Vaccine for protection against mycoplasma hyopneumoniae

Abstract

To provide a vaccine against Mycoplasma hyopneumoniae (Mhyo), which does not contain, as an adjuvant, a non-self compound having a site effect such as mineral and vegetable oils, non-natural polymers and aluminum hydroxide.SOLUTION: The vaccine comprises nanoparticles bound to Mycoplasma hyopneumoniae bacterin, wherein the nanoparticles comprise a cationic polysaccharide and an anionic phospholipid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) eumoniae bacterin-conjugated nanoparticles and a vaccine in pigs Concerning the use of this vaccine for the prevention of infection. [Background technology]

[0002] Mycoplasma hyopneumoniae (hereinafter referred to as Mhyo) is a cell wall-deficient cell Mhy is a bacterium that is the causative agent of porcine epizootic pneumonia, a contagious chronic disease in pigs. o infects the respiratory tract of pigs and grows in the trachea, bronchi, and bronchioles. It is found in epithelial lung cells They attach to the cilia of bacteria and eventually kill them, causing lung lesions and secondary infections [e.g., other mycobacteria]. Plasma species, such as Mycoplasma hyorhinis inis and Mycoplasma flocculare lare), as well as PRRSV (porcine reproductive and respiratory syndrome virus) and It causes infection with PCV2 (porcine virus type 2).

[0003] The disease is caused by airborne Mhyo pathogens excreted from the lungs of infected pigs. It is transmitted from pigs to pigs.

[0004] In particular, infection with Mhyo caused reduced weight gain and poor feed conversion, but This leads to significant economic losses on commercial pig farms.

[0005] Mhyo bacteria lack a cell wall, and most common antibiotics focus on cell wall synthesis. Because of the alignment of points, such antibiotics are ineffective.

[0006] Vaccines for use against Mhyo pathogens are known and commercially available, For example, RespiSure® (Zoetis), Ingelvac® M.hyo and MycoFLEX® (Boehringer Inge lheim), Hyoresp® (Merial), Stellamune® Registered trademark) Mycoplasma (Elanco Animal Health), Fo stera® PCV MH (Zoetis) and M+Pac® and Porcilis® Mhyo (both MSD Animal Health h).

[0007] All of these vaccines contain a combination of immunization strategies to boost the immune response and also optionally Mhy o Contains classical adjuvants to stabilize antigens, such as mineral oil and vegetable oil, Non-natural polymers and non-self compounds such as aluminum hydroxide as adjuvants The non-self compound enhances the immune response but causes undesirable site effects. In particular, pigs have a high tolerance to excipients such as mineral oil, alum particles, and other non-self compounds. It is highly sensitive to the effects of classical adjuvants and does not induce site effects typically associated with classical adjuvants. It was an object of the present invention to provide a fast and effective vaccine.

[0008] It is also a simple, safe, and effective drug used to prevent Mycoplasma hyopneumoniae infection. It was an object of the present invention to provide a vaccine that is

[0009] Additionally, commercially available vaccines generally require a first vaccination to ensure successful immunization. followed by a booster vaccination within 2-3 weeks of the initial dose. It is administered in a two-shot format. However, such two-shot applications are This is disadvantageous in terms of efficiency and cost.

[0010] Therefore, it is possible to provide a vaccine that is effective in a one-shot (single-dose) format. That was another purpose.

[0011] Furthermore, developing a combination vaccine is cumbersome because all the vaccines The safety and stability of the vaccine components must be ensured, and the development of a combination vaccine is simple. In particular, the combination of live attenuated vaccine and bacterin vaccine is stable. It is difficult to select an excipient that will impair the affinity and stabilize both types of antigens.

[0012] Therefore, it is important to provide a combination vaccine that is safe to use and / or stable. was another objective. Summary of the Invention

[0013] Overview of the invention These objects have surprisingly been achieved by the following aspects of the present invention.

[0014] The composition according to the present invention is an anti-Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) hyopneumoniae) bacterin-bound nanoparticles, wherein the nanoparticles The components include cationic polysaccharides and anionic phospholipids.

