CN106344920B - Adjuvant for vaccine and application thereof - Google Patents
Adjuvant for vaccine and application thereof Download PDFInfo
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- CN106344920B CN106344920B CN201510420446.4A CN201510420446A CN106344920B CN 106344920 B CN106344920 B CN 106344920B CN 201510420446 A CN201510420446 A CN 201510420446A CN 106344920 B CN106344920 B CN 106344920B
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Abstract
The invention provides an adjuvant for a vaccine, which comprises a polyacrylic acid polymer and cellulose ether, and can also comprise an immunopotentiator. The vaccine adjuvant can effectively promote the effect of the vaccine, and the adjuvant can be used as an adjuvant of an inactivated vaccine and a diluent of a live vaccine. The vaccine composition containing the adjuvant can achieve the effect of twice immunization of commercial products only by one immunization, thereby greatly simplifying the immunization process; side reactions can be avoided; the adjuvant of the present invention also stabilizes proteins without degradation or irreversible change, and can be used as a protein stabilizer.
Description
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to an adjuvant for enhancing antigen immune response.
Background
Bacterial, viral and parasitic infections are widely distributed in humans and animals, and diseases caused by these infectious agents are often resistant to chemicals such as antibiotics, and thus there is no effective treatment. Accordingly, the art increasingly uses vaccinology methods to control infection, specifically to induce specific immunity by vaccination with live pathogens, inactivated pathogens or their products, or genetically engineered vaccines prepared from pathogen protein subunits. However, it is difficult to produce sufficient stimulation of the immune system when certain antigens are administered alone. Therefore, an immunological adjuvant capable of increasing the immune response of the body must be added to obtain a sufficient amount of protective antibodies.
Immunological adjuvants are an essential component of vaccines, and not only can influence the strength of the immune response of the body to the vaccine, but also can induce the most effective type of immune response against a particular pathogen (Mlow ML, De Gregorio E, vaccine NM, et al. New adjituents for human vaccines. Current opinion in immunology,2010,22(3): 411-416). Currently, many types of immunologic adjuvants are studied, such as oil adjuvant, Freund's adjuvant, microorganism and its metabolites, nucleic acid and its analogs, cytokines, liposomes, etc. (Dey AK, Srivastava IK. novel adjuvants and delivery systems for enhancing immune responses. expression of vaccines,2011,10: 227), but are difficult to be practically applied due to some unavoidable defects such as various degrees of toxic side effects or safety hazards (Mnow ML, De Gregorio E, Valiante NM, Rappoli R. New administration for human immune responses. Current administration of vaccines 2010,22(3): 411. 416; Batista-DuEB A, Lindblad, Ovido-Orient. 2011 infection. vaccines, 2010, 97) and thus far from developing novel vaccines, 105. 12. environmental-infection vaccines, 105. 7. environmental vaccines, 105. 7. origin of vaccine, 105. origin of vaccine, and thus, the need for developing novel immunologic adjuvants. Therefore, the search for safe, effective and novel immunoadjuvants is a hotspot in the current vaccine research field (Schijin VEJC, Lavelle EC. terms in vaccine adjivants. expert review of vaccines,2011,10(4):539 550). Furthermore, immunologic adjuvant studies are now being listed as a priority field for vaccine studies (Harandi AM, Meaglini D, Shattock RJ. vaccine adjivants: A priority for vaccine research. vaccine 2010,28(12): 2363-.
Currently commercialized bacterins (e.g., mycoplasma hyopneumoniae bacterins, haemophilus parasuis bacterins) are prepared from organisms grown on serum-containing media, which cause side reactions induced by serum components (e.g., immune complexes or non-immunogenic specific proteins) present in the immune material (see chinese patent CN 104334186A).
In addition, in the research of clinical tests in the technical field of veterinary biological products, the repeatability and stability of the protein are found to be very important, and good stability is an important mark for data reliability. However, in clinical tests, some proteins have poor stability and are easily degraded or denatured under general conditions, and the accuracy of test results is directly influenced. Therefore, how to maintain the stability of the protein is a key technology in clinical trials.
Disclosure of Invention
The invention provides an effective adjuvant for vaccines, which can effectively enhance the immune efficacy of antigens. The adjuvant for the vaccine is used together with the antigen, so that the immunization program can be simplified, and the effect of twice immunization of the commercial vaccine can be achieved only by one immunization.
The invention relates to an adjuvant for a vaccine, which comprises an acrylic polymer and cellulose ether.
The invention also relates to an adjuvant for vaccines, which comprises an acrylic polymer, cellulose ether and an immunopotentiator; the immunopotentiator is a saponin-containing composition, preferably an immunostimulatory complex.
The invention also relates to a vaccine composition comprising an immunizing amount of an antigen and the acrylic polymer of the invention and a cellulose ether adjuvant.
