CN104271153B - PCV/ mycoplasma hyopneumoniaes/PRRS combination-vaccines - Google Patents

Abstract

The present invention provides trivalent immunogenic composition, and it includes the soluble parts of the full cellular preparations of mycoplasma hyopneumoniae (M.hyo)；Porcine circovirus 2 type (PCV2) antigen；With porcine reproductive and respiratory syndrome (PRRS) viral antigen, the wherein soluble part of M.hyo prepared products is combined the immune complex formed with immunoglobulin substantially free of (i) IgG and (ii) by antigen.

Description

PCV/ mycoplasma hyopneumoniaes/PRRS combination-vaccines

Invention field

The present invention relates to pig circular ring virus, mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae (M.hyopneumoniae or M.hyo)) and porcine reproductive and respiratory syndrome (PRRS) virus.More particularly it relates to three Valence immunogenic composition, it includes the soluble part of the full cellular preparations of M.hyo, PCV2 antigens and PRRS viral antigens, And the composition is for protecting pig to resist enzootic pneumonia and postweaning multisystem exhaustion syndrome (PMWS) Vaccine in application.

Background of invention

Enzootic pneumonia in pig, also referred to as Eaton agent pneumonia, are caused by M.hyo.The disease is one Chronic, the non-lethal disease of kind, influences the pig at various ages.Infected pig only shows to cough and have a fever slight Symptom, but this disease has economy important influence, because feed efficiency reduces and body weight increase is reduced.Place Property the air borne microbes that are discharged by the lung of infected pig of zoosis pneumonia propagated between pig by nasal meatus.M.hyo's is first Subinfection may be followed by other mycoplasma species (mycoplasma hyorhinis (Mycoplasma hyorhinis) and mycoplasma flocculare (Mycoplasma flocculare)) and other bacterial pathogens secondary infection.

M.hyo is small prokaryotic micro-organisms, can be existed to free living, although it is often found and eukaryocyte phase Contact, because it has absolute demand to exogenous sterol and aliphatic acid.These demands usually require raw in the culture medium containing serum It is long.M.hyo is by cell membrane rather than cell wall is coated with.

The physical bond of mycoplasma and host cell surface is that existing basis occurs and continues for enzootic pneumonia. The respiratory tract of M.hyo infected pigs builds group in trachea-bronchial epithelial cell, bronchiole.Mycoplasma generates cilia stagnation factor, causes Cilium stop motion on the inside of respiratory tract.Final cilium is degenerated, and pig is made to be susceptible to the infection of secondary pathogen.In infection animal In observe purple to the area of consolidation of grey typical lesions.The investigation of slaughtered animals is disclosed and is deposited in the pig of 30-80% In lesion.37 droves from 13 states the result shows that 99% drove, which has, meets enzootic pneumonia characteristic feature Pneumonia lesion.Therefore, very big to being effectively prevented and treated the demand of measure.

Antibiotic such as Tiamulin, methoxybenzyl aminopyrimidine, tetracycline and lincomycin have some benefits, but it is expensive simultaneously And it needs to be used for a long time.In addition, antibiotic is not yet proved to effectively eliminate propagation or the superinfection of M.hyo.By maintaining nothing Pathogen drove is sometimes feasible to prevent, but M.hyo is introduced back into frequent occurrence.Due to the serious warp of pig pneumonia Ji consequence is finding the vaccine for M.hyo.Contain the mycoplasma organism prepared product grown in serum-containing media Vaccine have been enter into market, but cause and serum component present in immune substance (such as immune complex or non-exempted from Epidemic focus specific proteins) induction side reaction concern.The existing winner of other trials of offer M.hyo vaccines, but this Kind disease is still widely current.

M.hyo and porcine circovirus 2 type (PCV2) are the two kinds of pathogen the most universal encountered in pig industry.By PCV2 The pig of infection shows the syndrome of commonly known as postweaning multisystem exhaustion syndrome (PMWS).The Clinical symptoms of PMWS is Weak, ochrodermia, thin and weak, respiratory distress, diarrhea, jaundice (icterus) and jaundice (jaundice).Other than PMWS, PCV2 includes pseudorabies, porcine reproductive and respiratory syndrome (PRRS), glasser ’ s disease (Glasser ' with several other infection S disease), streptococcus meningitis, salmonellosis, postweaning colibacillosis, diet liver disease and suppurative broncho-pulmonary It is scorching related.M.hyo is related to enzootic pneumonia, and it is porcine circovirus associated diseases (PCVAD) to be shown Main co-factor in generation.

Porcine reproductive and respiratory syndrome (PRRS) is caused by arteritis virus, and the virus and macrophage are especially in the presence of Macrophage (pulmonary alveolar macrophage) in lung has special affinity.These macrophages absorb and remove the bacterium of invasion And virus, but it is really not so in the case of PRRS viral (PRRSV).In the case of PRRS viruses, it is in macrophage Portion is bred, and is generated more viruses and is killed macrophage.Once PRRSV enters drove, it will indefinitely in the presence of and have Activity.Up to 40% macrophage is destroyed, this makes bacterium and other virus multiplications and is destroyed.The one of this event A common example is in childhood/old age group (grower/finisher units), when they are infected by PRRS viruses When, the severity of enzootic pneumonia dramatically increases.The pig negative more than the weaning period PRRS viruses of half is entering It is infected before market.

What is desired is that for the PCV2/M.hyo/PRRS trivalent vaccines of PCV2, mycoplasma and PRRSV infection in pig.It carries It will be desirable for the trivalent vaccine of single dose (single dose).Preferably, the PCV2/M.hyo components of vaccine It is provided in the form of single-use bottle of liquid compositions, it can be easily with PRRSV groups subassembly so that all antigen quilts It is administered simultaneously to pig.

Invention summary

The present invention provides trivalent immunogenic composition, and it includes the full cellular preparations of mycoplasma hyopneumoniae (M.hyo) Soluble part；Porcine circovirus 2 type (PCV2) antigen；With porcine reproductive and respiratory syndrome (PRRS) viral antigen, wherein The soluble part of M.hyo prepared products is combined what is formed to be immunized substantially free of (i) IgG and (ii) by antigen with immunoglobulin Compound.In one aspect, the soluble part of the full cellular preparations of M.hyo before being added into immunogenic composition It is handled with albumen-A or albumen-G.On the other hand, the soluble part of M.hyo prepared products and PCV2 antigens are to use The form of type liquid composition.

In one embodiment, PRRS viral antigens are the live virus of genetic modification.In another embodiment, base Because the work PRRS viruses of modification are the forms of freeze-dried composition.

In one embodiment, the soluble part of M.hyo prepared products includes at least one M.hyo proteantigens. In another embodiment, the soluble part of M.hyo prepared products includes two or more M.hyo proteantigens.

In one embodiment, PCV2 antigens are the forms of -2 type circovirus of chimeric 1 type, and embedded virus includes inactivation Expression Porcine circovirus type 2 Cap protein 1 type of recombinant porcine circovirus.In another embodiment, PCV2 antigens are Recombinate the form of ORF2 albumen.In yet another embodiment, recombination ORF2 albumen is expressed from baculovirus vector.

In some embodiments, trivalent composition of the invention causes the protection for M.hyo, PCV2 and PRRS virus Property immune response.In other embodiments, immunogenic composition of the invention further includes at least one other antigen. In one embodiment, at least one other antigen is directed to may cause the microorganism of disease to provide protection in pig.

In one embodiment, microorganism includes bacterium, virus or protozoan.In another embodiment, micro- Biology is selected from but not limited to following type：Pig parvoviral (PPV), haemophilus parasuis (Haemophilus parasuis), Pasteurella multocida (Pasteurella multocida), Streptococcus suis (Streptococcum suis), Staphylococcus hyicus (Staphylococcus hyicus), Actinobacillus pleuropneumoniae (Actinobacilllus pleuropneumoniae), branch Tracheitis byrd bacteria (Bordetella bronchiseptica), Salmonella choleraesuls (Salmonella Choleraesuis), Bacterium enteritidis (Salmonella enteritidis), erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae), mycoplasma hyorhinis (Mycoplama hyorhinis), mycoplasma hyosynoviae (Mycoplasma hyosynoviae), Leptospira bacteria, Lawsonia intracellularis (Lawsonia Intracellularis), swine influenza virus (SIV), bacillus coli antigen, Brachyspira hyodysenteriae (Brachyspira Hyodysenteriae), porcine respiratory coronavirus, pig epidemic diarrhea (PED) virus, rotavirus, thin circovirus virus (TTV), inclusion-body rhinitis virus of pigs, chitling virus, encephalomyocarditis virus, Ao Jieshijishi diseases, the pathogenic original of typicalness swine fever (CSF) With the pathogenic original of transmissible gastroenteritis of swine, or combinations thereof.

In some embodiments, composition of the invention further includes adjuvant.In one embodiment, adjuvant selects From but be not limited to following type：Oil-in-water adjuvant, polymer and water adjuvant, water-in-oil adjuvant, aluminum hydroxide adjuvant, vitamin E Adjuvant and combinations thereof.In another embodiment, composition of the invention further includes pharmaceutically acceptable carrier.

In certain embodiments, composition of the invention draws when the form being administered in a single dose is administered Protective immunological reaction of the hairpin to M.hyo, PCV2 and PRRS virus.

Method the present invention also provides immune swine to resist M.hyo, PCV2 and PRRS virus.This method includes being applied to pig Include the soluble fraction of the full cellular preparations of mycoplasma hyopneumoniae (M.hyo)；Porcine circovirus 2 type (PCV2) antigen；And PRRS The trivalent immunogenic composition of viral antigen, the wherein soluble part of M.hyo prepared products substantially free of (i) IgG and (ii) immune complex formed is combined with immunoglobulin by antigen.

In an embodiment of the method for the present invention, trivalent composition is by intramuscular, intradermal, percutaneous or subcutaneous administration. In another embodiment of the method for the present invention, trivalent composition is administered in the form of single dose.

In a further embodiment, composition is administered to have is directed to M.hyo, PCV2 and PRRS from parent The pig of at least one antibody of virus.In yet another embodiment, composition, which is administered to, has being directed to from parent The pig of the antibody of M.hyo, PCV2 and PRRS virus.

In one embodiment, composition is administered to the pig of 3 week old or bigger.

The present invention also provides the methods for preparing immunogenic composition according to the present invention.This method includes i) suitable The 18-144 hours times of M.hyo are cultivated in culture medium；Ii M.hyo cultures) are then inactivated；Iii) the culture of harvest inactivation Liquid, wherein the culture solution inactivated include the full cellular preparations of M.hyo, the full cellular preparations include soluble liquid fraction and Insoluble cellular material；Iv) soluble liquid fraction is detached from insoluble cellular material；V) from the soluble liquid fraction of separation IgG and antigen immunoglobulin compound are substantially removed, to form the soluble fraction of the full cellular preparations of M.hyo； And vi) then by the soluble part of the full cellular preparations of M.hyo and PCV2 antigens and PRRS virus antigen combinations.At one In embodiment, step vi) include ready-to-liquid composition and the freeze-drying that will include PCV2 antigens and M.hyo soluble parts PRRS virus antigen combinations.

In one embodiment, kit according to the present invention includes first bottle (or other suitable containers), packet Containing composition, the composition includes the soluble part of PCV2 antigens and the full cellular preparations of mycoplasma hyopneumoniae (M.hyo), The soluble part of the full cellular preparations of wherein M.hyo is substantially free of (i) IgG and (ii) antigen/immunoglobulin is compound Object；With second bottle, it includes PRRS viral antigens.In one embodiment, the composition in first bottle is with instant liquid The form of composition provides.In another embodiment, the PRRS virus antigen components in kit are with freeze-dried composition Form provides.In another embodiment, kit includes providing about by first bottle of content and second bottle of content The operation instructions of the explanation of object combination.In yet another embodiment, operation instructions further comprise will combine first Content with second bottle is administered to the explanation of pig.

Brief description

Fig. 1 is to show the M.hyo univalent vaccines prepared with the M.hyo antigens for carrying out different disposal (described in embodiment 3 T02-T10) the figure relative to the effect of placebo (T01).As a result it is expressed as % injury of lungs least square average values (%Lung Lesion Least Square Mean values)。

Fig. 2 is the PCV2 antigen efficacy outcomes for the M.hyo vaccines for showing that -2 type embedded virus of 1 types of PCV with inactivation combines The figure of (PCV2 antigen ELISAs).The initial level for the embedded virus for including in composition is about 1.6≤RP.Each sample As a result it is expressed as relative potency (RP).

Fig. 3 is to show the PCV2 viremia virusemia knots observed with the PCV/M.hyo vaccine preparations using different adjuvant platforms The figure of fruit (PCV2 quantitative PCRs).

Fig. 4, which is shown in, attacks poison the 1st, 20 and 42 day, is observed with the PCV/M.hyo vaccine preparations of different adjuvant platforms are used The figure of PCV2 antibody ELISAs (S/P) Serological arrived.

Fig. 5 is to show that the PCV2 excrement that the T02-T04 processing described in embodiment 7 is obtained relative to placebo (T01) is de- The figure fallen.As a result it is expressed as PCV2DNA copy numbers/ml.

Fig. 6 is to show that the PCV2 noses that the T02-T04 processing described in embodiment 7 is obtained relative to placebo (T01) fall off Figure.As a result it is expressed as PCV2DNA copy numbers/ml.

Fig. 7 (A＆B) is the interferon-γ (IFN-γ) that display measures PCV2- specific cell mediated immunities (CMI) response The figure of testing result.After inoculation/result attacked before poison is shown in Fig. 7 A, after inoculation/result attacked after poison is shown in Fig. 7 B.5x 10⁶The stimulation of a cell is considered significant.

Fig. 8 shows the M.hyo effect of the PCV2/M.hyo tentative bacterin preparations in SP- oil.T02- is handled using M.hyo The preparation of T08 scores relative to the lung of placebo (T01) to scheme to show in fig. 8 a.Table in Fig. 8 B shows processing T02- The comparison of T08 and placebo.

Fig. 9 is an implementation of the manufacturing process for showing the M.hyo antigens for being used to prepare the compatible albumen-A processing of PCV2- The flow chart of scheme.

Figure 10 is the table for showing the adjuvant evaluation for the viricidal activity of PRRS viruses.

Figure 11 is to show the PCV2 viremia virusemia results observed with PCV2/M.hyo/PRRS tentative bacterin preparations The figure of (PCV2 quantitative PCRs).

Figure 12 is shown in the 1st of research the, 7,13,20,28,35 and 42 day (it is the 21st day to attack poison), uses PCV2/M.hyo/ The figure for the PCV2ELISA results that PRRS tentative bacterin preparations are observed.