[0015] Surprisingly, polysaccharides (common dietary compounds) and phospholipids (the subject's own compounds, a vaccine comprising said nanoparticles containing a self-compound and Mhyobacterin; To provide a stable and effective vaccine for the prevention of Mycoplasma hyopneumoniae infection. It has been found that the compound provides a therapeutic effect while not inducing undesirable site effects.

[0016] Therefore, the present invention also provides a method for the prevention of Mycoplasma hyopneumoniae infection in pigs. In the following, this vaccine is a vaccine for use in the prevention of the disease of the present invention. It can also be referred to as a vaccine.

[0017] The present invention also provides a kit of parts comprising a first vaccine, a second vaccine, and a leaflet. The present invention relates to a kit of parts, wherein the first vaccine is a The second vaccine is a live attenuated porcine reproductive and respiratory syndrome virus (PRR). SV).

[0018] In another aspect, the present invention provides a method for the treatment of swine flu, comprising administering an effective amount of a vaccine. The present invention relates to a method for preventing Mycoplasma hyopneumoniae infection in humans.

[0019] In yet another aspect, the present invention provides a method for the treatment of Mycoplasma hyogenes in pigs. The present invention also relates to the use of a vaccine according to the invention for the manufacture of a medicament for the prevention of E. monnier infections.

[0020] In yet another aspect, the present invention provides a mixture of a first vaccine and a second vaccine. The present invention relates to a combination vaccine contained in a vial, wherein the first vaccine is a vaccine according to the present invention. The second vaccine is a live attenuated porcine reproductive and respiratory syndrome virus (PRR). The combined vaccine contains the so-called RTU (ready-to-use) It is a vaccine that is prepared in advance and therefore distributed to end users in a mixed form produced by the manufacturer. Alternatively, for example, the PRRS virus may be introduced into the target animal via an Mh It may be a vaccine that is mixed by dissolving in the yo vaccine.

[0021] Detailed Description The following definitions pertain to embodiments of the present invention.

[0022] The term "vaccine" refers to a pharmaceutical composition that protects against a pathogenic microorganism ("pathogen"). It can induce protective immunity in animals against infection by pathogens and and / or may induce effective prophylactic treatment against disorders or diseases resulting from this infection. .

[0023] An antigen generally refers to any substance that induces a specific immune response in a host animal. dead, attenuated or live whole organisms, subunits of organisms or In part, a recombinant vector containing a polynucleotide encoding an immunogen, a protein, may include polypeptides, peptides, epitopes, haptens, or any combination thereof Bacterins are specific antigens. The term "bacterin" refers to a suspension of dead bacteria. For example, bacterial cultures may be concentrated and then purified by treatment with binary ethyleneimine (BEI), with chemicals such as chlorocresol, formalin, or with UV light, or with cells It means that it is obtained by inactivating it by other types of inactivation that do not result in lysis. In contrast to lysates, the antigens are inactivated bacteria, e.g., frozen. Freeze-thaw cycles, sonication, French press, bead beating The cells are specially treated in a cell lysis or destruction process such as lysis.

[0024] "Nanoparticles" are particles with a particle size in the nm range, i.e., an average of 1 to 1000 nm. Volume particle diameter (D 50 In one embodiment, the nanoparticles are particles having Average volume particle size (D 50 For example, the average volume particle size is 10 to 4 00 nm, for example, 20 to 300 nm and 30 to 200 nm. Typically, the average The volume particle size is 35 to 150 nm. The average volume particle size can be determined by laser diffraction. In a preferred embodiment, the particle size is determined using a Malvern Autosizer (trademark). ) 4700 (Malvern Instruments SA, UK) It is measured by differential light scattering using a 488 nm laser beam at a fixed angle of 90°. It is preferable to carry out the measurement. Preferably, an aqueous solution of 15 mM NaCl is used as a dispersant. In this case, the nanoparticles are concentrated to 0.5 mg / ml. Preferably, measurements are performed in triplicate. cormorant.