The invention also relates to a vaccine composition comprising an immunizing amount of an antigen and the acrylic polymer of the invention, a cellulose ether and an immunopotentiator adjuvant.
Another technical problem to be solved by the present invention is to provide an antigenic protein stabilizing agent which can maintain the original molecular weight, structure and/or biological activity of the protein for a long time during the preparation process and under appropriate conditions.
The invention relates to an antigen protein stabilizer which comprises an acrylic acid polymer and cellulose ether.
The invention also relates to an antigen protein stabilizer which comprises an acrylic polymer, cellulose ether and a saponin-containing composition.
The adjuvant of the invention has lower antigen content, can make animals obtain better immune response level when being immunized once, reduces the stress reaction of pigs and reduces the occurrence of side reaction; the adjuvant can also be used for diluting the live vaccine and enabling the live vaccine to immunize pigs in an intramuscular injection mode, so that the operation is simple and convenient. In addition, the adjuvant can be used as a protein stabilizing solution to store protein, so that the protein is stable and is not degraded or irreversibly changed.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
The term "adjuvant" refers to a compound that, when administered in conjunction with an antigen, enhances the immune response of a subject to this antigen.
The term "acrylic polymer" refers to any polymer or copolymer containing acrylic moieties, including, but not limited to, polyacrylic acid, polymethacrylic acid, polymethacrylate, polyacrylamide, polyacrylate, polyacrylonitrile, and alkyl esters of polyacrylic acid. Examples of acrylic polymers include, for example, poly (acrylamide-co-butyl methacrylate), acrylic acid-methacrylic acid, acrylic acid-acrylamide, and poly (methacrylate). Examples of commercially available acrylic polymers include Carbopol (goodrich b.f. goodrich co., Cleveland Ohio, usa), cabesque Carboset (goodrich b.f. goodrich co., Cleveland Ohio, usa), niokneocryl (Avecia inc., Wilmington Del, terawa, usa) and Eudragit (Rohm technologies Rohm, Rohm technologies, inc., madden Mass, usa). Acrylic polymers particularly suitable for use in the adjuvants of the invention are carbomers (Carbomer, trade name Carbopol), also commonly referred to and known in the art as water-soluble polymers of acrylic acid cross-linked with polyallyl sucrose, see Phameuropa (1996,8(2)), and those skilled in the art can also see US2909462, which describes such acrylic polymers cross-linked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are substituted with unsaturated aliphatic radicals having at least 2 carbon atoms, preferably radicals containing from 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated radicals (ethylenically unsaturated radicals). The unsaturated groups may themselves contain other substituents, such as methyl. These products are sold under the name carbopol, particularly suitable under the name BF Goodrich (Ohio, USA), crosslinked with allyl sucrose or with allyl pentaerythritols (allyl pentaerythritols), among which mention may be made of carbopol 941, 980, 981, 940, 2020, 974P, 934P and 971P, the most preferred being carbopol 971P.
The term "cellulose ether" refers to ether derivatives of cellulose such as carboxymethyl cellulose CMC, hydroxyethyl cellulose HEC, hydroxypropyl methyl cellulose HMPC, and also includes its salts, i.e. carboxymethyl cellulose salts. The carboxymethyl cellulose salt includes, but is not limited to, sodium carboxymethyl cellulose, potassium carboxymethyl cellulose, calcium carboxymethyl cellulose, preferably sodium carboxymethyl cellulose.
The term "Sodium carboxymethylcellulose" (Sodium carboxymethylcellulose) is a cellulose-based biopolymer and is produced by esterification of cellulose in a Sodium hydroxide solution containing Sodium monochloroacetate (Feddesen et al, Sodium carboxymethylcellulose Sodium Sorboxymethyl cellulose, Industrial polysaccharides and derivatives thereof, Marcel Dekker Inc., New York, third edition (1993):537 578), written by Whistler RL, BeMiller JN, as a measure of the degree of esterification, the degree of substitution DS is given as the average number of esterified hydroxyl groups in a glucose unit. The presence of three reactive hydroxyl groups allows the introduction of three carboxymethyl sodium groups. The properties of sodium carboxymethylcellulose depend on its degree of substitution and its degree of polymerization (Beltz et al, Lehrbuch der Lebenstmitelchemie. Springer-verlag, Berlin, fourth edition, 1992), which is the average number of monomer molecules which are linked to form macromolecules during the polymerization process. According to the invention, the preferred sodium carboxymethylcellulose has an average molecular mass of 125000 g/mol. The term "sodium carboxymethylcellulose" also encompasses polysaccharides which are not sodium carboxymethylcellulose in the strict sense of their definition, but which can equally be used as they have the same or similar properties as sodium carboxymethylcellulose due to, for example, derivatization, e.g., by hydroxylation, carboxylation, esterification and/or etherification, of cellulose or other various sugars.