Figure 13 is T02 and T03 processing (the tentative vaccine systems of PCV2/M.hyo/PRRS shown with described in embodiment 14 Agent) figure that falls off of PCV2 excrement that is obtained relative to placebo (T01).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is an embodiment of the nucleotide sequence for the p46 for encoding the P-5722 strains from M.hyo；

SEQ ID NO:2 be an embodiment of the amino acid sequence of the p46 of the P-5722 strains from M.hyo；

SEQ ID NO:3 be an embodiment of the nucleotide sequence for the p97 for encoding the P-5722 strains from M.hyo；

SEQ ID NO:4 be an embodiment of the amino acid sequence of the p97 of the P-5722 strains from M.hyo；

SEQ ID NO:5 be an embodiment of the genome sequence of encoding chimera PCV1-2 viruses；

SEQ ID NO:6 are corresponding to an embodiment of the nucleotide sequence of pig circular ring virus ORF2；

SEQ ID NO:7 are corresponding to an embodiment of the amino acid sequence of pig circular ring virus ORF2 polypeptides；

SEQ ID NO:8 be an embodiment of the genome sequence of encoding chimera PCV1-2 viruses；

SEQ ID NO:9 are corresponding to an embodiment of the nucleotide sequence of pig circular ring virus ORF2；

SEQ ID NO:10 are corresponding to an embodiment of the amino acid sequence of pig circular ring virus ORF2 polypeptides；

SEQ ID NO:11 are corresponding to an embodiment of the amino acid sequence of pig circular ring virus ORF2 polypeptides；

SEQ ID NO:12 be coding SEQ ID NO:One embodiment of the nucleotide sequence of 11 amino acid sequence；

SEQ ID NO:13 are corresponding to an embodiment of the amino acid sequence of pig circular ring virus ORF2 polypeptides；

SEQ ID NO:14 be coding SEQ ID NO:One embodiment of the nucleotide sequence of 13 amino acid sequence；

SEQ ID NO:15 are corresponding to an embodiment of the amino acid sequence of pig circular ring virus ORF2 polypeptides；

SEQ ID NO:16 are known as the genome sequence of the non-strong toxic forms of the North American PRRS virus isolate of P129 One embodiment of row；And

SEQ ID NO:17 are corresponding to be referred to as the nucleotide of the ORF2 to ORF5 of the PRRS virus isolates of ISU-55 One embodiment of sequence.

SEQ ID NO:18 are corresponding to be referred to as the nucleotide of the ORF6 and ORF7 of the PRRS virus isolates of ISU-55 One embodiment of sequence.

Detailed description of the invention

The present invention provides trivalent immunogenic composition, and it includes the full cellular preparations of mycoplasma hyopneumoniae (M.hyo) Soluble part；Porcine circovirus 2 type (PCV2) antigen；With porcine reproductive and respiratory syndrome (PRRS) viral antigen, wherein The soluble fraction of M.hyo prepared products combined with immunoglobulin by antigen substantially free of (i) IgG and (ii) formed it is immune multiple Close object.In one embodiment, trivalent composition causes the protectiveness for PCV2, M.hyo and PRRS virus in pig and exempts from Epidemic disease is reacted.

The insoluble fraction that applicant is surprisingly found that the full cell cultures of M.hyo is non-immunogenicity.On the contrary, M.hyo Solubilised preparations without IgG have an immunogenicity, and can effectively with from other pathogen, such as PCV2 and The antigen combination of PRRSV, and there is no Analysis interference or immune interference between antigen.This make M.hyo Solubilised preparations at For the active platform of the polyvaccine of the present invention.Applicant, which has also surprisingly found that from M.hyo prepared products, removes immune globulin White and insoluble cell debris enhances the safety of immunogenic composition.

As used in specification and claims, singulative "one", "an" and "the" are drawn including plural number With unless the other clear stipulaties of context.For example, term " proteantigen " includes multiple proteantigens, including its mixing Object.

As it is used herein, term "comprising" refers to composition and method includes addressed element, but it is not excluded for it Its element.

As herein defined, the soluble part of the full cell cultures of M.hyo, which refers to, is detaching insoluble material and base The soluble liquid fraction of the full cell cultures of M.hyo after IgG and the immune complex of antigen binding is eliminated in sheet. M.hyo soluble parts are also referred to as supernatant fraction, culture supernatant etc. in the text.It includes from insoluble egg In vain, the M.hyo- that full bacterium and other insoluble M.hyo cellular materials pass through conventional method (such as centrifugation, filtering or precipitation) separation The soluble protein (M.hyo proteantigens) of expression.In addition to including soluble protein special M.hyo-, the full cell trainings of M.hyo The soluble part for supporting object also includes the heterologous protein contained in heterologous protein, such as the culture medium that uses of those M.hyo fermentations.

Term " antigen " refer to can animal moderate stimulation generate antibody or the compound of T- cell responses or the two, composition, Or immunogenic substance, include the composition injected to animal or absorbed by animal.Immune response can be directed to entire molecule or Person generates for a part (such as epitope or haptens) for molecule.

As herein defined, " immunogenic composition or immune composition ", refers to the combination for including at least one antigen Object, the antigen causes in host occurs cell and/or antibody-mediated immune response to interested composition or vaccine Caused by immune response.

Terms used herein " immune response " refer to the reaction caused in animal.Immune response can refer to cellular immunity (CMI)；Humoral immunity may include the two.Present invention further contemplates that being limited to the reaction of an immune system part.In general, " exempting from Epidemic disease is reacted " include, but are not limited to the one or more of following effects：Specificity is for institute in interested composition or vaccine Including one or more antigens antibody, B cell, T helper cell inhibits T cell and/or cytotoxic T cell and/or yd The generation or activation of T cell.Preferably, host will show therapeutic or protective immunological reaction, to which enhancing is to new The resistance of infection and/or the clinical severity for mitigating disease.It is this to protect the disease that typically exhibited by infected host The reduction or disappearance of shape, lower virus titer proves in faster recovery time and/or infected host.

As it is used herein, term " immunogenicity " refers to be generated in host animal for one or more antigens Immune response.This immune response constitutes the base of the protective immunity for specific infectious organisms caused by vaccine Plinth.

" adjuvant " used herein refers to the composition to the substance of antigen immune response comprising one or more enhancings.Adjuvant The mechanism of action also imperfectly understand.Think that some adjuvants enhance immune response by slow release antigen, and other adjuvants are certainly Body has strong immunogenicity, and is considered as being functioned by synergistic effect.

As it is used herein, it includes to come from the same species (the i.e. difference of mycoplasma hyopneumoniae that term " multivalence ", which refers to, Isolate) or from different species (come from pasteurella haemolytica (Pasteurella hemolytica) and kill more The isolate of both Pasteurellas (Pasteurella multocida)) be more than a kind of vaccine of antigen, or comprising from The vaccine of the combination for the antigen not belonged to is (for example, comprising from Pasteurella multocida (Pasteurella multocida), sand Door Salmonella (Salmonella), Escherichia coli (Escherichia coli), Haemophilus somnus (Haemophilus Somnus) and the vaccine of the antigen of clostridium (Clostridium)).

Terms used herein " pig " or " piggy " refer to the animal of pig origin, and " sow " refers in the childbearing age and have The female of fecundity." gilt " refers to the female pigs being never pregnant.

Term as used herein " toxicity " refers to the isolate for remaining its infection ability in animal reservoir.

" inactivated vaccine " refers to the vaccine composition for including the no longer infectious organisms or pathogen of reproducible or growth.Disease The source of substance can be bacterium, virus, protozoan or fungi.Inactivation can pass through accomplished in many ways, including freeze thawing, change Processing (such as being handled with thimerosal or formalin), ultrasound, radiation, heat any other are enough to prevent the organism from replicating Or grow but retain the conventional process of its immunogenicity.

Terms used herein " variant " refer to polypeptide or encode the nucleic acid sequence of polypeptide, have one or more conservative ammonia The variation of base acid or other small modifications, so that corresponding polypeptide has basically the same work(compared with wild polypeptide Energy.

" conservative variation " indicates to replace amino acid residue with the similar residue of another biology, or replaces nucleic acid sequence In nucleotide so that coding amino acid residue is constant or the similar residue of another biology.The reality of conservative variation Example includes that a hydrophobic residue (such as isoleucine, valine, leucine or methionine) is substituted by another hydrophobicity The substitution of residue or a polar residues, such as arginine are substituted by lysine, and glutamic acid is substituted by aspartic acid or paddy Glutamine is substituted by asparagine, etc..Term " conservative variation " further includes replacing unsubstituted parent with the amino acid of substitution Amino acid, condition are also immune response to occur with unsubstituted polypeptide with the antibody of the polypeptide excitation of substitution.

Term as used herein " pharmaceutically acceptable carrier " and " pharmaceutically acceptable medium " may be used interchangeably, Refer to the liquid vehicle for including vaccine antigen that can be injected into host and be free from side effects.It is known in the art suitable Pharmaceutically acceptable carrier includes but not limited to sterile water, brine, glucose, dextrose or buffer solution.Carrier may include helping Agent, including but not limited to diluent, stabilizer (i.e. sugar and amino acid), preservative, wetting agent, emulsifier, pH buffer, viscosity Enhance additive, dyestuff etc..

Term as used herein " vaccine composition " be included at least one of pharmaceutically acceptable medium antigen or Immunogene, for inducing immune response in host.Vaccine composition with dosage form and can pass through medicine or veterinary applications skill Technology well known to art personnel is administered, and considers these factors：The condition of age, gender, weight, species and receptor, and Administering mode.Administering mode can be percutaneous, mucosal (such as oral, intranasal, per anum, Via vagina) or pass through intestines (in intradermal, percutaneous, intramuscular, subcutaneous, intravenous or peritonaeum) is administered in stomach external square type.Vaccine composition can be administered alone, or It can be with other treatments or therapy co-administered or order of administration.The form of administration may include suspension, syrup or elixir, And for parenteral, the prepared product of subcutaneous, intradermal, intramuscular or intravenous administration (such as drug administration by injection), such as sterile suspension Or lotion.Vaccine composition can be used as spraying or be blended in food and/or water and be administered, or with suitable carrier, dilution Agent or excipient (such as sterile water, physiological saline, glucose) mixing administration.Composition can include that auxiliary substances are for example wet Moisten agent or emulsifier, pH buffer, adjuvant, gelling and or viscosity strengthen additive, preservative, flavoring agent, dyestuff etc., this depends on In administering mode and desirable prepared product.It can be with the pharmacy literature of reference standard, such as " Remington's Pharmaceutical Sciences, " 1990 prepare suitable prepared product, without excessive experiment.

" North American PRRS virus " refer to with any PRRS of the relevant hereditary feature of North American PRRS virus isolate virus, Such as, but not limited to, PRRS viruses that generation nineteen ninety early stage left and right is detached for the first time in the U.S. (see, e.g., Collins, J.E., et al.,1992,J.Vet.Diagn.Invest.4:117-126)；North American PRRS virus isolate MN-1b (Kwang, J.et al.,1994,J.Vet.Diagn.Invest.6:293-296)；PRRS viruses Quebec LAF-exp91 plants (Mardassi, H.et al.,1995,Arch.Virol.140:1405-1418)；With North American PRRS virus isolate VR 2385 (Meng, X.-J et al.,1994,J.Gen.Virol.75:1795-1801).Other examples of North American PRRS virus strain are described herein.It loses It passes feature and refers to genome nucleotide sequence similitude and amino acid sequence similarity between North American PRRS virus strain.Chinese PRRS Strain usually shows the nucleotide sequence similarity of about 80-93% with American pig.

" European PRRS virus " refers to special with the relevant heredity of PRRS viruses detached for the first time in Europe with 1991 or so Sign any PRRS Strain (see, e.g., Wensvoort, G., et al., 1991, Vet.Q.13:121-130)." Europe PRRS viruses " also refer to " lelystad virus (Lelystad virus) " sometimes in the art.European PRRS virus strain Other examples are described herein.

If the virus of genetic modification is smaller than unmodified parent plant toxicity, it is " attenuation ".If strain exists It determines and shows statistical significant decrease in one or more parameters of disease severity, then the strain is that " toxicity is smaller ".This parameter may include the level of viremia virusemia, and fever, the seriousness of respiratory distress breeds the seriousness or lung of symptom Quantity or seriousness of damage etc..

" infection clones " are the separation of pathogen (such as virus) or clone genomes, can be special in laboratory Surely it and is purposefully transformed, is subsequently used for the organism of the genetic modification of reproduction live body.The work generated from infection clones The organism of the genetic modification of body can be used in live virus vaccines.Alternatively, can by with inactivator such as formalin or Hydrophobic solvent, acid etc. are prepared by ultraviolet light or x-ray radiation by live virus of the processing such as heating from infection clones The viral vaccine of inactivation.

All currently available M.hyo and M.hyo combination-vaccines are all the full cell mycoplasma prepared product (bacterium with inactivation Seedling) manufacture.On the contrary, the present invention using the full cellular preparations of mycoplasma hyopneumoniae (M.hyo) soluble part and PCV2 and PRRSV antigen combinations, the wherein soluble fraction of M.hyo prepared products are substantially free of (i) IgG and (ii) by antigen and immune globulin It is white to combine the immune complex formed.

M.hyo has absolute demand for exogenous sterol and aliphatic acid.These demands are usually required in the culture medium containing serum, Such as M.hyo is grown in Swine serum.(such as pass through from the soluble protein of the full cellular preparations of M.hyo separation insoluble material Centrifugation, filtering or precipitation) pig IgG or immune complex can't be removed.In one embodiment of the invention, with albumen-A Or albumen-G handles M.hyo soluble parts substantially to remove the IgG and immune complex that include in culture supernatant.At this In embodiment, it should be understood that albumin A processing is happened at after M.hyo fermentations.This can also be referred to as downstream egg herein White A processing.In another embodiment, the upstream protein A processing to growth medium can be used (to ferment in M.hyo Before).The parts the Fc of albumin A and IgG combine.Protein G is preferentially combined with the parts the Fc of IgG, but can also be tied with the regions Fab It closes.From crude protein mixture, such as tissue culture supernatants, in serum and ascites the method for purifying/removing total IgG be this field Know.

In some embodiments, the soluble part of M.hyo prepared products includes at least one M.hyo proteantigens. In other embodiments, the soluble part of M.hyo prepared products includes two or more M.hyo proteantigens.

In one embodiment, M.hyo supernatant fractions include one or more following M.hyo specific proteins antigens： The M.hyo albumen of about 46kD (p46), 64kD (p64) and 97kD (p97) molecular weight.In another embodiment, supernatant grade Divide and includes at least p46, p64 and p97M.hyo proteantigen.The M.hyo albumen (p64) of about 64kD herein can also be claimed For the p65 surface antigens of M.hyo, by Kim et al. [Infect.Immun.58 (8):2637-2643 (1990)] and the U.S. it is special Sharp No.5,788,962 descriptions.