[0025] The term "zeta potential" refers to the charge between the dispersion medium and a fixed layer of dispersed particles. Thus, the zeta potential is determined by the surface charge of the particle, any adsorbed layer at the interface, and the surrounding suspending medium. In a preferred embodiment, the zeta potential is a function of the nature and composition of the body. Zetasizer Nano ZS (Malvern Instrument) determined by photon correlation spectroscopy using the nts (France). Preferably, measurements are carried out in triplicate.

[0026] The term "effective prophylactic treatment" or "prevention" of an infection (infectious disease) refers to the prophylaxis or prevention of an infection after treatment. is understood to include the prevention, amelioration, or alleviation of a disease or disorder resulting from a post-treatment infection. It should be.

[0027] "One-shot" or "single-dose" vaccines for use in prophylaxis The term "single dose" refers to a second vaccination or "boost" vaccination. It refers to a vaccination that does not require a seed and is still effective in conferring protective immunity. This does not exclude a single dose being administered as two or more separate doses administered simultaneously. It should be noted that

[0028] The word "comprises" means all of the features specifically mentioned and optionally should be construed to include any other, additional, or unspecified items, while The term "consisting of" includes only the features specified. As a specific example, the present invention includes a configuration specified by "consisting of."

[0029] As used herein, the term "pig" includes piglets, swine, pigs, porcine, sow, gilt, castration Barrow, boar, i.e., Suidae e) means all members of

[0030] The term "% by weight" refers to the percentage of a particular component relative to the total weight of the vaccine, unless otherwise indicated. means the weight of

[0031] Preferred embodiments according to the present invention are specifically described below. Furthermore, the following preferred embodiments are preferred in the overall scope of the present invention. vaccines, vaccines for use, kits of parts, methods of treatment, pharmaceutical preparations It should be understood to mean the use for the manufacture of vaccines and combination vaccines.

[0032] In one embodiment, the present invention provides a method for the treatment of Mycoplasma hyopneumoniae (Myc Containing nanoparticles bound to a bacterin from E. coli (Hyopneumoniae) and the nanoparticles are cationic polysaccharides and anionic phospholipids. Including quality.

[0033] The nanoparticles of the invention are known and methods for obtaining them are described, inter alia, in Pailla rd et al., Pharm.Res.2010,vol.27,pp.126-133, WO20 14 / 041427 and WO2018 / 104762 and references therein is described in.

[0034] In a preferred embodiment, the cationic polysaccharide is a solution of the respective polysaccharide (e.g., in 2N water) Dissolve the cross-linking agent (e.g., epichlorohydrin) in aqueous sodium hydroxide solution. hydrin) is added, followed by the addition of a cationic ligand (e.g., glycidyl-trimethylanhydrin). The pH can be adjusted by adding ammonium chloride; hydroxycholine. The polyol is neutralized, preferably with acetic acid, and the mixture is sheared, preferably under pressure, to form the cationic polyol. It is possible to obtain glyconanoparticles.

[0035] The resulting cationic polysaccharide is then loaded with anionic phospholipids, which are preferably Preferably, a cationic biopolymer and an anionic phospholipid (e.g., dipalmitoyl- phosphatidylglycerol) in a suitable solvent (e.g., ethanol for approximately 1 hour). This is done by mixing.

[0036] In one embodiment, the cationic polysaccharide is a porous cationic polysaccharide.

[0037] The nanoparticles are porous, i.e., they allow liquid or air to pass through the particles. The particles may have voids within the "bulk" of the particles so that the anions The ionic phospholipids are able to fill the pores, and the anionic phospholipids are present in the core of the nanoparticles. The cationic polysaccharide may be porous so that it is at least partially porous.