In a first aspect of the present invention, there is provided an adjuvant for a vaccine, the adjuvant comprising an acrylic polymer and a cellulose ether.
In one embodiment of the present invention, the acrylic polymer in the adjuvant for vaccine of the present invention includes polyacrylic acid, polymethacrylic acid, polymethacrylate, polyacrylamide, polyacrylate, polyacrylonitrile, and alkyl ester of polyacrylic acid.
In a preferred embodiment of the present invention, the acrylic polymer in the adjuvant for vaccine of the present invention comprises carbopol, cabbagel, Neocryl, Eudragit.
As a more preferred embodiment of the present invention, in the adjuvant for vaccine of the present invention, the acrylic polymer includes carbopol 941, 980, 981, 940, 2020, 974P, 934P and 971P.
As an embodiment of the present invention, the cellulose ether includes carboxymethyl cellulose, sodium carboxymethyl cellulose, potassium carboxymethyl cellulose, calcium carboxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl methyl cellulose.
In one embodiment of the present invention, in the adjuvant for a vaccine of the present invention, the acrylic polymer is 2 to 10mg/ml in terms of carbomer, and the cellulose ether is 0.1 to 8 mg/ml.
In a preferred embodiment of the present invention, the adjuvant for vaccine of the present invention comprises 4 to 10mg/ml of acrylic acid-based polymer and 4 to 8mg/ml of cellulose ether in terms of carbomer.
The term "immunopotentiator" (immunopotentiator) refers to a substance that is not immunogenic in itself, but can be used to stimulate the immune system to improve responses to other immunogenic substances. When used together with or administered separately to the same subject as the antigen of interest, the antigen may directly or indirectly modulate the immune response induced by the antigen or alter the type of immune response (including, e.g., humoral, cellular) to the antigen. The immunopotentiator includes, but is not limited to, saponin-containing compositions, active lactic acid bacteria, propolis, vitamin E, levamisole, transfer factor, interleukin, interferon, thymosin, transfer factor, globulin, bcg, or combinations thereof.
The term "saponin-containing composition" may comprise a purified or partially purified preparation such as QS21 (under the trade name Stimulon @)TM) And lipid formulations such as the immune stimulating complex ISCOM. The saponin (saponin) is also called saponin, saponin,Alkali soap or saponin, which is a heterologous group of sterol glycosides and triterpenoid glycosides, can be found in the bark, leaf, stem, root, and even flowers of various plants including, but not limited to, quillaja saponin, Ginseng saponin, Panax notoginseng saponin, Platycodon grandiflorum saponin, Astragalus saponin, Achyranthes saponin, polygla saponin, and the saponins mentioned in Sun et al (Sun HX, Xie Y, Ye yp, advanced in saponin-based adjivants, vaccine,2009,27(12): 1787-. The purified or partially purified preparations can be purified by HPLC and RP-HPLC, and specific purified fractions have been identified using these techniques, including at least one of QuilA, QS21, QS7, QS17, QS18, QH-A, QH-B, QH-C, VaxSap, SuperSap, GPI-0100, QP UF 1000.
The term "immunostimulatory complex" (ISCOM) means that an ISCOM-matrix, which is an immunostimulatory complex without an antigen, can also function as an immunoadjuvant when mixed with a vaccine, in glycosides such as triterpenoid saponins (in particular Quil a) and cholesterol, e.g. european publications nos. EPA109942 and 180564. Typical ISCOMs are unique particles containing saponin, cholesterol, and further including phospholipids such as cephalins or lecithins, and any known saponin may be used in the ISCOMs. Preferably, the ISCOM comprises QuilA (Quillaja saponin triterpene saponin, south America), cholesterol, lecithin, and is prepared by mixing and covalently bonding, and the immune complex is cage type or Golf ball type under electron microscope. Details of saponins and ISCOMs and methods of forming ISCOMs are described in Barr et al (Barr IG, Sjolander A, Cox JC. ISCOMs and other saponin based assays. advanced drug delivery reviews,1998,32(3): 247-. ISCOMs useful in the present invention are prepared using standard techniques, which are well known in the art, and described, for example, in U.S. Pat. Nos. 4981684, 5178860, 5679354, 6027732, European publications EPA109942, 180564, 231039, and Coulter et al (counter A, Wong TY, drift D, et al. students on experimental ad practical in fluorescent fuels: composites of animal stimulating compounds (ISxoms)TM)and oil-in-water vaccines.Vaccine,1998,16(11-12):1243-1253)。
In one embodiment of the present invention, the adjuvant for vaccine of the present invention further comprises an immunopotentiator, wherein the immunopotentiator comprises a saponin-containing composition, activated lactic acid bacteria, propolis, vitamin E, levamisole, transfer factor, interleukin, interferon, thymosin, transfer factor, globulin, bcg, or a combination thereof.