Futo et al. describe clone and the characterization of the 46kD surface proteins of M.hyo, can be used in the group of the present invention Close [J.Bact 177 in object:1915-1917(1995)].In one embodiment, M.hyo culture supernatants include p46, it From P-5722 plants of corresponding nucleotide and amino acid sequence respectively such as SEQ ID NO:Shown in 1 and 2.Further consider It is that the variant of this p46 can be used in the present compositions, as described below.

Zhang et al. describe the characterization [Infect.Immun.63 of the p97 adhesin antibodies of M.hyo:1013- 1019,1995].In addition, King et al. describe the 124kD albumen for being referred to as Mhp1 of the P-5722 strains from M.hyo, And it provides statistics indicate that Mhp1 and p97 are the same albumen [Vaccine 15:25-35(1997)].This p97 albumen can be used In the present compositions.In one embodiment, M.hyo culture supernatants include p97, it from P-5722 plants Corresponding nucleotide and amino acid sequence are respectively such as SEQ ID NO:Shown in 3 and 4.It is yet further contemplated that the variant of this p97 It can use in the present compositions, as described below.

M.hyo culture supernatants can further include the specific protein antigen of M.hyo, such as, but not limited to about 41kD (p41), the albumen of 42kD (p42), 89kD (p89) and 65kD (p65).Referring to, Okada et al., 2000, J.Vet.Med.B 47:527-533 and Kim et al., 1990, Infect.Immun.58 (8):2637-2643.In addition, M.hyo culture supernatants The specific protein antigen of the M.hyo of about 102kD (p102) and 216kD (p216) can be included.Referring to Minnion et al's United States Patent (USP) Nos.6,162,435 and 7,419,806.

The solvable of the M.hyo prepared products of any M.hyo plants composition that the present invention is generated as starting material can be used Property part.Suitable M.hyo plants can obtain from commercial source or academic sources, including preservation mechanism such as American Type culture Collection (ATCC) (Manassas, Va.) and NRRL culture collections (Agricultural Research Service,U.S.Department of Agriculture,Peoria,Ill.).ATCC individually lists following six M.hyo plants for selling：M.hyo ATCC 25095,M.hyo ATCC25617,M.hyo ATCC 25934,M.hyo ATCC 27714, M.hyo ATCC27715 and M.hyo ATCC 25934D.The preferred M.hyo plants of quilts for embodiment of the present invention It is considered P-5722-3 plants, the availability rule of the requirement of ATCC#55052,1990 Mays 30 according to U.S.Patent ＆ Trademark Office Preservation is then carried out.It, can also be by causing known to Eaton agent pneumonia from by pig in view of the wide-scale distribution of disease The pulmonary secretions or tissue recycling M.hyo of the pig of strain infection obtain strain.

It should be understood by those skilled in the art that the variant of M.hyo sequences can be used in the composition of the present invention.It is this Variant can have the up to variation of 10-20% on sequence identity, and still retain and can be used in immune composition Antigenic characteristic.Preferably, the full-length genome sequence of M.hyo plant of M.hyo variants and wild type at least 80%, preferably extremely Few 85%, more preferably at least 90%, more preferably at least 95% sequence identity.The antigenic characteristic of immune composition can be with example Such as estimated by the challenge test provided in embodiment.Moreover, the antigen when modification provides and wild type M.hyo albumen phases When than at least 70%, preferably 80%, more preferable by 90% protective immunity, then the antigenic characteristic for the M.hyo antigens modified is still Retain.

In one embodiment, M.hyo solubilities p46 antigens are comprised in the composition of the present invention, final concentration It is about 1.5 μ g/ml to about 10 μ g/ml, preferably about 2 μ g/ml to about 6 μ g/ml.It should be noted that p46 is to be used for The albumen (the embodiment part that see below) of M.hyo efficacy detections.In another embodiment, M.hyo antigens can be with Final quantity is that the amount of about 5.5% to about 35% of the supernatant of the full cell culture protein A processing of M.hyo is comprised in combination In object.

The M.hyo Solubilised preparations of the present invention are safe and are effective for M.hyo, and are suitable for single agent Amount administration.In addition, applicant be surprisingly found that M.hyo Solubilised preparations can effectively with from other pathogen, Antigen combination including PCV2 and PRRS viruses, and there is no immune interference between antigen.This makes M.hyo Solubilised preparations As polyvaccine, include the active platform of the PCV2/M.hyo/PRRS combination-vaccines of the present invention.PCV2 and PRRS viral antigens Can be administered simultaneously with M.hyo compositions (be used as three individual single vaccines), but preferably M.hyo Solubilised preparations with Form of the PCV2 antigen groups together into ready-to-liquid composition.This instant PCV2/M.hyo liquid compositions are right After can be with PRRS virus antigen combinations, so that all antigens can be concurrently administered to pig.In some embodiments, PRRS viral antigens are in lyophilised state, and PCV2/M.hyo liquid compositions can be used to make the PRRS viral antigens of freeze-drying again Aquation, to form trivalent vaccine.

In one embodiment, immunogenicity PCV2/M.hyo/PRRS compositions of the invention include it is at least one its Its antigen.In one embodiment, at least one other antigen is directed to can cause the microorganism of disease to provide protection in pig.

In some embodiments, at least one other antigen component is directed to the bacterium of known infected pigs, viral or primary Animal provides protection.The example of this microorganism includes, but are not limited to following types：Pig parvoviral (PPV), the bloodthirsty bar of secondary pig Bacterium (Haemophilus parasuis), Pasteurella multocida (Pasteurella multocida), Streptococcus suis (Streptococcum suis), Staphylococcus hyicus (Staphylococcus hyicus), Actinobacillus pleuropneumoniae (Actinobacilllus pleuropneumoniae), bronchitis byrd bacteria (Bordetella Bronchiseptica), Salmonella choleraesuls (Salmonella choleraesuis), Bacterium enteritidis (Salmonella enteritidis), erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae), hog snout branch Substance (Mycoplama hyorhinis), mycoplasma hyosynoviae (Mycoplasma hyosynoviae), Leptospira are thin Bacterium, Lawsonia intracellularis (Lawsonia intracellularis), swine influenza virus (SIV), bacillus coli antigen, swine dysentery Brachyspira (Brachyspira hyodysenteriae), porcine respiratory coronavirus, pig epidemic diarrhea (PED) virus, Rotavirus, thin circovirus virus (TTV), inclusion-body rhinitis virus of pigs, chitling virus, encephalomyocarditis virus, Ao Jieshijishi diseases, typicalness Pathogenic former and transmissible gastroenteritis of swine the pathogenic original of swine fever (CSF), or combinations thereof.

In one embodiment, the PCV2/M.hyo components of trivalent vaccine according to the present invention are with instant single bottle liquid The form of composition provides.This instant combination-vaccine need not mix independent PCV2 and M.hyo univalent vaccines, therefore not have The risk of pollution without the adjoint additional effort of hybrid working, and uses the requirement of mixture not within a few houres. Moreover, single bottle PCV2/M.hyo components make waste material and refrigerator storage space halve.

In some embodiments, the PCV2 antigen components of PCV2/M.hyo/PRRS combination-vaccines are chimeric 1 type -2 type circles The form of circovirus virus.Embedded virus includes 1 type of recombinant porcine circovirus of the expression Porcine circovirus type 2 Cap protein of inactivation. Chimeric porcine circovirus and preparation method thereof also has United States Patent (USP) Nos.7,279,166 and 7,575 in 03/049703 A2 of WO, Described in 752, its full text is merged into herein by reference.

In one embodiment, the full length DNA sequence for being fitted into the genome of PCV1-2 viruses corresponds to SEQ ID NO:5 Or its variant, as described below.In another embodiment, the immunogenicity ORF2 capsid genes for being fitted into PCV1-2 viruses are corresponding In SEQ ID NO:6.In another embodiment, by the amino of the immunogenicity ORF2 albumen of chimeric PCV1-2 expressing virals Acid sequence corresponds to SEQ ID NO:7.

In yet another embodiment, the full length DNA sequence for being fitted into the genome of PCV1-2 viruses corresponds to SEQ ID NO:8.In one embodiment, the immunogenicity ORF2 capsid genes for being fitted into PCV1-2 viruses correspond to SEQ ID NO:9. In another embodiment, it is corresponded to by the amino acid sequence for being fitted into the immunogenicity ORF2 albumen of PCV1-2 expressing virals SEQ ID NO:10。

But the PCV2 ORF2 DNA and albumen of chimeric PCV1-2 viruses are not limited to above-mentioned sequence, because being detached in PCV2 In object, PCV2 ORF2 DNA and albumen are highly conserved regions.

In some embodiments, the PCV2 antigen components of M.hyo/PCV2/PRRS combination-vaccines are recombination ORF2 albumen Form.In one embodiment, recombination ORF2 albumen is expressed from baculovirus vector.Alternatively, can use it is other The expression vector known, such as include, but are not limited to Parapoxvirus vectors.

In one embodiment, the sequence of recombination PCV2 ORF2 albumen is SEQ ID NO:11, by SEQ ID NO: 12 (GenBank Accession No.AF086834) are encoded.In another embodiment, the sequence of recombination ORF2 albumen is SEQ ID NO:13, by SEQ ID NO:14 codings.In yet another embodiment, recombination ORF2 albumen corresponds to SEQ ID NO:15.In a further embodiment, recombination PCV2 ORF2 albumen corresponds to SEQ ID NO:7.In further embodiment In, recombination PCV2 ORF2 albumen corresponds to SEQ ID NO:10.

But the present invention is not limited to above-mentioned specific ORF2 DNA and albumen.Since in PCV2 isolates, PCV2 ORF2 DNA and albumen are highly conserved region, sources of any PCV2 ORF2 as PCV2 ORF2 DNA and/or polypeptide It is all particularly likely that in chimeric PCV1-2 viruses or in recombination PCV2 albumen effective.

The example of suitable PCV2 isolates for generating PCV2 ORF2 DNA and protein sequence is that PCV2 isolates are compiled Number 40895 (on December 7th, 2001 is preserved in ATCC, and specified ATCC Patent Deposits are named as PTA-3914).PCV2 isolates Genome (nucleotide) sequence of number 40895 can be obtained from GenBank sequence numbers AF264042.For generating PCV2 Other suitable PCV2 isolates of ORF2 DNA and protein sequence include, but are not limited to following type：Imp.999, Imp.1010-Stoon, Imp.1011-48121 and Imp.1011-48285.Corresponding to the genome sequence of these PCV2 isolates The GenBank sequence numbers of row are AF055391, AF055392, AF055393 and AF055394 respectively.

The immunogenic portion of PCV2 ORF2 albumen is used as the antigen component in composition in the form of certain.For example, The truncation of PCV2 ORF2 albumen and/or substitution form can be used in the present compositions.

It should be understood by those skilled in the art that the variant of PCV2 sequences can be used in the present compositions.This variant There can be the up to variation of 10-20% on sequence identity, and still reservation can be used for resisting in immune composition Former characteristic.Preferably, the full-length genome sequence of PCV2 variants and wild type PCV2 isolates has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% sequence identity.The antigenic characteristic of immune composition can be with, such as Estimated by the challenge test provided in embodiment.Moreover, the antigen when modification provides and wild type PCV2 ORF2 albumen When protective immunity compared at least 70%, preferably 80%, more preferable 90%, then the antigenic characteristic for the PCV2 antigens modified is still Retain.

The PCV2 antigen components provided in immunogenic composition effectively to induce desirable immune response, that is, reduce by The incidence of clinical symptoms caused by PCV2 infection or the antigen for mitigating its seriousness include horizontal presence.

In one embodiment, PCV1-2 viruses are fitted into and the present invention is comprised in the level of at least 1.0≤RP≤5.0 Composition in, wherein RP be by relative to reference to vaccine carry out ELISA Antigen quantitative determinations (efficacy in vitro detection) determination Relative potency (Relative Potency) unit.In another embodiment, PCV1-2 viruses are fitted into about 0.5% The composition of the present invention is comprised in the final concentration of a large amount of PCV1-2 antigens of about 5% 20- times of (20X) concentration volume In.

In another embodiment, PCV2 ORF2 recombinant proteins are at least 0.2 μ g antigens/final immunogenicity groups of ml The level (μ g/ml) for closing object is comprised in the composition of the present invention.In another embodiment, PCV2 ORF2 recombinate egg It is in vain about 0.2 to about 400 μ g/ml comprising level.In yet another embodiment, PCV2 ORF2 recombinant proteins include water It is about 0.3 to about 200 μ g/ml to put down.In a further embodiment, PCV2 ORF2 recombinant proteins are about comprising level 0.35 to about 100 μ g/ml.In a further embodiment, PCV2 ORF2 recombinant proteins are about 0.4 to big comprising level About 50 μ g/ml.

In one embodiment, trivalent immunogenic composition of the invention is anti-comprising at least one M.hyo solubilities The creative combination of former (such as two or more), porcine circovirus 2 type (PCV2) antigen and PRRS viral antigens. In another embodiment, composition causes the protective immunological reaction for M.hyo, PCV2 and PRRS virus in pig.

In one embodiment, PCV2/M.hyo/PRRS combination-vaccines are provided in the form of single dose, 2 bottles of vaccines. For example, in some embodiments, PCV2/M.hyo combinations are provided in the form of the stable liquid compositions in first bottle, PRRS Virus is provided with the lyophilised state in second bottle.In some embodiments, other pig antigens be introduced into first bottle Or in second bottle.

In one embodiment, PRRS virus components are provided in the form of live virus be lyophilized, genetic modification.To Before medicine, the PCV2/M.hyo liquid forms in first bottle can be used to make the PRRS virus rehydration in second bottle, so that All three antigens can be administered in a single dose to animal.Although should be noted that PCV2/M.hyo/PRRS combination-vaccines are current Have, they be the single dose of the administration to need to be carried out at the same time three independent vaccines, 3- bottles of vaccines in the form of offer (such as IngelvacIngelvacWithPRRS MLV)。

PRRS pathogen is separated in Holland for the first time, and is named as lelystad virus.This virus is in WO Described in 92/21375 (Stichting Centraal Diegeneeskundig Instituut), point of European PRRS virus It is deposited in Paris Pasteur's Institute, preserving number I-1102 from object.North America type is almost in European type virus purification It is separated simultaneously, and described in WO-93/03760 (Collins et al.), the isolate of North America type virus contains In American type culture collection (ATCC), preserving number VR-2332.