[0038] Typically, the (porous) particles are at least 0.1 m 2 / g, at least 0.5m 2 / g, 5m 2 / g, at least 10m 2 / g, or at least 20m 2 / g BET (ratio Preferably, the nanoparticles have a BET of at least 5 m 2 / g. Ratio table The area was determined by placing the sample in a nitrogen / helium atmosphere at different pressures. When the sample is cooled, nitrogen molecules condense on the surface of the particles. is determined by the change in thermal conductivity of the nitrogen / helium mixture, and the surface area of ​​the sample is determined by the change in the thermal conductivity of the nitrogen / helium mixture. This value and the sample weight are used to calculate the specific surface area. The specific surface area can be measured using a BET soap meter, e.g., Monosorb™, Fa.Qu Uses antachrome [compliant with DIN ISO 9277 (published January 2014)] It can be obtained using

[0039] In one embodiment, the nanoparticles comprise a cationic polysaccharide and an anionic phospholipid. The weight ratio is preferably 0:1 to 1:20, 20:1 to 1:10, or 10:1 to 1:3. In a preferred embodiment, the nanoparticles comprise a cationic polysaccharide and an anionic phospholipid in a ratio of 10: Contains in a weight ratio of 1 to 1:3.

[0040] In one embodiment, the nanoparticles have a voltage of 0 to 70 mV, 5 to 65 mV, or 10 to The nanoparticles have a zeta potential of 10 to 50 mV. preferable.

[0041] Nanoparticles have an outer portion (i.e., the surface) and an inner portion (i.e., the core) In certain embodiments, the outer portion of the nanoparticle from the zeta potential is anionic phospholipids. It can be inferred that the outer portion is essentially free of cationic polysaccharides. In a preferred embodiment, the core comprises an anionic phospholipid. The thionic polysaccharide is porous. In this embodiment, anionic phospholipids are preferred. Therefore, in a preferred embodiment, the anionic phospholipid is In a preferred embodiment, the nanoparticles have a core. In one embodiment, the anionic phospholipid is present in the core. The nanoparticles do not contain a phospholipid layer surrounding the core. be.

[0042] Mhyo bacterins are commercially available and methods for obtaining bacterins are known to those skilled in the art. For example, a method is known in the art, in which Mhyo cells are cultured and then inactivated. The law is publicly known.

[0043] In one embodiment, the vaccine is 1:10 to 10:1, 1:5 to 5:1, or The weight ratio of nanoparticles to Mhyobacterin is 1:2 to 2:1. In the method, the weight ratio of nanoparticles to Mhyobacterin is 1:1 to 1:3, for example 1:1. do.

[0044] The Mhyobacterin and the nanoparticles are combined, i.e., they form a composition. where bacterins are bound by intermolecular forces such as van der Waals and electrostatic forces. Mhyobacterin is thought to be bound to the outside of the nanoparticles. In one embodiment, the nanoparticles bound to the Mhyo bacterin are 200~1500nm, 300~1200nm, or 400~1100nm Measurement D 50 values ​​(when using laser diffraction method) (observed values ​​are greatly affected by the measurement method) (Can be).

[0045] The cationic polysaccharide is selected from starch, dextran and maltodextrin. Preferably, the cationic polysaccharide core is obtainable from a polysaccharide. More preferably, the cationic polysaccharide core is obtainable from a starch. , dextran and maltodextrin, and a primary amine, a secondary amine and a ligand selected from amines and tertiary amines and quaternary ammonium salts. Crosslinking agents are known to those skilled in the art and are preferred. A suitable crosslinking agent is epichlorohydrin (1-chloro-2,3-epoxypropane).

[0046] In one embodiment, the anionic phospholipid is diacylphosphatidylglycerolipid. diacylphosphatidylserine and diacylphosphatidylinositol wherein the acyl in each instance can be derived from a carboxylic acid. The anionic phospholipid is dipalmitoyl-phosphatidylglycerol (DPPG) is.

[0047] By mixing the polysaccharide core with anionic phospholipids, anionic phospholipids can be added to the cationic polysaccharide core. It is possible to load anionic phospholipids. It should be understood that the present invention relates to a mixture of cationic phospholipids and cationic polysaccharides.