In a preferred embodiment of the present invention, the adjuvant for vaccine of the present invention, the immunopotentiator comprises a saponin-containing composition, and the saponin-containing composition is preferably an immunostimulatory complex. The saponin-containing composition comprises at least one of QuilA, QS21, QS7, QS17, QS18, QH-A, QH-B, QH-C, VaxSap, SuperSap, GPI-0100, QP UF1000, and an immune stimulating complex.
In one embodiment of the present invention, in the adjuvant for vaccine of the present invention, the acrylic acid-based polymer is 2 to 10mg/ml in terms of carbomer, the cellulose ether is 0.1 to 8mg/ml, and the saponin-containing composition is 0.1 to 10mg/ml in terms of QuilA.
In a preferred embodiment of the present invention, the adjuvant for vaccine of the present invention comprises 4 to 10mg/ml of acrylic acid-based polymer in terms of carbomer, 4 to 8mg/ml of cellulose ether, and 4 to 10mg/ml of saponin-containing composition in terms of QuilA.
The term "immunologically effective amount" also referred to as an immunoprotective amount or an amount effective to generate an immune response, is an amount effective to induce an immunogenic response in a recipient. The immune response may be sufficient for diagnostic purposes or other testing, or may be suitable for use in preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain, while the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity.
The term "antigen", also known as immunogen, refers to any substance that stimulates an immune response, is a molecule that can be bound by an antibody and is also capable of inducing a humoral and/or cellular immune response that produces B-and/or T-cells, and also has one or more epitopes (B-and T-epitopes). The antigen comprises a killed, inactivated, attenuated or modified live bacterium, virus or parasite, or a cultured cell preparation, supernatant thereof; also included are subunit antigens which are polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells, tissues, polysaccharides, lipids or fragments thereof, either individually or in any combination thereof, as well as antibodies such as anti-subject genotypic antibodies or fragments thereof and synthetic peptidomimetics which mimic an antigen or antigenic determinant (epitope). Wherein the killed, inactivated bacteria, virus or parasite is an infectious organism or pathogen that is no longer capable of replicating or growing, and the pathogen is inactivated by various means including freeze-thawing, chemical treatment (such as treatment with thimerosal or formalin), sonication, irradiation, heat or any other common method sufficient to prevent replication or growth of the organism while maintaining its immunogenicity; the modified live bacteria, viruses or parasites refer to antigens which cause variation under artificial conditions, lose pathogenicity but retain immunogenicity, multiplication capacity and residual toxicity, and reproduce or replicate in animals to a certain extent after inoculation without causing morbidity.
The term "Porcine circovirus antigen" refers to any composition containing at least one form of Porcine Circovirus (PCV) antigen that can induce, stimulate or enhance an immune response against infection by Porcine circovirus, including but not limited to inactivated, live or subunit antigens. The porcine circovirus antigen comprises an SH strain (with the preservation number of CGMCC No.23890, see Chinese patent CN101240264A), an SD strain (with the preservation number of CGMCC No.7707, see Chinese patent CN103421748A), a D strain (with the preservation number of CGMCC No.7245,see China patent CN103275938A), WH strain (with preservation number of CCTCC No. V201333, see China patent CN103409374A), ZJ/H strain (with preservation number of CGMCC No.6391, see China patent CN102787100A), DBN-SX07 strain (with preservation number of CGMCC No.3064, see China patent CN101549155A), V201312 strain (with preservation number of CCTCC No. V201312, see China patent CN103436498A), and any antigen of the following compositions such as Merial of Merria company of Merria
Pfizer of Pfizer, Pfizer
PCV2, porcine circovirus type 2 inactivated vaccine (LG strain) from Harbin Vitaceae Biotechnology developers, porcine circovirus type 2 inactivated vaccine (ZJ/C strain) from Nuo Wei Biotechnology GmbH, Zhejiang, Burlingger Vargehine Boehringer-Ingelheim
The term "Mycoplasma hyopneumoniae antigen" refers to any composition comprising at least one Mycoplasma hyopneumoniae (Mhp) antigenic form that can induce, stimulate or enhance an immune response against Mycoplasma hyopneumoniae infection, including, but not limited to, inactivated, live or subunit antigens, or culture supernatants thereof. The mycoplasma hyopneumoniae antigen comprises HN0613 strain (with the preservation number of CCTCC No. M2012230, see Chinese patent CN103083655A), J strain (purchased from American type culture Collection ATCC with the preservation number of 25934), NJ strain (with the preservation number of CCTCC No. M2012286, see Chinese patent CN103585622A), HDZK-Mhp57 strain (with the preservation number of CGMCC No.8096, see Chinese patent CN103484414A), AN306 strain (with the preservation number of CCTCC No. M2012431, see Chinese patent CN103740625A), DJ-166 strain (with the preservation number of CGMCC No.4545, see Chinese patent CN103184171A) and NM04/41259 strain (with the preservation number of NM04/41259, see Chinese patent CN102458462A), and can also comprise any one antigen of the following compositions, such as a group antigen of hamames, such as a hamames groupRedouche Respisure and Redouche-Wang Respisuerone of the same company, Anderk M + PAC of Intey corporation, and of the same company
Hyo, MycoGard of prestack, RespiFend MH of pfeik, refut-wang Suvaxyn MH-one of maotan (shanghai) animal care products, inc, prock SprintVac of merria, yinfeng Mypravac Suis of hebethley, live vaccine of strain Z produced domestically, live vaccine of strain 168, and live vaccine of strain RM 48.