Different strains is isolated from Europe and North America Virus Type.WO 93/07898 (Akzo) describes Europe Strain and vaccine from it, are deposited in CNCM (Pasteur's Institute), preserving number I-1140.Moreover, WO 93/ 14196 (Rhone-Merieux) describe a kind of new strain isolated in France, are deposited in CNCM (Pasteur's Institute), Preserving number is I-1153.Moreover, EP0595436 B1 (Solvay) describe a kind of strain of new North America type, retouched than initially That toxicity stated is stronger and its vaccine.The strain has been preserved in ATCC, but does not illustrate preservation in patent application Number.In addition, ES2074950BA (Cyanamid Iberica) and its GB2282811 B2 of the same clan describe it is a kind of so-called " Spain's strain ", be different from other Europe and North America strains.It is dynamic that this " Spain's strain " has been preserved in Europe Object Cell Culture Collection (EACCC), preserving number V93070108.

Include North America PRRS diseases for the suitable PRRS viral antigens in the PCV2/M.hyo/PRRS compositions of the present invention Malicious isolate, China's PRRS Strain and the genetic modification version of European PRRS virus strain and these isolate/strains. It is North American PRRS virus for the PRRS virus antigen components in composition according to the present invention in one embodiment.

In some embodiments, it is known as P129's for the PRRS virus antigen components in the composition of the present invention North American PRRS virus isolate or its work, the version of genetic modification.Preferably, the PRRS viruses of genetic modification cannot produce Raw pathogenic infection, but the effective immunoprotection reaction for wild-type PRRS virus infection can be caused.

The PRRS viruses of genetic modification for the present composition can be generated from infection clones.It is referred to as P129's The preparation of the infectious CDNA clones of North American PRRS virus isolate is in United States Patent (USP) No.6, described in 500,662, passes through reference It is completely integrated into herein.The sequence of P129 cDNA in Genbank sequence numbers AF494042 and United States Patent (USP) No.6,500, It is disclosed in 662.

In one embodiment, the nucleotide sequence of the non-toxic forms of the P129 of the present composition is used for by SEQ ID NO:16 indicate.But the present invention is not limited to the sequences.The sequence of the sequence and other non-toxic forms of P129 is 2011 Described in the international application No.PCT/IB2011/055003 submitted on November 9, by reference by its entire contents (including base In any American National phase application that the international application is submitted) it is merged into herein.Preferably, PRRS viruses are modified to prevent The Downstream regulatory of interferon-mediation function.

In other embodiments, it is known as ISU-55's for the PRRS virus antigen components in the present composition PRRS virus isolates.ISU-55 isolates are deposited in American type culture collection (ATCC), preserving number VR2430. The nucleotide sequence of ORF2 to the ORF5 genes of ISU-55 isolates is by SEQ ID NO:17 indicate.The ORF of ISU-55 isolates The nucleotide sequence of 6 and ORF7 genes is by SEQ ID NO:18 indicate.

It can be ISU-12 with the North American PRRS virus that another kind in the composition is suitble to, be deposited in ATCC, protect Tibetan number is VR2385 [3x is Plaque-purified] and VR2386 [without Plaque-purified].Also other available in the compositions of the present invention suitable The North American PRRS virus isolate of conjunction is those listed below：ISU-51, ISU-3927, ISU-1894, ISU-22 and ISU-79, it Be deposited in ATCC, preserving number is respectively VR2498, VR2431, VR2475, VR2429 and VR2474.These ISU isolates Any genetic modification version can be used in the present compositions.These ISU isolates and ISU-55 isolates are under Paul is stated, is described in detail in the United States Patent (USP) of et al：US 5,695,766、6,110,467、6,251,397、6, 251,404、6,380,376、6,592,873、6,773,908、6,977,078、7,223,854、7,264,802、7,264,957 With 7,517,976, the full text of all these patents is merged into herein by reference.

In a further embodiment, the PRRS virus antigen components in composition according to the present invention are to be deposited in American type culture collection (ATCC), preserving number are the North America type or its genetic modification version of VR-2332.For example, PRRS viruses can be the live virus of the modification based on the isolate for being confirmed as ATCC VR2332, be used in Boehringer Ingelheim Vetmedica, Inc'sPRRS ATP andIn PRRS MLV.

In a further embodiment, it is European PRRS virus separation with PRRS viral antigens in the compositions of the present invention Object or lelystad virus or its genetic modification version.The example of suitable PRRS Strain is confirmed as above-mentioned preserving number I-1102.Nucleotide and amino acid sequence corresponding to I-1102 preserved materials Wensvoort et al United States Patent (USP) no.5, Described in 620,691, it is completely integrated into herein by reference.European PRRS virus isolate or lelystad virus The preparation of infection clones is in United States Patent (USP) No.6, described in 268,199, is completely integrated into herein by reference.

Other examples of suitable PRRS virus isolates include, but are not limited to the above.Moreover, PRRS viruses point Work from object, the version of genetic modification can be used in the compositions of the present invention.Infection clones can be used for reproduction this The organism of kind genetic modification living.

It will be appreciated by those skilled in the art that the variant of PRRS virus sequences can be used in the composition of the present invention. This variant can have the up to variation of 10-20% on sequence identity, and still reservation can be used to that combination to be immunized Antigenic characteristic in object.Preferably, PRRS viral variants and the full-length genome sequence of wild-type PRRS virus isolate have At least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% sequence identity.Immune composition Antigenic characteristic can be with, such as the challenge test by being provided in embodiment is estimated.Moreover, when the antigen of modification provides and open country When raw type PRRS viral antigens compare at least 70%, preferably 80%, more preferable 90% protective immunity, then the PRRS modified is sick The antigenic characteristic of malicious antigen still retains.

In one embodiment, PRRS virus antigen components are the live virus of genetic modification, at least 2.1≤ TCID₅₀≤ 5.2 level is comprised in the composition of the present invention, wherein TCID₅₀It is by Antigen quantitative determination (external work( Effect detection) determine 50% Tissue Culture Infective Dose.

The PCV2 antigen components of the PCV2/M.hyo/PRRS compositions of the present invention can be -2 type circovirus of chimeric 1 type Form, embedded virus include inactivation expression Porcine circovirus type 2 Cap protein 1 type of recombinant porcine circovirus.Another In a embodiment, the PCV2 antigen components of PCV2/M.hyo/PRRS compositions of the invention are the forms for recombinating ORF2 albumen.

Suitable PCV2 antigens for PCV2/M.hyo/PRRS compositions can come from any of above PCV2 points From object and other PCV2 isolates.Included, but are not limited to suitable PCV2 antigens in the compositions of the present invention above-mentioned PCV2 sequences and its variant.

The vaccine of the present invention can follow generally acknowledged convention and be prepared, to be used for animal, including people comprising acceptable (if applicable) carrier, such as standard buffer solution, stabilizer, diluent, preservative and/or solubilizer, and can be with It is configured to conducive to the form of sustained release.Diluent includes water, brine, dextrose, ethyl alcohol, glycerine etc..Isotonic additive especially includes Sodium chloride, dextrose, mannitol, sorbierite, lactose.Stabilizer especially includes albumin.Other suitable vaccine mediums and Additive, including those live vaccines for being used in particular for preparing modification, be well known to the skilled artisan or Obviously.See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing is incorporated into herein by reference.

The vaccine of the present invention can further include especially one or more other immunological regulation components, such as adjuvant Or cell factor.Include following types with suitable adjuvant in the compositions of the present invention：Oil-in-water adjuvant, polymer and Shui Zuo Agent, water-in-oil adjuvant, aluminum hydroxide adjuvant, vitamin E adjuvant and combinations thereof.The specific example of some of adjuvant includes, but not It is limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, short corynebacterium parvum, BCG vaccine, gel aluminum hydroxide, glucan, sulfuric acid Portugal It is glycan, iron oxide, sodium alginate, Bacto- adjuvants, specific synthetic polymer such as polyaminoacid and amino acid copolymer, embedding Section copolymer (CytRx, Atlanta, Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M(Chiron,Emeryville Calif.)、Adjuvant, saponin(e, Quil A or other saponin(e grades Point, monophosphoryl lipid A and Avridine (Avridine) lipid-amine adjuvant (N, N- bis- octadecyl-N', N'-- bis- (2- hydroxyl first Base)-malonamide), " REGRESSIN " (Vetrepharm, Athens, Ga.), paraffin oil, RIBI adjuvant systems (Ribi Inc., Hamilton, Mont.), muramyl dipeptide etc..

The oil-in-water emulsion that can be used for vaccine of the present invention includes SEAM62 the and SEAM1/2 preparations of improvement.Improvement SEAM62 is containing 5% (v/v) squalene (Sigma), 1% (v/v)85 detergents (ICI Surfactants), 0.7% (v/v)80 detergents (ICI Surfactants), 2.5% (v/v) ethyl alcohol, 200 μ g/ml Quil A, the oil-in-water emulsion of 100 μ g/ml cholesterol and 0.5% (v/v) lecithin.The SEAM 1/2 of improvement is containing 5% angle (v/v) Squalene, 1% (v/v)85 detergents, 0.7% (v/v) Tween, 80 detergents, 2.5% (v/v) ethyl alcohol, 100 μ g/ The oil-in-water emulsion of ml Quil A and 50 μ g/ml cholesterol.

Another example that can be used for the adjuvant of the present composition is SP- oil.As made in description and claims , term " SP oil " refers to comprising polyox-yethylene-polyoxypropylene block copolymer, squalene, polyoxyethylene sorbitol acid anhydride list The oil emulsion of oleate and buffer salt solution.Polyox-yethylene-polyoxypropylene block copolymer is surfactant, helps to suspend Solid and liquid component.These surfactants are as trade namePolymer be commercially available. Preferred surfactant is Pluronic L121, is commercially available, trade nameL-121.Usually SP fat liquor is immune stimulatory adjuvant mixture, and it includes the block copolymers of about 1-3%vol/vol, about 2-6%vol/ The polyoxyethylene sorbitol acid anhydride of the squalene of vol, the more particularly squalene of about 3-6% and about 0.1-0.5%vol/vol Monoleate, remaining is buffer salt solution.In one embodiment, SP- fat liquor in the final composition with about 1% to 25%, preferably approximately 2% to 15%, more preferably from about 5% to 12%v/v amount (v/v) exists.

It is AMPHIGEN with another example of appropriate adjuvants in the compositions of the present invention^TMAdjuvant, by being dissolved in oil Deoiled lecithin composition in (being typically light liquid paraffin).

It can be following proprietary adjuvant with the example of other adjuvants in the compositions of the present invention：Microsol DiluvacDouble emulsion adjuvant systems, Emunade adjuvants and Xsolve adjuvants.Emunade and Xsolve adjuvants are all that water packet is light The lotion of mineral oil, but Emunade includes also aluminium glue, and d, l- alpha-tocopherol acetate are a parts for XSolve adjuvants. It is ImpranFLEX with another example of appropriate adjuvants in the compositions of the present invention^TMAdjuvant (water-in-oil adjuvant).Suitable assistant Another example of agent is to be based on CarbomerAdjuvant.PreferablyAdjuvant includes934 polymer and941 polymer.

In one embodiment, adjuvant or adjuvant mixture are added with the amount of every dosage about 100 μ g to about 10mg Enter.In another embodiment, adjuvant/adjuvant mixture is added into the amount of every dosage about 200 μ g to about 5mg. In another embodiment, adjuvant/adjuvant mixture is added into the amount of every dosage about 300 μ g to about 1mg.

The amount (v/v) of the adjuvant or adjuvant mixture that are typically present in vaccine composition of the present invention is about 1% to 25%, Preferably approximately 2% to 15%, more preferably from about 5% to 12%v/v.

Other " immunomodulators " that can include in vaccine include, such as one or more interleukins, interferon or its Its known cell factor.In one embodiment, adjuvant can be cyclodextrine derivatives or polyanionic polymer, such as It is described respectively in United States Patent (USP) No.6,165,995 and 6,610,310.

It is related to the method for preparing immunogenic composition according to the present invention on the other hand.This method includes i) suitable Culture medium in cultivate the 18-144 hours times of M.hyo；Ii M.hyo cultures) are then inactivated；Iii) the training of harvest inactivation Nutrient solution, wherein the culture solution inactivated includes the full cellular preparations of M.hyo, the full cellular preparations include soluble liquid fraction And insoluble cellular material；Iv) soluble liquid fraction is detached from insoluble cellular material；V) from the soluble liquid grade of separation Divide and substantially remove IgG and antigen immunoglobulin compound, to form the soluble fraction of the full cellular preparations of M.hyo Point；And vi) then by the soluble part of the full cellular preparations of M.hyo and PCV2 antigens and PRRS virus antigen combinations. In one embodiment, step vi) include will include PCV2 antigens and M.hyo soluble parts ready-to-liquid composition with The PRRS virus antigen combinations of freeze-drying.

The example of suitable culture medium for cultivating M.hyo is PPLO meat soups (mycoplasma Mycoplasma Broth Base), when its addition When fortification substance, it can be used for detaching and cultivate mycoplasma.

In some embodiments, the growth of M.hyo cultures is inactivated up to logarithmic growth later stage, then culture. In some other embodiments, by improving pH (such as reaching about 7.8) inactivated culture.Pass through the culture that will be generated It is exposed to inactivation reagent, as binary ethylenimine (BEI) makes inactivation.The L- bromine ethylamine hydrobromides in productive culture object (BEA) in-situ preparation BEI in incubation period.Then, the pH of inactivated culture is neutralized, such as the neutralization by the way that equivalent is added The reagent of reagent is inactivated in solution.In some embodiments, inactivation reagent is BEI, and neutralization reagent is sodium thiosulfate.One In a embodiment, the pH of inactivated culture is adjusted to about 7.4 by the way that sodium thiosulfate is added.

In some embodiments, the full cellular preparations of M.hyo are detached from insoluble cellular material using conventional method Soluble liquid fraction.In one embodiment, this separation is to pass through filtration step.In another embodiment, this Kind separation is to pass through centrifugation step.In yet another embodiment, this separation is to pass through settling step.

In one embodiment, inactivation, neutralize the full cellular preparations of M.hyo with Protein A resin handled with Substantially remove IgG therein and antigen immunoglobulin compound.In other embodiments, protein G resin can quilt For substantially removing the IgG contained in soluble liquid fraction and antigen immunoglobulin compound.With albumin A or It is well known in the art that protein G resin, which removes IgG and the method for antigen immunoglobulin compound,.

According on the other hand, the method for preparing trivalent immunogenic composition according to the present invention include prepare it is above-mentioned Soluble M .hyo antigens, and itself and PCV2 antigens, PRRS viral antigens, suitable adjuvant and one or more pharmacy can be connect The carrier mixing received.This method is optionally included PCV2 antigens and soluble M .hyo antigen combinations to form bivalent composition, And then this bivalent composition is added in monovalent PRRS virus antigen combinations object to form trivalent composition.

Another aspect of the present invention is related to kit." kit " refers to the multiple components combined.In a reality It applies in scheme, kit according to the present invention includes first bottle (or other suitable containers), it includes composition, the combination Object includes the soluble part of PCV2 antigens and the full cellular preparations of mycoplasma hyopneumoniae (M.hyo), the full cell systems of wherein M.hyo The soluble part of standby object is substantially free of (i) IgG and (ii) antigen immunoglobulin compound；With second bottle, it includes PRRS viral antigens.In one embodiment, kit further comprises operation instructions.