[0048] In one embodiment, the cationic polysaccharide is a glycidyl- crosslinked maltodextrin. Cationic polymaltodextrins obtainable by reaction with trimethylammonium It's a string.

[0049] In one embodiment, the vaccine comprises a pharmaceutically acceptable solvent, whereby The vaccine is provided as a so-called ready-to-use vaccine. Preferably, the solvent comprises water and the bacterin is diluted so that it retains its effectiveness. In a preferred embodiment, the solvent is saline and / or phosphate buffered saline ( In another embodiment, the vaccine is administered in a pharmaceutically acceptable carrier prior to administration. The compound may be provided as a lyophilized product mixed with a suitable solvent.

[0050] In one embodiment, the vaccine contains a classical adjuvant, such as an oil and / or Essentially containing aluminum hydroxide, and / or saponin and / or carbopol No.

[0051] "Essentially free" means a concentration of less than 0.1% (v / v) of the total composition of the vaccine. Refers to the degree.

[0052] Adjuvants can generally be classified according to the immunological events they induce. The ras contain, inter alia, ISCOMs (immunostimulating complexes), saponins (or fractions thereof and derivatives thereof), Conductors, e.g., Quil A), aluminum hydroxide, liposomes, coclate (coc hleate), polylactic / glycolic acid, and antigen uptake, transport and presentation [A The second class, among others, is oil emulsions. ions, gels, polymer microspheres, non-ionic block copolymers, and possibly also contain aluminum hydroxide, which provides a depot effect. The third class, among others, , CpG-rich motif, monophosphoryl lipid A, mycobacteria (muramyldipeptide) It contains preserved microbial structures, so-called pathogen-associated microorganisms, including cholera toxin, yeast extract, and cholera toxin. Based on the recognition of biological patterns (PAMPs) (defined as signal 0). The agent contains, among other things, an oil emulsion, a surfactant, aluminum hydroxide, and hypoxic conditions. The ability of the immune system to distinguish between dangerous and harmless (which are not necessarily the same as self and non-self) The fifth class includes, inter alia, cytokines and is based on stimulating the expression of co-expressing cytokines on APCs. Adjuvants are based on the upregulation of stimulatory molecules (signal 2). Although the present nanoparticles do not specifically belong to one of these classes, Although it is not possible to determine the effect, an adjuvant effect may still be obtained in some specific configurations. It seems.

[0053] The vaccine may optionally contain auxiliary substances such as wetting agents, pH buffers, viscosity additives and preservatives. Suitable auxiliary substances are, for example, those listed in the "Pharmaceutical Products eformulation and Formulation”(Mark Gibso n, 2nd edition).

[0054] A vaccine should be understood to contain an immunologically effective amount of Mhyo bacterin. Such amounts can be easily established based on general knowledge and can be obtained, for example, from commercially available Porclis MyoID ONCE (MSD Animal Health h) or other commercially available bacterin products. It can be the amount of phosphorus.

[0055] In one embodiment, the vaccine is administered in a volume of 0.1 to 3 ml per dose, preferably 0.2 to 2 In one embodiment, the vaccine is administered to the target animal in a volume of 100 ml / dose. 2μg to 10mg, 10μg to 5mg, 20μg to 2000μg, or The vaccine contains 200 μg to 2000 μg of nanoparticles. Typically, the vaccine Each dose contains 1-5 mg of nanoparticles.

[0056] In one embodiment, the vaccine is for Mycoplasma hyopneumoniae in pigs. It is used to prevent infection with nie.

[0057] In one embodiment, the vaccine used is administered to pigs between the first and fifth week of life (postnatal). In another embodiment, the vaccine used is administered in weeks 2 to 4. Chin is administered as a one-shot dose between the first and fifth weeks of life.

[0058] In another embodiment, the vaccine is administered systemically. In contrast to intragastric administration (intramuscular administration or intragastric administration), systemic administration involves delivering the vaccine into the subject's circulatory system and absorbing its entire volume. Examples of systemic administration include intramuscular (IM), intravenous (IV), and (IV), intradermal (ID), transdermal (TD) and subcutaneous (SC).