The term "Haemophilus parasuis antigen" refers to any composition containing at least one form of Haemophilus Parasuis (HPS) antigen which upon inoculation of a pig induces, stimulates or enhances an immune response against Haemophilus parasuis infection, including but not limited to inactivated, live or subunit antigens, or culture supernatants thereof. The haemophilus parasuis antigens include clinically isolated wild strains well known to those skilled in the art, haemophilus parasuis containing 1-15 serotypes identified so far or no serotype measurable, mixed antigens containing at least two serotypes such as type 4 (e.g. JS strain, accession number CCTCC No. m2011172, see chinese patent CN103083655A) and type 5 (e.g. ZJ strain, accession number CCTCC No. m2011173, see chinese patent CN103083655A), and mixed antigens of any of the following compositions such as Ingelvac HP-1 of buerger neglighn, hiprasis-Glasser of boelli, Hiprasuis-Glasser of midge stock company ltd, and haemophilus parasuis inactivated vaccine (type 4 MD0322 strain +5 SHO165 strain) of pre-wuhan animal biologics ltd liability company.
A second aspect of the invention provides a vaccine composition, wherein the vaccine composition comprises an immunizing amount of antigen and an adjuvant of the invention.
As an embodiment of the invention, in the vaccine composition of the invention, the antigen includes porcine circovirus antigen, mycoplasma hyopneumoniae antigen, haemophilus parasuis antigen, porcine reproductive and respiratory syndrome virus antigen, porcine parvovirus antigen, pasteurella multocida antigen, streptococcus suis antigen, staphylococcus suis antigen, bordetella bronchiseptica antigen, actinobacillus pneumoniae bullosa antigen, escherichia coli antigen, porcine atrophic rhinitis antigen, porcine pseudorabies virus antigen, salmonella cholerae antigen, salmonella enteritidis antigen, erysipelothrix rhusiopathiae antigen, mycoplasma hyorhinis antigen, mycoplasma hyosynoviae antigen, leptospira suis bacterial antigen, porcine respiratory coronavirus antigen, porcine epidemic diarrhea virus antigen, porcine rotavirus antigen, porcine transmissible gastroenteritis virus antigen, porcine torque virus antigen, porcine circovirus antigen, porcine streptococcus pneumoniae antigen, porcine epidemic infectious coryza antigen, porcine, One or more of porcine cytomegalovirus antigen, porcine enterovirus antigen, encephalomyocarditis virus antigen, swine influenza virus antigen and swine fever virus antigen; and the adjuvant comprises an acrylic polymer and a cellulose ether.
As an embodiment of the present invention, in the vaccine composition of the present invention, the antigen includes one or more of the above-mentioned antigens; and the adjuvant comprises an acrylic polymer, a cellulose ether, and a saponin-containing composition.
As a preferred embodiment of the present invention, in the vaccine composition of the present invention, the antigen is composed of a porcine circovirus antigen, a mycoplasma hyopneumoniae antigen, and a haemophilus parasuis antigen, and the adjuvant comprises an acrylic polymer and cellulose ether.
As a preferred embodiment of the present invention, in the vaccine composition of the present invention, the antigen is composed of a porcine circovirus antigen, a mycoplasma hyopneumoniae antigen, and a haemophilus parasuis antigen, and the adjuvant comprises an acrylic polymer, a cellulose ether, and a saponin-containing composition.
As a most preferred embodiment of the present invention, in the vaccine composition of the present invention, the antigen is composed of inactivated antigen of SH strain of porcine circovirus, inactivated antigen of HN0613 strain of mycoplasma hyopneumoniae, inactivated antigen of JS strain 4 and ZJ strain 5 of haemophilus parasuis, and the adjuvant comprises acrylic polymer, cellulose ether and immunostimulating complex.
As an embodiment of the present invention, in the vaccine composition of the present invention, the adjuvant is contained in an amount of 5% to 30% V/V.
In a preferred embodiment of the present invention, the adjuvant is present in the vaccine composition of the present invention in an amount of 10% to 25% V/V.