In some embodiments, the PCV2/M.hyo in first bottle of kit is combined with ready-to-liquid composition Form provides.In other embodiments, PRRS viral antigens are the forms of the live virus of genetic modification, with the state of freeze-drying It is provided.In the present case, operation instructions will include in the liquid in first bottle combined containing PCV2/M.hyo The explanation of the tolerant PRRS virus component rehydration made in second bottle.Operation instructions further preferably include will combine first and Two bottles of content is administered to the explanation of pig.

In some embodiments, immunogenic composition according to the present invention is administered to being directed to from parent The pig of at least one antibody of M.hyo, PCV2 and PRRS virus.In other embodiments, immunogenicity group of the invention It closes object and is administered to the pig with the antibody for M.hyo, PCV2 and PRRS virus from parent.

In some embodiments, trivalent immunogenic composition according to the present invention is administered to 3 week old or bigger Piggy.But, it is considered that trivalent vaccine composition according to the present invention can be also used for carrying out to the gilt before breeding It inoculates.Road as known in the art, gilt is the female pigs being never pregnant.The gilt of inoculation can will be female by colostrum The antibody of body is transmitted to the newborn cub of nursing period.

It is yet further contemplated that the trivalent vaccine of the present invention can be used for being inoculated to kind of a swinery every year.Preferably, Trivalent vaccine according to the present invention is administered to pig (such as piggy or gilt) with single dose.In one embodiment, Polyvaccine according to the present invention need not mix independent PCV2 and M.hyo univalent vaccines, i.e. PCV2/M.hyo groups before administration Divide and is provided in the form of the instant preparation in single bottle.In another embodiment, need will be in first bottle for multivalent formulation Including divalent PCV2/M.hyo vaccines mixed with the monovalent PRRS vaccines for including in second bottle.Optionally, other antigens can To be added into any of these bottles.

In some embodiments, that immunity is initially the 2- after being inoculated with trivalent vaccine composition according to the present invention 3 weeks.In other embodiments, the duration of immunity be with trivalent vaccine composition according to the present invention inoculation after about 17-23 weeks.

Following embodiments describe preferred material according to the present invention and program.It is to be understood, however, that these embodiments are only It is provided in the illustrated manner, any of which content is all not construed as the limitation to invention entire scope.

Embodiment

Embodiment 1：PCV2 combines the mycoplasma hyopneumoniae production method of M.hyo antigens

M.hyo ferments and inactivation

Seed scale and the culture medium of antigen production press following preparations.Pig is prepared according to the explanation (i.e. 21g/L) of manufacturer Pleuropneumonia sample organism (PPLO) meat soup (BD Biosciences catalogue No.21498) derived from the heart, and by yeast extract It is prepared into 21g/L, in USP.Then yeast extract solution is added to 6.25% concentration in PPLO, is heated to 121 DEG C simultaneously>Mixture is set to sterilize within 30 minutes.Cysteine hydrochloride is prepared into 90g/L and filtration sterilization.It is added in every liter of USP water 450g dextroses, then heat sterilization, prepares dextrose solution.In order to prepare final culture medium, add into basal medium Enter 10% Swine serum, 0.01% cysteine and 1.0% dextrose is then added.Culture medium 10%v:The pig lung of v Scorching mycoplasma (P-5722-3 plants) exponential phase culture inoculation.Culture is placed in 37 DEG C, and pH and dO are kept respectively In 7.0 and 25%.In logarithmic growth late period, by binary ethylenimine (BEI), a kind of nitrogen generated by 2- bromine ethylamine hydrobromides The inactivation of heterocycle propane compounds makes culture inactivate.Specifically, extremely final concentration of by the way that 2- bromine ethylamine hydrobromides (BEA) are added 4mM is simultaneously incubated 24 hours, and pH is improved makes inactivation to 7.8.By with 1:Sodium thiosulfate and again is added in 1 molar ratio 24 hours are incubated to neutralize BEI.The culture solution of inactivation is placed in 2-8 DEG C until being further processed.

Embodiment 2：Chimeric porcine circovirus (cPCV) 1-2 production methods

By the way that the immunogenicity capsid gene of pathogenic porcine circovirus 2 type (PCV2) is cloned into non-pathogenic pig annulus CPCV1-2 is built in the genome skeleton of viral 1 type (PCV1).The building process of chimeric DNA clone is for example in United States Patent (USP) No.7 described in 279,166, is incorporated herein by reference in its entirety.The infectious stock of embedded virus from Dr.X.J.Meng,Virginia Polytechnic Institute and State University,Blacksburg,VA Place obtains, and is being added to 0.05% lactalbumin hydrolysate (LAH), 30 μ g/mL gentamicin sulphates and 5% tire for infecting - 15 cell of pig kidney (PK) grown in the minimum essential medium (MEM) of cow's serum.The PK-15 of obtained cPCV1-2 infection is thin Born of the same parents are expanded for 4 times using identical growth medium by continuous passage, in addition to containing 2-3% fetal calf serums in culture medium. 5th passage is frozen, melts and filters, obtained lysate be used for prepare before main seed (pre-master seed) and Subsequent main seed.

For producing the culture medium of viral seed such as use in the viral stock of production.For growth medium, MEM, OptiMEM or equivalent are to can be used for cultivating the basal medium that PK-15 cell lines are grown.Growth medium can be with Add most 10% cow's serum, most 0.5% lactalbumin hydrolysate, most 0.5% bovine serum albumin(BSA) and at most The gentamicin of 30 μ g/mL.For virus breeding culture medium, MEM, OptiMEM or equivalent are used.Virus breeding culture medium can With add most 0.5% lactalbumin hydrolysate, most 2% cow's serum, most 0.5% bovine serum albumin(BSA)s and at most The gentamicin of 30 μ g/mL.Most glucose of 5g/L and the L-Glutamine of most 5mmol/L can be added into as needed To in growth medium and/or virus breeding culture medium to maintain cell.

The main seed virus of cPCV1-2 is added into the cell suspension of PK-15 cells and adsorbs most 3 hours.Seed disease Poison is diluted in growing basal medium to provide the infection multiplicity (MOI) of 0.1-0.0001.

PK-15 cell cultures in cell seeding, or cell reach about 20% to 50% converge when with work kind Sub- virus carries out initial inoculation.This initial passage can be referred to as " step infects method ", for generating antigen stock, Or continuous passage can be further used in.For continuous passage, the PK-15 cells of cPCV1-2 infection pass through with 1:5-20 Ratio continuously division further expanded to the 7th generation to carry out virus breeding.It is outstanding containing the infection cell for coming from previous generation The culture medium of liquid is as follow-on seed object.The cell that cPCV1-2 per a generation infects incubates 3-14 at 36 ± 2 DEG C It is until cell reaches >=90% and converges.In virus replication, cPCV1-2 viruses cause observable cell characteristic of disease to change Become.When harvest, cell rounding and the fragment of considerable floating are observed.Culture is also observed by bacterium or fungal contamination Tangible proof.Incubative time between the harvest of cPCV antigens provides in following tables 1：

Table 1 harvests the most short and maximum duration of cPCV antigens

CPCV1-2 culture solutions are harvested into sterile chamber, and sampling carries out detection of mycoplasma with known method.Repeatedly receive Obtaining can be carried out with roller bottle, bioreactor and perfusion container.

Before the cPCV1-2 viruses to harvest inactivate, (such as most 60X) one can be concentrated by ultrafiltration Or multiple antigen batches.Concentrate can be with the brine of balance to reduce haemocyanin.

CPCV1-2 inactivation of virus, attenuation or the method for detoxification will now be described.After the concentration of cPCV antigens, to merging Beta-propiolactone (BPL) is added in cPCV1-2 viral materials, reaches the concentration of about 0.2%v/v.Then by combined virus liquid Body stirs minimum 15 minutes, then goes to a large amount of antigen liquid of inactivation in second sterile chamber.The antigen liquid being transferred Body is maintained at 2-7 DEG C, continuously stirs, and continues minimum 24 hours.After minimum 24 hours, it is added for second into combined suspension The BPL of 0.2%v/v.It is subsequently agitated for content, and is gone in third container, 2-7 DEG C is maintained at, continuously stirs, is in addition continued No less than 84 hours.In general, the time of complete inactivation is no less than 108 hours and is no more than 120 hours.Ablation method is summarised in In following table 2.

2 ablation method of table

Inactivation is terminated by the way that hypo solution of the final concentration no more than 0.1M is added.The pH of inactivated antigen storage is used NaOH or HCl is adjusted to about 6.8.After inactivation, taking-up representative sample is detected the completeness of inactivation from consolidated material. The cPCV1-2 antigen products of inactivation are standardized to meet target of effect ELISA measurement more than 1.0RP.

Embodiment 3：The downstream processes of M.hyo antigens are detected with these by the analysis of processing antigen

The downstream processes of M.hyo antigens：

For the group pointed by each, processing inactivation fermented liquid as described below (such as prepared by above-described embodiment 1).These Processed M.hyo antigens are used in following embodiments 4.

T02：(entirety) is untreated.

T03：(10X UF concentrations) is concentrated by 100KDa molecular cut offs film (doughnut) through tangential flow filtration.Finally Volume reduce be equal to 10X.

T04 & T05：(10X UF are concentrated and centrifuged) is collected the bovis cells (coming from T03) of concentration and is passed through with PBS It washed once in~20,000xg centrifugation (Sorvall model RC5B).

T06 & T07：(10X centrifugations) inactivate fermentation fluid~20,000xg centrifugation (Sorvall RC5B) and pass through by Cell is resuspended in PBS and then centrifuges again washed once.Final volume, which is reduced, is equal to 10X.

T08：(10X is centrifuged and heated) concentrates according to T06 and washs bovis cells, and is heated to 65 DEG C and is kept for 10 points Clock.

T09：The supernatant that (cell free supernatant) is collected by centrifugation for the first time as described in T06 passes through 0.2 micron of filter (Nalgene) it is sterile filtered.

T10：(cell free supernatant-albumen-A processing) sterile supernatant (being prepared according to T09) and Protein A resin (albumin A Sepharose, Pharmacia Inc) with 10:1 volume ratio mixes 4 hours.It is sterile filtered and removes resin, and by the liquid of filtering Body is stored in 2-8 DEG C.This processing has used " downstream " albumin A after fermentation to handle to remove antibody and immune complex.Though The right present invention is not excluded for upstream protein A processing, the inventors discovered that in the case of M.hyo, the upstream protein A of growth medium Processing causes the result of p46 relatively low and inconsistent compared with untreated culture medium (data are not shown).

The analysis of M.hyo downstream processes antigens detects

Returning for M.hyo specificity p46 antigens is carried out to the M.hyo antigen preparations (preparing as described above) of downstream processes It receives, the existing detection of PCV2 antibody.In addition, carrying out the existing inspection of thin circovirus virus (TTV) to these M.hyo antigen preparations It surveys, including genotype 1 (g1TTV) and genotype 2 (g2TTV).As a result it is shown in following Table 3.

The characterization of 3 M.hyo downstream processes antigens of table

About above-mentioned table 3, M.hyo specificity p46 antigen quilts are proved for each M.hyo downstream processes antigen preparation Recycling.In addition, following successfully remove PCV2 antibody：10X UF are concentrated and are centrifuged, 10x centrifugation, 10X centrifuge and heat with Cell free supernatant (albumen-A processing).It is following successfully to remove g1TTV about TTV：10X UF are concentrated and are centrifuged, 10x from The heart and heating and cell free supernatant (albumen-A processing).Only it is referred to as the processing removing g2TTV that 10X UF are concentrated and centrifuged. Its full text is merged into this by ringlet virus isolates (including genotype 1 and 2) described in US20110150913, by reference Text.

Due to albumin A combination IgG known in the art, those of ordinary skill in the art should understand that not only PCV2 is anti- Body, also other pig antibody, including PRRS antibody, HPS antibody and SIV antibody will effectively be removed by albumen-A processing.This makes The M.hyo supernatants for obtaining acellular albumen-A processing of the invention are not only compatible with PCV2 antigens, also compatible with other pig antigens, Reason is do not have immune interference between antigen.In addition, the removal of non-protective cell fragment and immunoglobulin and antigen/ The removal of immunoglobulin complex can be prepared safer vaccine by rational expectation.

Embodiment 4：The preparation of M.hyo tentative bacterin preparations

All tentative M.hyo vaccines are prepared with final concentration of 5% Amphigen (Amphigen) adjuvant.In addition, all Vaccine is standardized with p46ELISA and uses thimerosal anti-corrosion.Examination is prepared with the M.hyo antigens handled according to above-mentioned processing T02-T10 The property tested vaccine preparation.In addition, processing T01 corresponds to placebo (no M.hyo antigens, only 5% Amphigen adjuvant), but handle T11 is positive control, corresponding to it is overdue based on vaccine M.hyo vaccines (Pfizer Animal Health).These preparations are described in following table 4.

4 M.hyo tentative bacterin preparations of table

* experiment veterinary drug (IVP) series

Embodiment 5：The assessment of the internal effect of the M.hyo vaccines of M.hyo antigens with different downstream processes

The research is carried out to evaluate the mycoplasma hyopneumoniae (M hyo) of the M.hyo antigens with different downstream processes (DSP) The internal effect of vaccine.The intramuscular different vaccine preparations being vaccinated described in the above-mentioned table 4 of single dose of the pig of 3 week old.Each Processing group includes 16 animals.It carries out attacking poison with the fields toxicity M.hyo isolate within 21 days after inoculation.28 days are attacked after poison to dynamic Object is autopsied, and takes out lung and pair infects consistent consolidation with M.hyo and scores.Poison is attacked for M.hyo, and protection is provided Main standard be lung's consolidation scoring.Generally acceptable is the size of the injury of lungs caused by enzootic pneumonia There is relationship with the adverse effect in growth rate.Following table 5 includes that the injury of lungs of each processing group scores.Pass through the lung to every group Scoring carries out mixed model analysis and determines significance,statistical.

5 injury of lungs result of table

About above-mentioned table 5, the M.hyo antigens of different downstream processes the result shows that all tentative other than T04 Vaccine is all dramatically different with placebo.The result of these M.hyo damages is indicated in Fig. 1 in a manner of scheming.As shown in Figure 1, T04 Give unacceptable result.All other processing is all dramatically different with placebo (T01).Pulmonary consolidation scoring shows T02, T03 and T09-T11 attacks poison for M.hyo and provides most effective protection.