[0059] In one embodiment, the vaccine is administered intradermally. Preferably, the vaccine is administered intradermally. It is administered by needle-free injection using the AL™ (Intradermal Liquid Application) Injection System. DAL is a pressure-based application system (from MSD Animal Health) (These are the ones).

[0060] In one embodiment, the vaccine used is administered as a single dose. Therefore, vaccines may be administered in multiple doses (e.g., a primary vaccination and a booster vaccination). It is administered as a one-shot dose rather than a two-shot application (including vaccination).

[0061] In a preferred embodiment, the vaccine used is administered intradermally to the target animal as a single dose. It is given.

[0062] Typically, vaccines are used to reduce lung lesions caused by Mycoplasma hyopneumoniae infection. Compared to sham-vaccinated control pigs, lung lesions were reduced by 10-100% and even It was found that the reduction was 50 to 100%.

[0063] Lung lesion scores (LLS) using a common scoring system were significantly higher in the pulmonary stenosis compared to untreated control pigs. , can be reduced by 10 to 100%, typically by 50 to 100%.

[0064] LLS was measured after 3 weeks of challenge, as described in more detail in Example 1. The reduction in lung lesions and LLS was observed in pigs treated with the vaccine of the present invention compared to untreated pigs. LLS refers to the comparison of pigs treated with the vaccine. This is a useful index. The proportion of lung lesions was measured and compared with that of Goodwin and Whittlestow. Goodwin & Whittlestone score (Goodwin et al., Vet erinary Record,“The detection of enzooti c pneumonia in pig herds.I.Eight years g enal experience with a pilot control s The LLS can be determined by converting the LLS to

[0065] In one embodiment, the present invention provides a first vaccine, a second vaccine and a leaflet wherein the first vaccine is a vaccine according to the invention. The second vaccine is a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV). The leaflet contains instructions for use. The parts kit includes needles and / or syringes. It may further comprise

[0066] A live attenuated pathogen is a viable, replicative form of a pathogen that has reduced virulence. The process of attenuation uses an infectious agent and transforms it into, for example, a cell line. by multiple passage of the pathogen through bacteria or by genetically modifying the pathogen to render it harmless or or to make it less pathogenic.

[0067] In one embodiment, the present invention provides a mixture comprising a first vaccine and a second vaccine. The present invention relates to a combination vaccine, wherein the first vaccine is a vaccine according to the present invention. The second vaccine contains live attenuated porcine reproductive and respiratory syndrome virus (PRRSV).

[0068] Instead of being administered simultaneously, the first and second vaccines are administered in adjunctive doses for combination administration. It is also possible to provide the vaccine as a non-mixed vaccine. Without mixing, the mixture is mixed for 1 hour, preferably within 30 minutes, 25 minutes, 20 minutes, or 15 minutes. Within, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 The drugs are administered to the pigs separately within minutes, two minutes, or one minute.

[0069] However, to minimize the amount of injections administered to each pig and further increase efficiency, two Combinations of vaccines are generally advantageous. However, when two or more vaccines are combined, The vaccine requires different excipients and therefore may be instable, lose activity, or be more susceptible to the effects of one vaccine. Stability issues are common, as the presence of ATP can lead to degradation and / or breakdown. The stability of PRRSV was enhanced in the presence of the nanoparticles of the invention.

[0070] In one embodiment, the combination vaccine according to the invention is for the prevention of Mhyo infection and P It is used to prevent RRSV infection.

[0071] In one embodiment, the present invention provides a vaccine according to the present invention or a combination vaccine according to the present invention. The vial is preferably a PET vial. The vaccines are available in 2ml, 20ml, 50ml, 100ml, 200ml or 500ml. It is preferred that the vaccine contains a flu vaccine or combination vaccine.

[0072] The present invention will now be described in more detail with the aid of the following examples.

[0073] Example Example 1: Lung Lesion Scores Challenge Materials: Bacterin mycoplasma frozen in 1 ml aliquots and stored below -50°C M. hyopneumoniae strain 98 was used.

