. Such host expression systems may be vectors from which the coding sequence of interest may be produced and subsequently purified, or cells which are capable of displaying the Mycoplasma hyopneumoniae gene product of the invention in situ when transformed or transfected with the appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., E.coli, B.subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing the sequence encoding mhp 3; yeast (e.g., saccharomyces, pichia) transformed with a recombinant yeast expression vector containing a sequence encoding a mycoplasma hyopneumoniae gene product; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing mycoplasma hyopneumoniae coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing Mycoplasma hyopneumoniae coding sequences; or a mammalian cell system (e.g., COS, CHO, BHK, 293, 3T3) transformed with a recombinant expression construct containing a promoter (e.g., metallothionein promoter) or a mammalian viral promoter (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter) taken from the genome of a mammalian cell. In a preferred embodiment, the expression vector is a bacterial system.

Mycoplasma hyopneumoniae polypeptides and proteins and immunogenic fragments thereof may also be used with live recombinant viral and bacterial vectors such as adenovirus or Salmonella. The vectors actually used are also well known and readily available in the art or can be constructed by those skilled in the art using methods well known in the art.

Dosage and method of administration

According to the present invention, administration of a single effective dose of mycoplasma hyopneumoniae to pigs aged about 3-10 days provides effective immunity against subsequent challenge with mycoplasma hyopneumoniae. Preferably, swine from about 6 to about 8 days of age are administered a mycoplasma hyopneumoniae vaccine. Most preferably, a mycoplasma hyopneumoniae vaccine is administered to pigs at about 7 days of age.

An effective dose of a vaccine of Mycoplasma hyopneumoniae bacterin in a single dose administration is about 1X 10 per dose⁶To about 5X 10¹⁰Color Changing Unit (CCU). Preferably, the mycoplasma hyopneumoniae bacterin vaccine single dose capable of providing effective immunity comprises 1 × 10⁸To about 5X 10¹⁰CCU and more preferably, about 5X 10⁸To 5X 10¹⁰CCU。

According to the present invention, when the preferred vaccine product RESPISURE-1 is used, the amount of RESPISURE-1 administered in a single dose is about 0.5-3.0ml, preferably about 1.5ml to about 2.5ml, more preferably about 2 ml.

An effective amount of a mycoplasma hyopneumoniae subunit vaccine of the invention comprising one or more proteins or polypeptides, or immunogenic fragments of said proteins or polypeptides, is from about 0.01 μ g to about 200 μ g.

An effective amount of a mycoplasma hyopneumoniae vaccine of the invention comprising one or more mycoplasma hyopneumoniae genes or nucleic acids (preferably DNA) encoding an immunogenic protein or polypeptide, or an immunogenic fragment of said protein or polypeptide, is from about 0.1 μ g to about 200 mg.

According to the present invention, the vaccine can be administered by routes well known in the art, including oral, intradermal, intramuscular, mucosal topical, transdermal and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular). Needle-free delivery devices may also be used for administration. Administration can also be accomplished by a combined route, e.g., the first administration is by the parenteral route and the subsequent administration is by the mucosal route. A preferred route of administration is intramuscular.

The effective dose (immunization dose) of the vaccine of the invention can also be extrapolated from the dose-response curve in a model test system.

The vaccination method of the invention provides protective immunity against mycoplasma hyopneumoniae for both seropositive and seronegative piglets. The seropositive piglet refers to the piglet containing the anti-mycoplasma hyopneumoniae antibody in the serum. A seronegative piglet is one that does not contain detectable levels of anti-mycoplasma hyopneumoniae antibodies in the serum.

The invention is further described in, but not limited to, the following examples.

Example 1

Preparation of Mycoplasma hyopneumoniae bacterin

Mycoplasma hyopneumoniae NL1042 strain was inactivated with binary aziridine (BEI)).

At the end of the growth period, the pH of the culture was adjusted up to 7.8. + -. 0.2 and maintained within this range for at least one hour. During this time, a filter-sterilized aqueous solution of 2-bromoethyl ammonium hydrobromide (2-bromoethyl amine hydrobromide) (BEA) was added to a final concentration of about 4.0 mM. At elevated pH BEA will be chemically converted to BEI. The cultures were incubated at 37. + -. 2 ℃ for at least 24 hours with continuous stirring.

After 24 hours of incubation, a filtered, sterile aqueous solution of sodium thiosulfate was added to a final concentration of about 4mM to neutralize excess BEI. The culture was incubated at 37. + -. 2 ℃ for 24 hours with continuous stirring.