Use the p46 relative potencies of double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) evaluation test vaccine. The p46DAS shown in above-mentioned table 5 ELISA's the result shows that all tentative vaccines have been above targeted efficacy.In addition, p46 Relative potency more than one month vaccine storage during be kept or increased (data are not shown).In the antigen of centrifugation In observe as time goes by, effect have noticeable increase, in addition to those by heating vaccine.However it is not intended to It being bound by any theory, in the case where centrifuging antigen, it is likely that cell " corpse " ruptures with time going by, from And release more film combination p46 antigens.

Embodiment 6：The evaluation of tentative M.hyo vaccines and PCV2 antigen compatibilities

The tentative vaccines of M.hyo and PCV2 that the research is carried out to evaluate the M.hyo antigens with different downstream processes are anti- Former compatibility.M.hyo tentative bacterin preparations are as described in above-mentioned table 4 and 5.The p46 of these observed vaccines is with respect to work( Effect is as described in above-mentioned table 5.The tentative vaccines of these M.hyo each with PCV2 antigen combinations.In this embodiment, PCV2 antigens It is -2 type embedded virus of 1 types of PCV (Fostera PCV) of inactivation prepared as described in above-described embodiment 2.Embedded virus is wrapped Containing in the composition, initial level is about 1.6≤RP, and wherein RP is that (efficacy in vitro is examined by PCV2ELISA Antigen quantitative determinations Survey) it is identified relative to the effective relative potency unit for referring to vaccine.

Tentative M.hyo/PCV2 combination preparations are evaluated by PCV2ELISA.As a result it shows in fig. 2.Such as Fig. 2 institutes Show, the M.hyo antigen preparations of only following downstream processes are compatible with PCV2 antigens：Ultrafiltration simultaneously centrifuges (T04＆T05), centrifugation (T06＆T07), supernatant (T10) of the centrifugation plus heating (T08) and albumin A processing.Among them, with comprising embedded virus and Amphigen adjuvant, but the placebo without M.hyo antigens compares, the supernatant and PCV2 antigens most phase of the processing of M.hyo albumin As Hold.Compared with the 1.69RP of placebo, the chimeric PCV virus levels in the supernatant of albumin A processing are 1.5RP.It speculates There is no or only minimum exempt between the M.hyo insoluble antigen preparations and the PCV2 antigens of embedded virus of albumin A processing Epidemic disease is interfered.

The internal effect of the M.hyo supernatants of the albumen-A processing proved in above-described embodiment 5 and the present embodiment description The result shows that the supernatant of albumen-A processing is the active platform of potential M.hyo-PCV2 combinations.

Embodiment 7：The evaluation of the PCV2 effect of the single bottle PCV2/M.hyo combination-vaccines of different adjuvant prescriptions

Design the research and be the PCV2 effect of the single bottle PCV2/M.hyo combination-vaccines in order to evaluate different adjuvant prescriptions. In the present embodiment, PCV2 antigens are -2 type embedded viruses of 1 types of PCV (Fostera PCV) of inactivation.Embedded virus with substantially not M.hyo insoluble antigen preparations (i.e. the supernatant of albumin A processing) combination containing IgG.

The processing of liquid：

For the group pointed by each, the M.hyo zymotic fluids (described in above-described embodiment 1) of inactivation are as described below It is handled.

T02-T04：Whole fermentation broth (above-mentioned) containing mycoplasma hyopneumoniae cell living is in~20,000xg centrifugations (Sorvall RC5B) collects supernatant and is sterilized by 0.2 μM of filter.RProtein A Sepharose (part number 17- 5199-03, GE Healthcare) it is loaded into the chromatographic column of 1L.Remove storage buffer solution and with the 1M's of 2 times of column volumes After acetic acid processing, with the 50mM NaPO4/1M NaCl buffer solutions of 5 times of volumes, pH 7.04 balances resin.Make about 2 liters of clarifications / filtering the liquid containing mycoplasma hyopneumoniae antigen Protein A resin is flowed through with the flow velocity of 100cm/hr.Collect efflux simultaneously It is sterilized by 0.2 μM of filter.

T05：This is corresponding to the positive control of Fostera PCV- samples preparation (being free of M.hyo antigens).This Fostera The level of embedded virus is approximately minimum immune dosage (MID) preparation level in PCV- sample preparations.Embedded virus is with similar system Agent level is comprised in the tentative vaccines of PCV2/M.hyo.

All tentative PCV2/M.hyo vaccines are prepared with different adjuvant prescriptions.With according to above-mentioned processing T02-T04 The M.hyo antigens handled prepare tentative bacterin preparation.In addition, processing T01 corresponds to placebo (Sterile Saline).

All vaccines are standardized with p46ELISA and use thimerosal anti-corrosion.

Described in following table 6, wherein symbol * is indicated at complete M.hyo seeds, albumin A these tentative preparations The M.hyo antigens of supernatant are managed, symbol * * indicate experiment veterinary drug (IVP) series.

Table 6 is used for the PCV2/M.hyo tentative bacterin preparations of PCV2 effect research

Different vaccine preparations described in above-mentioned table 6 of the pig of 3 week old by intramuscular inoculation single dose.In each processing group Including 16 animals.Animal is carried out with toxicity PCV2 field isolates to attack poison within 21 days after inoculation.

Fig. 3 is the figure for showing the PCV2 viremia virusemias result (PCV2 quantitative PCRs) observed with different adjuvant platforms.It should Note that PCV2 viremia virusemias are used as main efficacy variable.PCV2 viremia virusemia results are shown as DNA copy number/ml.Such as figure Shown in 3, the 28th, 35 and 42 day (it is the 21st day to attack poison), all processing all significantly reduced viral blood compared with placebo Disease.At the 28th and 35 day, 10%SP- oil adjuvants substantially reduced viremia virusemia compared with 5% Amphigen.At the 28th and 35 day, 5% Amphigen substantially reduces viremia virusemia plus 5%SLCD adjuvants compared with 5% Amphigen.At the 28th, 35 and 42 day, 20%SLCD Adjuvant platform substantially reduces viremia virusemia compared with 5% Amphigen.

Also monitoring PCV2 serology, PCV2 excrement falls off, PCV2 noses fall off, cell-mediated immunity (CMI) reaction, lymph damage Consumption and immunohistochemistry (IHC) are used as the second efficacy variable.These results will be described below.

Fig. 4 is shown in the figure of the PCV2ELISA results of the 1st of research the, 20 and 42 day (it is at the 21st day to attack poison).Each The state of sample is expressed as sample to positive ratio (S/P).As shown in figure 4,20%SLCD is uniquely at the 20th day and the 42nd Its dramatically different processing with placebo (T01).Moreover, at the 20th day, 5% Amphigen is uniquely not notable with placebo Different processing.

Fig. 5 is the figure for showing the PCV2 excrement that T02-T04 processing is obtained relative to placebo (T01) and falling off.These results It is expressed as PCV2DNA copy numbers/ml.It is in Fig. 5 the result shows that at the 42nd day, all processing all substantially reduce compared with placebo Excrement falls off.In addition, at the 42nd day, 5% Ai Feijin ＆5%SLCD (T04) substantially reduces excrement compared with 5% Amphigen (T03) It falls off.Other processing differences are not found.

Fig. 6 is the figure for showing the PCV2 noses that T02-T04 processing is obtained relative to placebo (T01) and falling off.These result tables It is shown as PCV2DNA copy numbers/ml.It is in Fig. 6 the result shows that at the 42nd day, all processing all substantially reduce nose compared with placebo It falls off.In addition, at the 42nd day, 20%SLCD (T05) substantially reduces nose compared with 5% Amphigen (T03) and falls off.Do not find Other processing differences.

Fig. 7 (A＆B) is the interferon-γ (IFN-γ) that display measures PCV2- specific cell mediated immunities (CMI) response The figure of testing result./ after attacking (Fig. 7 A) before poison and being inoculated with/the CMI results of (Fig. 7 B) after poison are attacked after display inoculation.In these figures In, 5x 10⁶The stimulation of a cell is considered as significant (...).All tentative vaccines of PCV2/M.hyo provide detectable Inoculation after IFN-γ response.10%SP- oil (T02) drives IFN-γ response after strongest inoculation.20%SLCD (T05) is lured It leads and replys earlier, but it is minimum in response in the 20th day.Have and largely attack response after poison, is especially observed in placebo 's.In addition, in the pig processing group of inoculation attack poison after response it is relatively low compared with placebo.

Following table 7 shows that compared with placebo, the lymph obtained with tentative processing consumes.

7 PCV2 histopathologies of table (lymph consume)

It is being shown in above-mentioned table 7 the result shows that all vaccines to lymph consume strong protective effect is all provided.Moreover, not having Observe statistically significant vaccine processing comparison.

Following table 8 is shown compared with placebo, with the immunohistochemistry of tentative processing acquisition.

8 PCV2 histopathologies (immunohistochemistry) of table

It is in table 8 the result shows that all vaccines provide strong protective effect to the PCV2 proved by immunohistochemistry the groups that found a capital. Moreover, statistically significant vaccine processing comparison is not observed.

In short, the result of the present embodiment proves that M.hyo insoluble antigen preparations do not interfere the effect of PCV2.As a result table is gone back Bright all PCV/M.hyo tentative bacterin preparations provide the effect that poison is attacked for PCV2.In addition, the results showed that matched with different adjuvants Side obtains some statistical and numerical value differences, and 10%SP- oil generates strongest effect.

Embodiment 8：The evaluation of the M.hyo effect of the single bottle PCV2/M.hyo combination-vaccines of different adjuvant prescriptions

Design the research and be the M.hyo effect of the single bottle PCV2/M.hyo combination-vaccines in order to evaluate different adjuvant prescriptions. M.hyo antigens and pig circular ring virus (- 2 type chimera of 1 type or PCV1-2, inactivation of viruses) are combined in single bottle.

The processing of liquid：

For the group pointed by each, the M.hyo zymotic fluids (described in above-described embodiment 1) of inactivation are as described below It is handled.

T02-T04：Its processing is identical as described in processing group T02-T04 in above-described embodiment 7.

T05：Matched with the M.hyo cells (M.hyo vaccines) of the inactivation described in 1 title of above-described embodiment " fermentation and inactivation " System.

All tentative PCV2/M.hyo vaccines are prepared with different adjuvant prescriptions.With according to above-mentioned processing T02-T04 The M.hyo antigens handled prepare tentative bacterin preparation.In addition, processing T01 corresponds to placebo (Sterile Saline).Processing T05 is corresponding to overdueVaccine, the vaccine (Pfizer Animal Health) based on M.hyo vaccines Positive control.

Described in following table 9, wherein symbol * is indicated at complete M.hyo seeds, albumin A these tentative preparations The M.hyo antigens of supernatant are managed, symbol * * indicate experiment veterinary drug (IVP) series.

The PCV2/M.hyo tentative bacterin preparations for M.hyo effect research of the different adjuvant prescriptions of table 9

Different vaccine preparations described in above-mentioned table 9 of the pig of 3 week old by intramuscular inoculation single dose.Placebo and 10% Include 14 animals in SP- oil groups, positive controls include 13 animals, 5% Amphigen and 5% Amphigen+5% Include 16 animals in SLCD groups.

Animal is carried out with toxicity M.hyo field isolates to attack poison within 21 days after inoculation.It is autopsied within 28 days after attacking poison, It takes out lung and pair infects consistent consolidation with M.hyo and score.Following table 10 includes that the injury of lungs of each processing group scores.It is logical It crosses and the determining significance,statistical of mixed model analysis is carried out to every group of lung's scoring.

10 M.hyo injury of lungs of table

Processing	# animals	LS is averaged injury of lungs	Injury of lungs range %
				Placebo (T01)	14	13.1%	0.1-50.5
10%SP- oil (T02)	14	4.3%	0.0-50.8
				5% Amphigen (T03)	16	4.7%	0.0-38.5
5% Amphigen+5%SLCD (T04)	16	12.0%	0.1-55.8
				Overdue RSO (T05)	13	2.28%	0.0-34.5

As shown in Table 10 above, the 10%SP- oil to score with the Mean pulmonary for being respectively provided with 4.3% and 4.7% and 5% love Luxuriant and rich with fragrance gold processing group is compared, and placebo scores with 13.1% average injury of lungs.10%SP- oil and 5% Amphigen preparation are equal Reduce and/or prevent injury of lungs.Therefore, the tentative PCV/M.hyo vaccines prepared with 10%SP- oil or 5% Amphigen are recognized To be effective.PCV2 antigens seem that the M.hyo effect of these preparations will not be interfered.

On the contrary, 5% Amphigen+5%SLCD groups score with 12.0% average injury of lungs, this is unacceptable knot Fruit, because of no difference compared with placebo.Therefore, not with the PCV/M.hyo vaccine preparations of 5% Amphigen+5%SLCD preparations It is considered effective.

It should be noted that due to the medium-altitude changeability of size of animal and injury of lungs scoring of reduction, do not have in our current research Statistical treatment effect proves with being determined.For this reason, determine another research of design to detect in 10%SP- oil The tentative preparations of PCV/M.hyo M.hyo effect.This repetition research is described in following embodiments 9.

Embodiment 9：The evaluation of the M.hyo effect of single bottle PCV2/M.hyo combination-vaccines in 10%SP- oil

This research is Proof of Concept, is designed to evaluate by the M.hyo of the different removals for carrying out IgG using albumin A Four kinds of tentative PCV2/M.hyo vaccines prepared by manufacturing method (serial number L0711RK11, L0711RK12 in following table 11, L0711RK13 and L0711RK14) M.hyo fraction effect compared with the control vaccine prepared with standard M.hyo manufacturing methods. Four kinds of tentative PCV2/M.hyo vaccines each all include 10%SP- oil as adjuvant.

The processing of liquid：

T02：The mycoplasma hyopneumoniae antigen of inactivation as described in above-described embodiment 1 " fermentation and inactivation ".

T03 and T04：It is prepared with the mycoplasma hyopneumoniae cell of the inactivation described in above-described embodiment 1 " fermentation and inactivation ".

T05：Albumin A processing for the culture medium for growing mycoplasma hyopneumoniae.According to the explanation (i.e. 21g/L) of manufacturer PPLO (derived from Pigs Hearts) is prepared, and yeast extract is prepared into 21g/L, in USP.Then by yeast extract solution with 6.25% concentration is added in PPLO, and being heated to 121 DEG C and >=30 minutes makes mixture sterilize.It prepared by cysteine hydrochloride At 90g/L and filtration sterilization.450g dextroses are added in every liter of USP water, then heat sterilization, prepare dextrose solution.For Prepare final culture medium, 10% Swine serum be added into basal medium, be then added 0.01% cysteine and 1.0% dextrose.By handling the antibody removed in complete PPLO culture mediums with albumin A.In simple terms, by 1 liter RProtein A Sepharose (part number 17-5199-03GE Healthcare) are loaded into glass column (10X 11.5cm) In.After removing storage buffer solution, column is handled with the acetic acid of the 1M of 2 times of column volumes.With 50mM NaPO4, the 1M NaCl of 5 times of volumes Buffer solution (pH 7.0) balances resin.15 liters of complete PPLO culture mediums were loaded into resin with 140cm/ hours linear flow rates On.It collects column efflux and is sterilized by 0.2 micron membrane filter (Sartorius).Processed culture medium be used to breed as above State the mycoplasma hyopneumoniae cell described in " fermentation and inactivation ".Whole inactivated cultures (including cell) are configured to final epidemic disease Seedling.