[0074] Preparation of challenge material: Mycoplasma hyopneumoniae (M. hyopneumoniae) strain 98, The culture was diluted 2000-fold in FRIIS + 20% SPF serum. After 4 and 5 days of incubation, A sample of this culture was taken for challenge. 4.5 ml of FRIIS broth + 20% SPF (specific pathogen free) serum was inoculated with 0.5 ml of culture, mixed, and Transfer 0.5 ml of culture from one tube to the next, then add 10 ml of -10 CCU( The tubes were incubated at 37°C for 3 weeks before challenge. and later viable counts were performed.

[0075] How to take part: Seventy-two SPF pigs aged 3 weeks (±3 days) were assigned to six groups of 12 pigs each. Four animals per group were added to groups 1 to 4, for a total of 16 animals per group. All animals were challenged intratracheally with 10 ml of pure culture on two consecutive days using a 500-mL syringe pump.

[0076] Dosage and Administration: The vaccine has a D of 37 nm. 50 dipalmitoyl palmitate, with a zeta potential of +37 mV Porous cationic maltodextrin loaded with methyl-phosphatidylglycerol The test vaccine contained 2.9 mg of particles per 0.4 ml dose. The latter type was produced using Mhyo bacterin and Mhyo lysate. antigens are typically more effective when used with nanoparticles in the prior art The bacterin vaccine was prepared by mixing particles with a bacterin preparation (BEI inactivated Mhyo bacteria) at 1: The mixture was then allowed to stand for 24 hours. The lysate vaccine was prepared by lysing the cells and precipitating the mixture to achieve binding of the particles to the antigen. The difference was that the bacterin was subjected to ultrasonic treatment and was prepared accordingly. The zeta potential of the particles after binding to the hyo antigen is +14 for the bacterin vaccine. 9mV for the lysate vaccine and +15.6mV for the lysate vaccine.

[0077] At 3 and 5 weeks of age, topically (intranasally) or systemically in the neck according to Table 1 [The IDAL® vaccine dispenser was used to administer the vaccine partially into the dermis and partially into the skin. The piglets were vaccinated with the vaccine, which was administered by depositing the vaccine in the muscle tissue. Use the product Porcilis® Myo ID ONCE according to the manufacturer's instructions. did.

[0078] The new vaccine did not induce any unwanted site effects and was considered safe. Pigs were euthanized on day 21 after challenge to determine lung lesion scores. The proportion of lung lesions was determined during the biopsy and scored according to the Goodwin and Whittlestone score. The lung lesion scores were obtained by converting the values ​​into the above formula. The results are shown in Table 1. [Table 1]

[0079] As can be deduced from Table 1, the systemic efficacy of the vaccine containing Mhyo bacterin and nanoparticles In the case of oral administration, lung lesion scores were significantly reduced compared to topical application. The telin vaccine was substantially more effective than Mhyo lysate, which was due to the Compared with the technique in which the nanoparticles are typically applied for topical vaccination using lysates. It is surprising that

[0080] Example 2: Comparison of single and double doses The LLS was determined for four groups of pigs, each containing 20 pigs, in the same manner as in Example 1 above. did.

[0081] In Group 1 (One Shot ID), pigs received a single dose of 0.2 mL of vaccine at week 4. Group 2 (two-shot ID) In this study, pigs were given a primary vaccination of 0.2 mL intradermally at week 1, followed by a second vaccination at week 4. In group 3, pigs received a 0.4 mL booster vaccination at week 4. In Group 4, the mice were vaccinated intradermally with a single dose (one shot) of the vaccine. The vaccines in groups 1 to 3 were the same, and included nanoparticles and Mhy. bacterin (weight ratio 1:1), where each 0.2 ml dose contains 2.18 mg The zeta potential of the formulated particles was +15.2 mV.