After inactivation, but before neutralization with sodium thiosulfate, samples were taken and tested for completion of inactivation. Fresh medium containing 0.0026% phenol red was inoculated with 5-20% inoculum and incubated at 37 ± 2 ℃ for at least one week before detecting the color change, which was indicative of failure to inactivate. A large number of samples were tested for sterility in mercaptoacetate medium at 37. + -. 2 ℃ and tryptic soy medium at room temperature. The inactivated cultures were transferred to sterile storage containers and stored at 2-8 ℃ prior to assembly.

The antigen in the final container was quantified by in vitro serological experiments to determine the titer. The potency of the vaccine used in the efficacy studies determines the minimum activity that the vaccine must exhibit at the time of expiration.

Whole or final container samples of the finished product of each series or first subline were tested for Mycoplasma hyopneumoniae using the following procedure.

The vaccine was stored in 100ml vials at-50 ℃. After thawing the vials were aliquoted into 15ml and stored at 5+/-2 ℃ before use.

To test the titer of an assembled series, a sample of the assembled series is compared to a reference and the RP unit value of the assembled series is determined. The assembly series or assembly sublay preferably comprises at least 6.33RP at the start and at least 5.06RP throughout.

RP refers to relative potency. RP values can be determined using the relative amount of antigen compared to a reference vaccine. In the present application, the reference vaccine RP value is determined to be 1. The single dose product of the invention preferably has a 6.33RP value, i.e. 6.33 times that of the reference vaccine.

Thimerosal (Merthiolate) was added as a preservative at a final concentration of not more than 0.01% (w/v).

A10% solution of ethylenediaminetetraacetic acid (EDTA, disodium or tetrasodium salt) was added as a preservative to a final concentration of about 0.07% (w/v).

Example 2

Animal(s) production

Pigs of approximately 1 week of age were selected for vaccination. The serum status of mycoplasma hyopneumoniae was determined by ELISA assay. Pigs with ELISA value less than or equal to 0.50 are negative for Mycoplasma hyopneumoniae. Pigs with ELISA values greater than 0.50 were seropositive for mycoplasma hyopneumoniae.

Vaccine

Mycoplasma hyopneumoniae vaccine RESPISURE-1 (Peyer) was used to immunize pigs. Before using the vaccine, the titer was determined by the relative amount of antigen compared to the reference mycoplasma hyopneumoniae bacterin. The reference vaccine (RP ═ 1.0) contained approximately 8000 units of antigen per dose (approximately 1-2 x 10 of viable cells harvested prior to inactivation)⁸CCU) using a probe to detect porcine lung in vaccineSolid phase immunization experiment of mycoplasma inflammation antigen detects the mycoplasma inflammation antigen.

The same liquid Adjuvant (AMPHIGEN) used in the preparation of RESPISURE-1 was used as a placebo (i.e., no bacterial cells.)

Challenge inoculum (challenge inoculum)

Challenge inoculum, provided as a 10ml aliquot of lung homogenate, was frozen at-70 ℃ and considered a derivative of mycoplasma hyopneumoniae strain 11 (L136). The inoculum was thawed and diluted 1: 25 with Fris Mycoplasma Broth and kept in an ice bath prior to inoculation. Each pig received a 5ml dose (2.5 ml of a 1: 25 suspension per nostril) nasally at the particular time of day in each of the following examples. Each day of challenge, an aliquot of the lung inoculum was incubated to determine the absence of bacterial infection. Thereafter, on each of the three days, a second aliquot was titrated and the results showed that the inoculum contained about 10⁶-10⁷Mycoplasma hyopneumoniae in Color Change Units (CCU).

Experimental procedure

The pigs were identified on the sows using ear tags [ (-1) day ]. The pigs were assigned to pens and treatment groups according to the general random grouping method. Pig grouping was performed on a litter and weaning pen basis.

On day 0, the pigs were intramuscularly vaccinated with a 2ml dose of mycoplasma hyopneumoniae vaccine RESPISURE-1 (fevery), or with a 2ml dose of placebo. Each pig received a 5ml dose of 1: 25 suspension nasally at the specified time of day in each of the examples below. All pigs were monitored and examined daily for clinical signs of disease.

At a specific time after day 1 of challenge, all pigs were euthanized and necropsied. Lungs were removed and evaluated. Autopsy assays involve the assessment of the degree of pathology associated with mycoplasmal respiratory disease.

Each lung lobe was examined and a sketch of the lesion was drawn to evaluate its percentage of each lung lobe. And record the total lesion size.

Data analysis

Efficacy was assessed as a percentage of typical lung lesions infected with mycoplasma hyopneumoniae. The percentage of total lung lesions was significantly less (P.ltoreq.0.05) in pigs in the treatment group (vaccinated) than in the placebo group.