The term "protein" includes chemically synthesized proteins, naturally synthesized proteins encoded by genes of cultured cells, and recombinant proteins secreted by cells. The recombinant proteins are those encoded by transgenes introduced into the cell by molecular biotechnology and may be modified by chemical means or by enzymes in post-translational processes. Such proteins include animal proteins, those produced by cell culture, as well as proteins expressed from other sources, such as insects, bacteria, plants, and the like, as well as mutant, artificial, synthetic, fusion, or chimeric proteins. According to the invention, said proteins include, but are not limited to, VP60 protein of rabbit viral hemorrhagic disease virus RHDV, VP2 protein of canine parvovirus PPV, Cap, Rep protein of porcine circovirus PCV2, P46, P65, NrdF, P97R1, P102, PdhA, XylF, P78, Eutd, Mhp145, P132, Mhp389 protein of Mycoplasma hyopneumoniae, PilA, OMP, HbpA, OppA, PalA protein of porcine reproductive and respiratory syndrome virus PRRSV, Nsp2, GP3, GP4, GP5, M, N protein of porcine parvovirus PPV, VP2 protein of porcine parvovirus PPV, Pasteurella multocida P, HyaE protein of porcine streptococcus suis, 38KDa, OmoA, HM A, HM6, GAPDH 2, PDH 2, Exphb 8656, Exphb protein of porcine parvovirus, OmhpI protein 2, OmhpA 368672, OmhpA protein of porcine reproductive and Omphb protein of porcine respiratory syndrome virus, Omphb 2, Omphb protein of porcine streptococcus, LTB, Orf353, TolC protein, PMT of porcine atrophic rhinitis AR, gB, gC, gD, gE of porcine pseudorabies virus PRV, Hfq protein of Salmonella enterica, SpaA, Lipo protein of porcine erysipelothrix rhusiopathiae, P37 protein of porcine mycoplasma rhinotracheale Mhr, S, N protein of porcine respiratory coronavirus PRCV, S, M, N protein of porcine epidemic diarrhea virus PEDV, VP4, VP6, VP7 protein of porcine rotavirus PRV, S, M, N protein of porcine transmissible gastroenteritis virus TGEV, Cap protein of porcine torque teno virus TTSuV, gB protein of porcine cytomegalovirus PCMV, VP1, VP2, 3AB protein of porcine encephalopathy virus EMCV, myocarditis proteins of porcine influenza virus, NP, NA, M, CSNS protein, E0, E1, 48325 protein of porcine virus FV.
The term "protein stabilizing solution" means a solution that provides a significant improvement in the stability of the immunogenic protein component, maintains at least 80%, preferably at least 90%, of the original molecular weight, structure and/or biological activity of the protein, maintains the protein biostability, physical stability, chemical stability, does not form chemical degradation products, does not undergo irreversible changes (e.g., aggregation, precipitation or denaturation) during the manufacturing process and after long-term (e.g., months to years) storage under appropriate conditions, preferably for more than 12 months (e.g., 24 months or 36 months). The chemical degradation products have potentially lower bio-potency and/or potentially lower immunogenicity compared to the native protein structure.
In a third aspect of the present invention, there is provided an antigenic protein stabilizing agent.
As one embodiment of the present invention, the antigenic protein stabilizing agent of the present invention comprises an acrylic acid-based polymer and cellulose ether.
As a preferred embodiment of the present invention, the antigenic protein stabilizing agent of the present invention comprises an acrylic acid-based polymer, a cellulose ether and a saponin-containing composition.
As a preferred embodiment of the present invention, the antigenic protein stabilizing agent of the present invention comprises an acrylic acid-based polymer, a cellulose ether and an immunostimulatory complex.
As one embodiment of the present invention, in the antigenic protein stabilizing agent of the present invention, the acrylic acid-based polymer is 2 to 10mg/ml in terms of carbomer, the cellulose ether is 0.1 to 8mg/ml, and the saponin-containing composition is 0.1 to 10mg/ml in terms of QuilA.
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
In the embodiment of the invention, the porcine circovirus antigen is an inactivated porcine circovirus type 2 SH strain, the porcine mycoplasma pneumoniae antigen is an inactivated porcine mycoplasma pneumoniae HN0613 strain and a live mycoplasma hyopneumoniae 168 strain, and the haemophilus parasuis antigen is an inactivated haemophilus parasuis type 4 JS strain and an inactivated haemophilus parasuis type 5 ZJ strain.