[0082] The results are shown in Table 2 below. [Table 2]

[0083] As can be seen, quite surprisingly, a single dose significantly reduced LLS compared to two doses. Furthermore, LLS appeared to be further reduced. With this vaccine, a single vaccination is all that is needed to obtain effective protection against Myo. This means that can be used.

[0084] Example 3: Stability of PRRSV vaccine To determine the stability of a live-attenuated PRRSV vaccine in the presence and absence of nanoparticles PRRSV was then incubated with the common vaccine diluent Diluv in the presence and absence of nanoparticles. Mixed in ac Forte® (MSD Animal Health). The TCID50 (50% tissue culture infectious dose) of PRRSV on MARC cells was measured at 0, 45, and 9 TCID50 corresponds to the efficacy of the PRRSV antigen. For this experiment, in each case 1.67 mg of nanoparticles (see Example 1) were used. Add approximately 7.2 ml of the diluted solution to an Eppendorf tube. * 10 ∧ 5 TCID5 A dose of live PRRS virus in diluent is added to the tube so that a 0 is achieved. The following Table 3 shows the TCID50 values ​​at each measurement interval ( * 10 ∧ 5) As can be seen from the results, the stability of PRRSV in the presence of nanoparticles is significantly greater than that in the absence of nanoparticles. The effect was enhanced compared with the existing formulation. [Table 3]

[0085] As can be seen from the results shown in Table 3, the stability of PRRSV in the diluent was The efficacy of the combination vaccine was surprisingly increased in the presence of the PRRSV component. This also means increasing the

Claims

1. Mycoplasma hyopneumoniae iae) a vaccine comprising nanoparticles bound to a bacterin, the nanoparticles being cationic; The vaccine comprises an anionic polysaccharide and an anionic phospholipid.

2. 2. The vaccine of claim 1, wherein the cationic polysaccharide is a porous polysaccharide.

3. Anionic phospholipids include diacylphosphatidylglycerol, diacylphosphatidyl 3. The compound according to claim 1, wherein the compound is selected from the group consisting of lucerin and diacylphosphatidylinositol. The vaccine described.

4. The cationic polysaccharide is cross-linked with maltodextrin and glycidyl-trimethylammonium The vaccine according to any one of claims 1 to 3, obtainable by reacting

5. The vaccine according to any one of claims 1 to 4, which comprises a pharmaceutically acceptable solvent.

6. The vaccine contains oil and / or aluminum hydroxide and / or saponin and / or The vaccine according to any one of claims 1 to 5, which is essentially free of carbopol.

7. Mycoplasma hyopneumoniae in pigs 10. The method of claim 1 for use in the prevention of S. neumoniae infections. The vaccine listed.

8. The vaccine for use according to claim 7, wherein the vaccine is administered systemically.

9. 9. The vaccine for use according to claim 7 or 8, wherein the vaccine is administered intradermally.

10. Use according to any one of claims 7 to 9, wherein the vaccine is administered as a single dose. vaccine.

11. Mycoplasma hyopneumoniae 11. The method according to claim 7, wherein the pulmonary pathology caused by the pulmonary inflammatory disease (pulmonary inflammatory disease) is reduced by the pulmonary inflammatory disease (pulmonary inflammatory disease) caused ... A vaccine for the use described.

12. A kit of parts comprising a first vaccine, a second vaccine and a leaflet, wherein the first vaccine is the vaccine according to any one of claims 1 to 6, and the second vaccine is The kit comprises a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV).

13. A method for the treatment of pigs comprising administering an effective amount of the vaccine according to any one of claims 1 to 6. Mycoplasma hyopneumoniae niae) infection prevention method.

14. Mycoplasma hyopneumoniae in pigs 10. The method according to claim 1, for the manufacture of a medicament for the prevention of S. neumoniae infections. Use of the vaccine described.

15. A combination vaccine comprising a mixture of a first vaccine and a second vaccine, wherein the first vaccine The vaccine according to any one of claims 1 to 6 is a live attenuated vaccine. The combination vaccine further comprises PRRSV.

16. A vaccine according to any one of claims 1 to 6 or a combination vaccine according to claim 15. vial.

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