Percentage of total diseased lung

The percentage of each lobe is weighted average with the following ratio of individual lobes to total lung mass: left cephalic leaf 10%, left middle leaf 10%, left caudal leaf 25%. 10% of right cephalic leaf, 10% of right middle leaf, 25% of right caudal leaf and the balance 10%. The weighted lobe values are then summed to give the total diseased lung percentage (Pointon et al, 1992).

Example 3

The protection of pigs against virulent Mycoplasma hyopneumoniae challenge was evaluated, said pigs being Mycoplasma hyopneumoniae serotype positive pigs administered a single dose of Mycoplasma hyopneumoniae bacterin RESPISURE-1 (Peyer) at 3-8 days of age.

Five replication titer assays were performed on RESPISURE-1 at or before vaccination. Relative titers (RP) were determined using the relative amount of antigen compared to the vaccine reference. The RP of the vaccine reference was 1.0 and contained approximately 8000 units of mycoplasma hyopneumoniae antigen. The RP values for the five experiments were 5.42, 3.96, 4.71, 5.49 and 4.36, respectively.

On day 0, pigs in the T02 treated group (see Table 1 below) were intramuscularly vaccinated with 2ml of Mycoplasma hyopneumoniae vaccine RESPISURE-1 (Peyer). Pigs in the T01 group were intramuscularly vaccinated with 2ml placebo. On days 178, 179 and 180, each pig was inoculated nasally with 5ml of a 1: 25 challenge inoculum suspension. Each of the three days, an aliquot of the challenge was cultured at the time of inoculation to determine the absence of bacterial contamination. The second aliquot was back-titrated to determine that the challenge stock contained about 10⁷CCU/ml Mycoplasma hyopneumoniae. All pigs were tested daily and examined for clinical signs of disease.

All pigs were euthanized 30 days after the first day of challenge and necropsied. Lungs were removed and evaluated. Autopsy assays involve the assessment of the degree of pathology associated with mycoplasmal respiratory disease. Each lung lobe was examined and a sketch of the lesion was drawn to evaluate its percentage of each lung lobe. And the total lesion percentage was recorded.

TABLE 1

¹Toxic mycoplasma hyopneumoniae inoculum

²Pigs 71 and 73 were removed from the study before challenge due to loss of ear markers, as these pigs resulted in an inability to determine the identity of each animal. Pig No. 36 died on day 178 due to anesthetic complications. Pig No. 31 died on day 179 due to anesthetic complications.

The lung foci results are summarized in table 2. The results show that the minimum mean square value of lung lesions due to pneumonia was significantly lower in vaccinated pigs (T02) (P ═ 0.0385) than in placebo-administered pigs (T01) (2.0 vs 4.5%).

TABLE 2 summary of Total diseased Lung percentages

^a,bDifferent superscript values have significant statistical significance (P ═ 0.0385).

The results show that administration of mycoplasma hyopneumoniae bacterin RESPISURE-1 to a single vaccination of pigs at about 1 week of age induces resistance to subsequent challenge with virulent mycoplasma hyopneumoniae.

Example 4

The protective effect of pigs against virulent mycoplasma hyopneumoniae challenge was evaluated, said pigs being mycoplasma hyopneumoniae serotype negative pigs administered a single dose of a mycoplasma hyopneumoniae bacterin RESPISURE-1 at an age of 3-8 days.

Five replication titer experiments were performed against the vaccine at or before vaccination. RP was determined by the relative amount of antigen compared to the vaccine reference. The RP of the vaccine reference was 1.0 and contained approximately 8000 units of mycoplasma hyopneumoniae antigen. The RP values for the five experiments were 5.42, 3.96, 4.71, 5.49 and 4.36, respectively.

On day 0, pigs in the T02 treated group were intramuscularly vaccinated with a dose of 2ml of Mycoplasma hyopneumoniae vaccine RESPISURE-1. Pigs in the T01 group were intramuscularly vaccinated with 2ml placebo. On days 173, 174 and 175, each pig was inoculated nasally with 5ml of a 1: 25 challenge inoculum suspension. Each of these three days, an aliquot of the challenge was cultured at the time of inoculation to determine the absence of bacterial contamination. The second aliquot was back-titrated to determine that the challenge stock contained about 10⁶CCU/ml Mycoplasma hyopneumoniae. All pigs were tested daily and examined for clinical signs of disease.

All pigs were euthanized 29 days after the first day of challenge and necropsied. Lungs were removed and evaluated. Autopsy assays involve the assessment of the degree of pathology associated with mycoplasmal respiratory disease. Each lung lobe was examined and a sketch of the lesion was drawn to assess the total percentage added per lobe. And record the total lesion size.