T06：Such as above-described embodiment 1 " fermentation and inactivation " the mycoplasma hyopneumoniae cell for preparing inactivation.The hair of inactivation For zymotic fluid in~20,000xg centrifugations (Sorvall RC5B) 30 minutes, supernatant passed through 0.2uM filtration sterilizations.115ml's RProtein A resins (part number 12-1279-04, MAbSelect, GE Healthcare) are loaded into chromatographic column (5x6cm) In.It is slow with the 50mMNaPO4/1M NaCl of 5 times of volumes after removing storage buffer solution and being handled with the acetic acid of the 1M of 2 times of column volumes Fliud flushing, pH 7.01 balance resin.Make about 1.2 liters it is clear/filtering the liquid containing mycoplasma hyopneumoniae antigen with The flow velocity of 120cm/hr flows through Protein A resin.It collects efflux and is sterilized by 0.2 μM of filter.

T07：Such as above-described embodiment 1 " fermentation and inactivation " the mycoplasma hyopneumoniae cell for preparing inactivation.By tangential Flowing through filter makes the zymotic fluid of inactivation clarify.In simple terms, the polyether sulfone filter (GE with 0.2 μM of normal pore size HealthCare, part number 56-4102-71) it is sterilized with 0.5N sodium hydroxide solutions, then largely rinsed with sterile USP water.It will The mycoplasma culture solution of inactivation introduces the device, transmembrane pressure 2- with the recirculation rate that target is 14.6L/ minutes 3.4PSI.It is clarified in room temperature.It collects filtering permeate and is stored in 2-8C until being further processed.115ml's RProtein A resins (part number 12-1279-04, MAbSelect, GE Healthcare) are loaded into chromatographic column (5x6cm) In.After removing storage buffer solution and being handled with the acetic acid of the 1M of 2 times of column volumes, with the 50mM NaPO4/1M NaCl of 5 times of volumes Buffer solution, pH 7.01 balance resin.Make about 2.3 liters it is clear/filtering the liquid containing mycoplasma hyopneumoniae antigen with The flow velocity of 120cm/hr flows through resin.It collects efflux and is sterilized by 0.2 μM of filter.

T08：Such as above-mentioned " fermentation and inactivation " the mycoplasma hyopneumoniae cell for preparing inactivation.The zymotic fluid of inactivation~ 20,000xg centrifugations (Sorvall RC5B) 30 minutes, supernatant passes through 0.2uM filtration sterilizations.The rProtein A resins of 115ml (part number 17-5199-03, GE Healthcare) is loaded into chromatographic column (5x6cm).Remove storage buffer solution and with 2 times After the acetic acid processing of the 1M of column volume, with the 50mM NaPO4/1M NaCl buffer solutions of 5 times of volumes, pH 7.01 balances resin.Make The liquid containing mycoplasma hyopneumoniae antigen of about 1.2 liters of clear/filterings flows through albumin A tree with the flow velocity of 120cm/hr Fat.It collects efflux and is sterilized by 0.2 μM of filter.

With the M.hyo antigen test preparation vaccines handled according to above-mentioned processing T02-T08.T02, T03 and T04 are corresponded to Positive control.In addition, processing T01 corresponds to placebo (Sterile Saline).

These tentative preparations are described in following table 11.M.hyo antigens, which correspond to, comes from complete M.hyo seeds, albumin A The M.hyo antigens of the supernatant of processing.Information during " albumin A processing " one arranges indicates that M.hyo supernatants are before sterilization or after sterilizing It is no to be handled with albumin A.

The PCV2/M.hyo tentative bacterin preparations for M.hyo effect research in 11 SP- oil adjuvants of table

Different vaccine preparations described in above-mentioned table 11 of the pig of 3 week old by intramuscular inoculation single dose.Each processing group Include 18 animals.Animal is carried out with toxicity M.hyo field isolates to attack poison within 21 days after inoculation.Corpse is carried out after attacking poison within 28 days Body is dissected, and is taken out lung and pair is infected consistent consolidation with M.hyo and scores.Fig. 8 (A＆B) shows the injury of lungs of each processing group Scoring.Significance,statistical is determined by carrying out mixed model analysis to every group of lung's scoring.

Injury of lungs in Fig. 8 A and 8B is the result shows that in all processing, 100% pig of only two groups (T07 and T08) It belongs to<The classification of 5% injury of lungs.It should be noted that observing strong significant difference in our current research.

Result in the present embodiment proves the M.hyo supernatants for using albumin A to handle and uses SP- oil as the single bottle of adjuvant The tentative preparations of PCV2/M.hyo have significant M.hyo effect.In addition, above-described embodiment 7 is proved using albumin A processing M.hyo supernatants simultaneously use SP- oil as the PCV2 effect of the tentative preparations of single bottle PCV2/M.hyo of adjuvant.In general, The effect of both M.hyo and PCV2 is demonstrate,proved in the single bottle PCV2/M.hyo combinations of the M.hyo supernatants handled using albumin A It is bright.

Embodiment 10：The internal safety of tentative PCV2/M.hyo experimental vaccines

Row this research to evaluate the animal for giving minimal ages (3 week old) when, different adjuvant prescriptions with maximum antigen-agent Measure internal safety of the tentative PCV2-M.hyo vaccines prepared in host animal.Based on temperature, injection site reaction and Clinical observation evaluates different adjuvant platforms to determine which platform provides acceptable safety.20%SLCD/10%SP- oil Preparation is used as positive (" unsafe ") control, this is because once there is the note once observed by this research group and other people Penetrate the historical events of position reaction.

The processing of liquid：

All vaccines are prepared with the inactivation mycoplasma hyopneumoniae antigen described in " fermentation and inactivation " in embodiment 1.M.hyo Whole antigen is used, because it is known that it by albumin A in addition to comprising will handle the immunoglobulin that is removed and immune multiple It closes except object, also includes soluble and insoluble M.hyo antigens.Reasonably infer insoluble cell debris and immunoglobulin and The removing of immune complex can only further enhance the safety of vaccine preparation.Object of this investigation is scrupulously to detect to include The safety of the different adjuvant formulations of PCV2 antigens and M.hyo antigens.PCV2 and M.hyo antigens with maximum emission levels prepare with Further assess safety.These tentative preparations are described in following table 12.IVP indicates experiment veterinary drug (IVP).

Table 12 is used for the PCV2/M.hyo tentative bacterin preparations of safety research

* M hyo antigens=come from complete M hyo seeds (whole antigen).

The security parameters used in this research are rectal temperature distribution and injection site reaction.Result of the study demonstrate that Acceptable safety is all provided according to all candidate adjuvant platforms of rectal temperature distribution and clinical observation (result is not shown).Only There is 20%SLCD+10%SP- oily (i.e. positive control) to be markedly different from placebo vaccine and there are many serious injection sites It reacts (result is not shown).

Embodiment 11：The preparation for the M.hyo antigens that albumin A for important research is handled

Fig. 9 is flow chart, and display is used to prepare an embodiment of the M.hyo antigens of the compatible albumin A processing of PCV2. The full cultures of M.hyo of inactivation carry out cell clarification by tangential flow filtration.In simple terms, poly- with 0.45 μM of normal pore size Ether sulfone filter (GE HealthCare, part number 56-4102-49) is sterilized with 0.5N sodium hydroxide solutions, then with sterile USP Water largely rinses.The mycoplasma culture solution of inactivation is introduced into the device, cross-film with the recirculation rate that target is 11.0L/ minutes Pressure is~5PSI.It is clarified in room temperature.It collects filtering permeate and is stored in 2-8 DEG C until being further processed.

After clarification, the liquid containing antigen is handled with Protein A resin to reduce antibody level.In simple terms, MAbSelect Protein A resins (GE Healthcare) are loaded into the glass column that height is 12cm.With 5 times of column volumes 50mM sodium phosphates, 250mM NaCl buffer solutions (pH 7.0) balance resin.The antigen of 10 times of column volumes be would be equivalent to 100cm/ The linear flow rate of hour is loaded on resin.It collects efflux and is sterilized by 0.2 micron membrane filter.By flowing through 3 times of column volumes 25mM pH3.7 acetate solution, then passing through the acetic acid solution of the 1M of 4 times of column volumes makes column regeneration.Pass through PCV2 respectively Specific antibody ELISA and p46 antigen quantitative ELISA measures anti-PCV2 antibody and mycoplasma hyopneumoniae in final antigen liquid Antigen levels.

Embodiment 12：For the evaluation of the viricidal activity of PRRS viruses

Research in the present embodiment is designed to evaluate the viricidal activity that different adjuvant platforms are directed to PRRS viruses.Most First experiment concentrates on individual adjuvant (i.e. preparation does not include PCV or M.hyo antigens).The adjuvant of PRRS viricidal activities is commented Valence is shown in Fig. 10.Preliminary assessment of killing the virus shows 10%SP- oil, 0.2% carbomer and 2.5% Amphigen to PRRS diseases Malicious effect of not killing the virus.Opposite 20%SLCD adjuvants show the effect of killing the virus to PRRS viruses.

Whether conduct further research right to evaluate the PCV/M.hyo preparations for using different adjuvant platforms to be prepared as adjuvant PRRS viruses are not killed the virus effect.These results are shown in table 13, and wherein symbol * indicates those vaccine series to PRRSV With the effect of killing the virus.

Table 13 is killed the virus the result of detection with the PRRS that different preparations carry out

* indicate be kill the virus (>0.7 logarithm loses)

A-, which kills the virus, detects control GMT~5.53log/mL

B-, which kills the virus, detects control GMT~6.42log/mL

It is in above-mentioned table 13 the result shows that 10%SP- oil does not kill the virus effect to PRRS viruses.Use 10%SP- oil as Adjuvant prepares other PCV/M.hyo vaccine series (table 14).The result shown in following table 14 further demonstrates that 10%SP- oil is right It does not kill the virus effect in PRRS viruses.Each of detection sample value in table 14 compares high (+symbol) than detection of killing the virus, right According to geometric mean titer (GMT) be about 5.9 ± 0.5log/ml.

The result of the detection of killing the virus for the different PCV/M.hyo preparations that table 14 is used 10%SP- oil as adjuvant and prepared

Kill the virus detection control GMT~5.9 ± 0.5log/ml

Result of this example indicate that 10%SP- oil does not kill the virus effect to PRRS viruses.The result of the present embodiment into One step proof, which uses 10%SP- oil to belong to those as the PCV/M.hyo preparations that adjuvant is prepared, to be considered not killing PRRS viruses The vaccine series (table 13 and table 14) of virus function.Therefore, the PCV/M.hyo preparation quilts for using 10%SP- oil to be prepared as adjuvant It is considered effective platform, which can be as the basis of the Trivalent Combinations including PCV, M.hyo and PRRS virus.

Embodiment 13：The preparation of PCV/M.hyo/PRRS combination-vaccines

It uses and the do not kill the virus adjuvant platform of effect of PRRS viruses is prepared as adjuvant (see above-mentioned table 13 and 14) PCV/M.hyo preparations are provided in single bottle liquid composition in a manner of instant.This single bottle PCV/M.hyo preparations make With the M.hyo supernatants of albumin A-processing.In the PCV2/M.hyo preparations of this supernatant using M.hyo albumin As-processing, The effect of M.hyo and PCV2 has been proven (see embodiment 7-9).In the present embodiment, this divalent PCV2/M.hyo preparations With monovalent PRRS virus antigen combinations.

In one embodiment, in 10%SP- oil and corresponding in above-mentioned table 11 vaccine series L0711RK11, The PCV/M.hyo of one of L0711RK12, L0711RK13 and L0711RK14 are combined in single bottle liquid composition with instant Mode is provided.Result in above-described embodiment 12 proves that 10%SP- oil does not kill the virus effect to PRRS viruses.Embodiment 12 Also confirm that using 10%SP- oil to belong to those as the PCV2/M.hyo preparations that adjuvant is prepared is considered not killing disease to PRRS viruses The vaccine series of toxic action.In the present embodiment, this single bottle PCV2/M.hyo liquid compositions be used to make to contain in second bottle The work PRRS virus composition rehydration of the genetic modification of some freeze-dryings, so that all antigens are in the pig to the suitable age It is comprised in single bottle before (such as 3 week old or bigger) administration.

In one embodiment, PRRS viruses, which have, corresponds to SEQ ID NO:16 genome sequence or its variant. In another embodiment, the PRRS viruses used in trivalent composition are known as the PRRS virus isolates of ISU-55, It is deposited in ATCC, and preserving number is VR 2430.The suitable amount of each antigen is described herein.It is desirable that All antigens are administered to pig with single dose.

Embodiment 14：The PCV2 effect evaluations of the PCV2/M.hyo/PRRS combination-vaccines of poison are attacked with PCV2

This research is designed with the PCV1-2 chimeras of evaluation test PCV2/M.hyo/PPRS combination-vaccines, inactivation disease The effect of malicious fraction, it is primary to the pig of 3 week old by intramuscular administration, and attack poison with toxicity PCV2 isolates within three weeks after inoculation. These trivalent vaccines include -2 type chimera of 1 type of pig circular ring virus, inactivation of viruses, breathing and reproductive syndrome vaccine, breathing shape Formula, the live virus of modification and mycoplasma hyopneumoniae bacterial extract.

The Trivalent Combinations are by with including -2 type chimera of 1 type of pig circular ring virus, inactivation of viruses and M.hyo bacterial extracts (PCV2/M.hyo) the single bottle liquid preparation of combination makes viral (PRRS-MLV) rehydration of the work PRRS of the genetic modification of freeze-drying It prepares, the single bottle liquid preparation uses 10%SP- oil to prepare (referring to above-described embodiment 13) as adjuvant.In the mistake of this research The tentative preparation being administered in journey is as described in following tables 15.

Table 15 is used for the PCV2/M.hyo/PRRS tentative bacterin preparations of PCV2 effect research

IVP=tests veterinary drug

CP=reference products

IM=is intramuscular

IN=is intranasal

¹%=PCV2 antigens, RU/mL=M hyo antigens, log10 TCID50=PRRSV antigens

Different vaccine preparations described in above-mentioned table 15 of the pig of 3 week old by intramuscular inoculation single dose.Each processing group Including 24 animals.Animal is carried out with toxicity PCV2a isolates to attack poison within 21 days after inoculation.