The lung disease index change in the embodiment of the invention is judged by a 28-point method. The 28-point method is to quantitatively judge the degree of the disease according to typical lung eye lesions of the mycoplasma pneumonia of swine, and comprises the substantial lesions of the lung cuspid, heart lobe, diaphragm lobe and middle lobe, such as 'flesh-like change' and 'pancreas-like change'. The pneumonia lesion scoring standard is as follows: the total of 7 lung lobes are 2 sharp lobes, 2 heart lobes, 2 diaphragm lobes and 1 middle lobe, and each lung lobe is divided into 4 parts and 28 parts. Each lobe is scored separately according to the proportion of the lobe area that has undergone substantial lesions to that lobe. The lesion without the pneumonia is marked as 0 point; the ratio of the lesion area is 1% -25%, and is marked as 1 point; the ratio of the lesion area is 26-50 percent and is marked as 2 points; the proportion of the lesion area is 51-75 percent and is marked as 3 points; the ratio of the lesion area is 76% -100%, and is marked as 4 points. For example, both the front and back sides of the lung lobes are diseased, and the side with large diseased area is scored. The sum of the scores of all the lung lobes is the pneumonia lesion score of the diseased plant. After the immune group pigs and the control group pigs are respectively scored, the lung lobe lesion reduction rate of the immune group pigs is calculated according to the following formula:
the statistical analysis method of the embodiment of the invention comprises the following steps: and (5) counting lung disease index of 7 lung lobes to determine the lesion degree. ANOVA analysis was performed using SPSS computer software to compare differences between groups and determine the effectiveness of the lesion differences.
In the inventionThe formulation of the used PBS solution with pH 7.2is as follows: NaCl 8g, KCl 0.2g and Na are added into 800ml of distilled water2HPO41.42g、KH2PO40.27g, pH adjusted to 7.4, volume to 1000ml, and use after autoclaving at 121 ℃ for 30 minutes, but this embodiment should not be construed as limiting the scope of the invention in any way.
The chemical reagents used in the present invention are all analytically pure and purchased from the national pharmaceutical group, but the present embodiment is not limited to the present invention in any way.
In order that the invention may be more readily understood, reference will now be made to the following examples. It should be understood that these examples are only for the purpose of the present invention and are not intended to limit the scope of the present invention. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
EXAMPLE 1 preparation and characterization of antigens
1.1 preparation and content determination of porcine circovirus antigen
1.1.1 preparation of porcine circovirus type 2 SH Strain
Diluting porcine circovirus type 2 SH strain with MEM liquid culture medium (prepared by MEM dry powder culture medium purchased from Invitrogen corporation, USA according to the instruction) at 1:9V/V, inoculating PK15(ATCC, deposition number is CCL-33) monolayer cells according to 5% of cell culture solution volume, adsorbing at 37 deg.C for 30 min, adding cell maintenance solution (adding 4% calf serum and 2mmol/L D-glucosamine hydrochloride in MEM liquid culture medium), culturing at 37 deg.C for 4 days, freezing and thawing for 2-3 times, and harvesting virus with virus titer of 106.5TCID50/ml。
Culturing virus liquid by spinner flask cell culture method, removing cell culture solution (adding 6% calf serum and 2mmol/L D-glucosamine hydrochloride into MEM liquid culture medium), mixing with virus seed liquid at a ratio of 0.1-0.2TCID50The inoculation amount of each cell is inoculated on PK15 cells, the cell bottle is rotated for 2 weeks, the cells are adsorbed for 30 minutes at 37 ℃, cell maintenance solution is added, and the cells are placed at 37 ℃ for rotating (10-12 r/h) for culture. Observing for 1-2 times daily, culturing at 37 deg.CHarvesting cells and cell sap in 4 days, freeze-thawing for 3 times, and storing virus liquid at-20 deg.C or below.
The prepared virus solution was filtered through a hollow fiber filter column (Millipore Corp. with a pore size of 10 μm and 0.45 μm) to remove cell debris, and then concentrated 5-fold V/V with a Millipore membrane (Millipore Corp.) pack (molecular cut-off 300 Kda).
1.1.2 measurement of the content of SH Strain 2 Virus fluid of porcine circovirus
Diluting the virus solution with MEM liquid culture medium by 10 times, and collecting 10 times-5、10-6、10-73 dilutions were used, each dilution was inoculated into a monolayer of PK15 cells in 96-well culture plates (0.1 ml per well), and negative controls were set at 37 deg.C and 5% CO2Continuously culturing for 24 hours in the incubator, changing the cell maintenance liquid, and continuously culturing for 24 hours; cells were fixed with cold acetone, the number of wells containing PCV2 positive cells (in green) was determined for each dilution by the indirect immunofluorescence assay IFA, and the virus TCID was calculated according to the Karber method50. The calculation result shows that: the content of porcine circovirus type 2 SH strain is 5 multiplied by 106.0TCID50/ml。
1.1.3 inactivation and examination of virus liquid of SH strain 2 of porcine circovirus
Adding qualified virus liquid into formaldehyde solution (pure by analysis of chemical reagent factory in Luoyang, with content of 37% -40% V/V) to make final concentration of the formaldehyde solution be 0.2% V/V, inactivating at 37 deg.C for 18 hr, stirring every 4 hr for 1 time, each time for 10 min, and storing the inactivated virus liquid at 2-8 deg.C after inactivation.