TABLE 3

¹Virulent Mycoplasma hyopneumoniae

²No. 123 pig with chronic pus toxicityPolyarthritis was sacrificed painlessly on day 19.

³Pig number 222 died on day 40. Necropsy revealed massive pericardial effusion and epicardial hemorrhage. Pig # 102 was euthanized on day 95 due to rectal prolapse. Pig 204 died on day 145. Necropsy was not performed due to high putrefaction of the carcass.

⁴Pig 244 died on day 174 after the first day challenge due to anesthetic complications.

⁵Scoring 3 pigs with NEEA

The lung foci results are summarized in table 4. Analysis showed that the minimum mean square value of lung lesions due to pneumonia was significantly lower in vaccinated pigs (T02) (P0.0001) than in placebo-administered pigs (T01) (0.3 vs 5.9%).

TABLE 4 summary of percentage of total lung foci

Percentage of diseased lung

^a，bDifferent superscript values have significant statistical significance (P ═ 0.0001).

The results show that administration of mycoplasma hyopneumoniae bacterin RESPISURE-1 to pigs for single vaccination induces resistance to subsequent virulent mycoplasma hyopneumoniae challenge.

Example 5

Assessing the protective effect of a swine against virulent Mycoplasma hyopneumoniae challenge, said swine being a Mycoplasma hyopneumoniae serotype negative swine to which a single dose of a Mycoplasma hyopneumoniae bacterin RESPISURE-1 has been administered at an age of 3-8 days.

Five replication titer experiments against bacterins were performed at or before vaccination. RP was determined by the relative amount of antigen compared to the vaccine reference. The RP of the vaccine reference was 1.0 and contained approximately 8000 units of mycoplasma hyopneumoniae antigen. The RP values for the five experiments were 5.42, 3.96, 4.71, 5.49 and 4.36, respectively.

On day 0, pigs in the T02 treated group were intramuscularly vaccinated with a dose of 2ml of mycoplasma hyopneumoniae bacterin. Pigs in the T01 group were intramuscularly vaccinated with 2ml placebo. On days 76, 77 and 78, each pig was inoculated nasally (2.5 ml per nostril) with a 1: 25 dose of 5ml challenge inoculum suspension. Each of these three days, an aliquot of the challenge was cultured at the time of inoculation to determine the absence of bacterial contamination. The second aliquot was back-titrated to determine that the challenge stock contained about 10⁶CCU/ml Mycoplasma hyopneumoniae. All pigs were tested daily and examined for clinical signs of disease.

All pigs were euthanized 29 days after the first day of challenge and necropsied. Lungs were removed and evaluated. Autopsy assays involve the assessment of the degree of pathology associated with mycoplasmal respiratory disease. Each lung lobe was examined and a sketch of the lesion was drawn to evaluate its percentage of each lung lobe. And the total lesion percentage was recorded.

The experimental design is summarized in table 5.

TABLE 5

¹Toxic mycoplasma hyopneumoniae inoculum

²Pigs 237 and 139 were positive for mycoplasma hyopneumoniae by day-1. It was removed from the study on day 14 and euthanized without pain. Pig number 220 died on day 3 due to sow crushing.

³Pigs 238, 240 and 277 were positive for mycoplasma hyopneumoniae on day-1. It was removed from the study on day 14 and euthanized without pain. No. 280 pigs are depressed and cachectic due to reduced appetiteOn day 7, the sacrifice was painless. Pig number 177 died on day 40 due to chronic failure syndrome.

Table 6 summarizes the lung foci results. All analyses showed that the minimum mean square value of the percentage of pneumonia-caused lung lesions in vaccinated pigs (T02) was significantly lower (P ═ 0.0001) than in placebo-administered pigs (T01) (0.5 vs 9.9%).

TABLE 6 Total diseased Lung percentage summary

Percentage of diseased lung

^a，bThe different superscript values are statistical differences (P ═ 0.0001)

Claims (3)

1. Use of an effective amount of a single dose of a mycoplasma hyopneumoniae vaccine in the manufacture of a medicament for treating or preventing a disease or disorder caused by mycoplasma hyopneumoniae infection in an animal of 3 to 10 days of age, wherein the mycoplasma hyopneumoniae vaccine is a subunit vaccine and the subunit vaccine does not comprise Mph 3.

2. The use of claim 1, wherein the subunit vaccine comprises one or more immunogenic polypeptides or proteins or immunogenic fragments of said polypeptides or proteins.

3. The use according to claim 2, wherein the polypeptide or protein is selected from the group consisting of: p46, P65, P97, P102, P70, P50 and P44.