The PCV2 viremia virusemias result (PCV2 quantitative PCRs) observed in this research is shown in Figure 11.It should be noted that PCV2 viremia virusemias are used as main efficacy variable.PCV2 viremia virusemia results are shown as DNA copy number/ml.Such as Figure 11 institutes Show, the 28th, 35 and 42 day (it is the 21st day to attack poison), all processing all had the viral blood substantially reduced compared with placebo Disease (P<0.001).

Also monitoring PCV2 serology, PCV2 excrement fall off, lymphatic loss and immunohistochemistry (IHC) become as the second effect Amount.These results will be described below.

PCV2 Serologicals are shown in fig. 12, (are attacked within the 1st, 7,13,20,28,35 and 42 day which show this research Poison is at the 21st day) PCV2ELISA results.The state of each sample is expressed as sample to positive ratio (S/P).These knots Fruit shows, relative to placebo, two processing groups all have significantly higher PCV2 antibody titers (P after attacking poison<0.0345).

T02 and T03 processing falls off display in fig. 13 relative to the PCV2 excrement that placebo (T01) obtains.These results It is expressed as PCV2DNA copy numbers/ml.It is in Figure 13 the result shows that at the 35th and 42 day, when compared with placebo, T02 and T03 All there is significantly less excrement to fall off (P for processing<.0001).

Following table 16 is shown, compared with placebo, the notable guarantor for lymphatic loss is obtained with tentative processing (T02) Shield.

16 PCV2 histopathologies of table (lymph consume)

It is in following table 17 the results show that compared with placebo, with tentative processing (T02) obtain for histocyte The notable protection replaced.

17 PCV2 histopathologies of table (histocyte replacement)

Following table 18 is shown, compared with placebo, with the immunohistochemistry of tentative processing acquisition.Two vaccines (T02 and T03 the notable protection (P for building group in lymphoid tissue for PCV2 antigens) is all shown<0.0059).

18 PCV2 histopathologies (immunohistochemistry) of table

In short, the present embodiment the results show that tentative vaccine in this research provides the protection for attacking poison for PCV2.Two The effect of planting vaccine level builds group to primary variables and PCV2 and provides significantly protection.But T02 groups are damaged also directed to PCV2 (lymphatic loss and histocyte are replaced) provides significantly protection.

Embodiment 15：The M.hyo effect evaluations of the PCV2/M.hyo/PRRS combination-vaccines of poison are attacked with M.hyo

This research is designed with the effect of the M.hyo fractions of evaluation test PCV2/M.hyo/PPRS combination-vaccines, quilt Intramuscular administration is primary to the susceptible pig of 3 week old, and attacks poison with toxicity mycoplasma hyopneumoniae isolate within three weeks after inoculation.These three Valence vaccine includes -2 type chimera of 1 type of pig circular ring virus, inactivation of viruses, breathing and reproductive syndrome vaccine, respiratory form, modification Live virus and mycoplasma hyopneumoniae bacterial extract.

The processing of liquid：

For each processing group as described below, inactivation, clear M.hyo zymotic fluids (such as above-described embodiment 11 is used It is described).

T01：The negative control processing being made of the PCV1-2 vaccines without mycoplasma hyopneumoniae antigen, in freeze-drying It is used as diluent in the live vaccine of PRRSV modifications.

T02：The mycoplasma hyopneumoniae antigen of inactivation and pig circular ring virus (- 2 type chimera of 1 type or PCV1-2, inactivation disease Poison) it is combined into single bottle form.PCV1-2/M.hyo is combined is used as diluent in the live vaccine that the PRRSV of freeze-drying is modified.

T03：The inactivation that antigen is concentrated to as described in above-described embodiment 11 and in additional step by molecular filtration 20X Mycoplasma hyopneumoniae antigen is combined into single bottle form with pig circular ring virus (- 2 type chimera of 1 type or PCV1-2, inactivation of viruses). PCV1-2/M.hyo is combined is used as diluent in the live vaccine that the PRRSV of freeze-drying is modified.

These tentative preparations are described in following table 19.In table 19, CP is reference product, and IVP is experiment veterinary drug. M.hyo antigens correspond to the M.hyo antigens of the supernatant from complete M.hyo seeds, albumin A processing.

Table 19 is used for the PCV2/M.hyo/PRRS tentative bacterin preparations of M.hyo effect research

Different vaccine preparations described in above-mentioned table 19 of the pig of 3 week old by intramuscular inoculation single dose.20 days after inoculation Animal is carried out with toxicity M.hyo field isolates to attack poison.Research is completed with 25 animals in T01 and T03 groups, In T02 groups research is completed with 24 animals.Attack 28 days after poison and carry out ptomatopsia, take out lung and pair with M.hyo infection unanimously Consolidation scores.Following table 20 includes that the injury of lungs of each processing group scores.By being mixed to every group of lung's scoring Model analysis determines significance,statistical.

20 M.hyo injury of lungs of table

Compared with negative control group (T01), processing group T03 is proved compared with T01 on the percentage of the lung with damage It substantially reduces (P≤0.05).Injury of lungs percentage and the T01 or T03 of T02 is not significantly different.

The present embodiment the results show that used in this research tentative trivalent vaccine preparation (T03 processing) offer be directed to M.hyo attacks the notable effect of poison.

Embodiment 16：The evaluation of the PRRSV effect of PCV/M.hyo/PRRS combination-vaccines

This research is designed to the effect of the PRRSV fractions of evaluation test PCV2/M.hyo/PPRS combination-vaccines.

Research overview：

At the 0th day, select about 102 clinical healths, three week old, to PRRSV, SIV and mycoplasma hyopneumoniae for Seronegativity and the pig of no PCV viremia virusemias is checked by PCR, and assign to (block farrowing) four (every group of processing groups 24) one of in or label (NTX) group (6) in.Pig is by the tentative pig circular ring virus of intramuscular (IM) dosage of the 2mL of carry out single - 2 type chimera of 1 type, inactivated virus vaccine-mycoplasma hyopneumoniae bacterial extract (T01) or tentative pig circular ring virus 1-2 Type chimera-breathing and reproductive syndrome vaccine, respiratory form, modification work and inactivation virus-mycoplasma hyopneumoniae it is thin The administration of fungus extract (T02, T03 and T04).In the vaccine inoculation stage of this research, NTX groups animal is housed in not with processing group In same fence.Surrounding after inoculation is implemented to be euthanized and carries out ptomatopsia, with true before stable breeding processing group again to NTX pigs Recognizing does not have PRRSV injury of lungs.The pig of all processing attacks strain (NADC20) with toxicity PRRSV and carries out attacking poison.10 days after attacking poison All remaining pigs are implemented to be euthanized and carry out ptomatopsia.When ptomatopsia, to each lobe of the lung (left side head, the left side of lung Middle side part, left side tail portion, right side head, right side central, right side tail portion and attachment) consolidation percentage scored and recorded For the percentage for the lobe of the lung with damage observed.PRRSV negative status (the IDEXX of T01 pigs is detected before attacking poison ELISA).Clinical observations being recorded daily is primary in studying time-continuing process, weighs when before attacking poison with ptomatopsia.

Tentative reagent is described in following table 21.M.hyo antigen controls batch is prepared as described in above-described embodiment 11. PCV2 antigens are the cPCV1-2 antigens of the inactivation prepared as described in above-described embodiment 2.Before embedded virus inactivation, PCV2 is anti- Former batch is concentrated 20X and with balanced salt solution washing concentrate.PCV/M.hyo single vial formulations (use 10%SP oil as assistant Agent prepare) be used to make freeze-drying modification or PRRSV rehydration.

T01：Height is for -2 type chimera of 1 type of pig circular ring virus, inactivation of viruses (1.65% 20X concentrated antigens batch) and pig Mycoplasma pneumoniae bacterial extract (dosage -9.0RP；Tentative prepared product 153RU/mL).T01 prepared products correspond to series number L0912RK12 (PCV/M.hyo) and be negative control (no PRRSV antigens).

T02：Height is for -2 type chimera of 1 type of pig circular ring virus, inactivation of viruses (1.65% 20X concentrated antigens batch) and pig Mycoplasma pneumoniae bacterial extract (dosage -9.0RP；Tentative prepared product and the breeding of Gao Dai pigs 153RU/mL) is integrated with breathing Levy vaccine respiratory form, modification live virus (the tentative prepared product of MID (≤2.5logs).T02 prepared products are corresponding to series Number L0912RK12 (PCV/M.hyo)+(PPRS MLV, with MID≤2.5log).

T03：Height is for -2 type chimera of 1 type of pig circular ring virus, inactivation of viruses (1.65% 20X concentrated antigens batch) and pig Mycoplasma pneumoniae bacterial extract (dosage -9.0RP；Tentative prepared product and the breeding of Gao Dai pigs 153RU/mL) is integrated with breathing Levy vaccine respiratory form, modification live virus (the tentative prepared product of MID (≤3.0logs).T03 prepared products are corresponding to series Number L0912RK12 (PCV/M.hyo)+(PPRS MLV, with MID≤3.0log).

T04：Height is for -2 type chimera of 1 type of pig circular ring virus, inactivation of viruses (1.65% 20X concentrated antigens batch) and pig Mycoplasma pneumoniae bacterial extract (dosage -9.0RP；Tentative prepared product and the breeding of Gao Dai pigs 153RU/mL) is integrated with breathing Levy vaccine respiratory form, modification live virus (the tentative prepared product of MID (≤3.5logs).T04 prepared products are corresponding to series Number L0912RK12 (PCV/M.hyo)+(PPRS MLV, with MID≤3.5log).

Described in following table 21, M.hyo antigens are corresponded to from complete M.hyo seeds, albumin A these tentative preparations Handle the M.hyo antigens of supernatant.The serial number of PRRSV prepared products has (TBD) to be determined.

21 research and design of table

¹The pig circular ring virus 1-2 types chimera (PCV2) of experiment veterinary drug (IVP)=10%SP oil is used to be prepared as adjuvant, Inactivated virus vaccine-mycoplasma hyopneumoniae (M hyo) bacterial extract (diluted)-porcine reproductive and respiratory syndrome vaccine, is exhaled Suction form, the live virus (PRRSV) of modification

Reference product (CP)=pig circular ring virus 1-2 types chimera (PCV2), inactivated virus vaccine-mycoplasma hyopneumoniae (M Hyo) bacterial extract-is without porcine reproductive and respiratory syndrome vaccine fraction；10%SP oil is used to be prepared as adjuvant

IM=is intramuscular

NA=is not applicable.

Claims (22)

1. a kind of trivalent immunogenic composition, it includes：

The supernatant of mycoplasma hyopneumoniae (M.hyo) culture；

Porcine circovirus 2 type (PCV2) antigen；

Porcine reproductive and respiratory syndrome (PRRS) viral antigen；With

SP- oil adjuvants,

The wherein supernatant of M.hyo cultures

Including antigen special M.hyo-, and

It is detached from insoluble cellular material, and is free of IgG and antigen immunoglobulin compound,

The supernatant of the wherein described M.hyo cultures used before being added into immunogenic composition albumen-A or albumen-G into Row processing.

2. the composition of claim 1, wherein PCV2 antigens described in the supernatant of the M.hyo cultures are instant liquid groups Close the form of object.

3. the composition of claim 1, wherein the PRRS viral antigens are the live virus of genetic modification.

4. the composition of claim 3, wherein the work PRRS viruses of the genetic modification are the forms of freeze-dried composition.

5. the composition of claim 1, wherein the composition causes the guarantor for M.hyo, PCV2 and PRRS virus in pig The immune response of shield property.

6. the composition of claim 1, wherein the PCV2 antigens are the forms of -2 type circovirus of chimeric 1 type, it is described chimeric 1 type of recombinant porcine circovirus of expression Porcine circovirus type 2 Cap protein of the virus comprising inactivation.

7. the composition of claim 1, wherein the PCV2 antigens are the forms for recombinating ORF2 albumen.

8. the composition of claim 7, wherein the recombination ORF2 albumen is expressed from baculovirus vector.

9. the composition of claim 1, wherein the composition further includes pharmaceutically acceptable carrier.

10. the composition of any one of claim 1 to 9, wherein the composition is carried out when the form being administered in a single dose When administration, cause the protective immunological reaction for M.hyo, PCV2 and PRRS virus.

11. the composition of claim 1 is being prepared for protecting pig to resist mycoplasma hyopneumoniae (M.hyo), PCV2 and PRRS Purposes in the drug of virus.

12. the purposes of claim 11, wherein the drug is by intramuscular, intradermal, percutaneous or subcutaneous administration.

13. the purposes of claim 11, wherein the drug is administered in the form of single dose.

14. the purposes of claim 11, wherein the drug be administered to from parent for M.hyo, PCV2 and The pig of at least one antibody of PRRS viruses.

15. the purposes of any one of claim 11~14, wherein the drug is administered to the pig of 3 week old or bigger.

16. a kind of kit, it includes：

First bottle, described first bottle includes composition, and the composition includes

PCV2 antigens,

The supernatant of mycoplasma hyopneumoniae (M.hyo) culture and

SP- oil adjuvants,

The wherein supernatant of M.hyo cultures

Including antigen special M.hyo-, and

It is detached from insoluble cellular material, and is free of IgG and antigen immunoglobulin compound,

The supernatant of the wherein described M.hyo cultures used before being added into immunogenic composition albumen-A or albumen-G into Row processing；With

Second bottle, described second bottle includes PRRS viral antigens.

17. the kit of claim 16, wherein the composition in described first bottle of the kit is with instant liquid The form of body composition provides.

18. the kit of claim 16, wherein the PRRS viral antigens in described second bottle are the shapes of freeze-dried composition Formula.

19. the kit of any one of claim 16 to 18, wherein the kit further comprises providing about by first The operation instructions for the explanation that the content of bottle is combined with second bottle of content.

20. the kit of claim 19, wherein the operation instructions further comprise first and second bottles will combined Content is administered to the explanation of pig.

21. a kind of method preparing immunogenic composition, the method includes：

(i) 18~144 hours times of M.hyo are cultivated in suitable culture medium；

(ii) M.hyo cultures are then inactivated；

(iii) the M.hyo cultures of the inactivation are centrifuged, filtered or is precipitated, to obtain the supernatant of M.hyo cultures, The supernatant of the wherein described M.hyo cultures includes the special antigens of M.hyo-；

(iv) by handling the supernatant of the M.hyo cultures with albumen-A or albumen-G come from the upper of the M.hyo cultures IgG and antigen immunoglobulin compound are removed in removing；And

(v) then by the supernatant and PCV2 antigens, SP- oil adjuvants of the M.hyo cultures handled with albumen-A or albumen-G With PRRS virus antigen combinations.

22. the method for claim 21, wherein (v) including will locate comprising the PCV2 antigens, the albumen-A or albumen-G The PRRS viral antigen groups of the ready-to-liquid composition of SP- oil adjuvants and freeze-drying described in the supernatant of the M.hyo cultures of reason It closes.