Performing inactivation test on the inactivated virus solution, inoculating a small amount of inactivated virus solution to PK15 cells which grow into a monolayer, adsorbing the virus solution at 37 ℃ for 1 hour, then abandoning the virus solution, adding new cell maintenance solution, culturing at 37 ℃ for 2 days without cytopathic CPE (CPE), continuously conducting blind propagation for 3 times, changing the cell maintenance solution after growing into a cell monolayer, culturing at 37 ℃ for 2 days, detecting by using an indirect immunofluorescence IFA (fluorescence immunoassay) method, and generating no green PCV2 positive cells, thereby indicating that the porcine circovirus type 2 SH strain virus solution is successfully inactivated.
1.2 preparation and content determination of Mycoplasma hyopneumoniae antigen
1.2.1 preparation and content determination of Mycoplasma hyopneumoniae HN0613 strain
After the freeze-dried strain is unsealed, inoculating a liquid culture medium according to the inoculation amount of 10%, carrying out shaking culture at 37 ℃ for 3-7 days, harvesting when the pH value is reduced from 7.5 to 6.8, and carrying out pure inspection to obtain the first-stage production seeds. Inoculating the first-stage seeds with liquid culture medium at 5% of inoculum size, performing shake culture at 37 deg.C for 3-7 days, harvesting when pH value is reduced from 7.5 to 6.8, and performing pure inspection to obtain second-stage production seeds.
The formula of the mycoplasma hyopneumoniae liquid culture medium is as follows: 300ml of bovine heart extract (BD Co., Ltd.), double distilled water 360m1, adjusted to pH 7.4, and sterilized at 121 ℃ for 15 minutes. The following filter sterilized components were added: hank's balanced salt solution (10X) 40m1, 0.25% W/V phenol red 10m1, horse serum 200m1, 5% W/V hydrolyzed milk protein 100m1, 25% W/V yeast extract 20m1, 10000IU/ml penicillin 10 ml.
Inoculating qualified secondary seeds of the mycoplasma hyopneumoniae HN0613 strain into a liquid culture medium according to 5% V/V, performing shake culture at 37 ℃ for 3-7 days, and harvesting a bacterial solution when the pH value is reduced from 7.5 to 6.8. The collected bacterial solution was concentrated 10-fold V/V using Millipore (Millipore) membrane (molecular cut-off 300 Kda).
The content of the mycoplasma hyopneumoniae bacterial liquid is determined according to a bacterial liquid viable count method, a liquid culture medium containing a phenol red indicator is subpackaged into small test tubes, two rows are arranged, 13 cells are arranged in each row, 1.8 ml/cell is obtained, 0.2ml of a culture to be detected is inoculated into a first small test tube, the culture is evenly mixed and then diluted to a 12 th small test tube by 10 times in sequence, a 13 th tube is a control tube and is placed at 37 ℃ for shake culture, the maximum tube number of color change generated until 21 days is recorded to judge the titer of CCU, and the average value of the two rows of results is obtained. The results show that: the content of Mycoplasma hyopneumoniae HN0613 strain is 1 × 109CCU/ml。
1.2.1.1 inactivation and identification of Mycoplasma hyopneumoniae HN0613 strain liquid
Adding formaldehyde solution into the harvested bacterium solution to enable the final concentration of the formaldehyde solution to be 0.3% V/V, inactivating the formaldehyde solution for 24 hours at 37 ℃, stirring the solution for 1 time every 4 hours and 10 minutes every time, and storing the inactivated bacterium solution at 2-8 ℃ after inactivation is finished.
And (3) performing inactivation inspection on the inactivated bacterial liquid, taking 1ml of the inactivated bacterial liquid, inoculating 50ml of liquid culture medium, culturing at 37 ℃, transplanting once in each of 5 th and 10 th days, continuously culturing and observing for 11 days after the last transplantation, wherein the pH value of the culture medium is not reduced, and the coloring of the culture medium is not changed.
1.2.2 Mycoplasma hyopneumoniae 168 strain antigen
A freeze-dried live vaccine powder of mycoplasma hyopneumoniae 168 strain is purchased from Nanjing Tianbang Biotech Co., Ltd, and each live vaccine powder containing mycoplasma hyopneumoniae 168 strain is 1 × 107CCU。
1.3 preparation and identification of Haemophilus parasuis antigens
1.3.1 preparation of Haemophilus parasuis type 4 JS Strain and type 5 ZJ Strain
The Haemophilus parasuis type 4 JS strain and type 5 ZJ strain are streaked and inoculated on a tryptic soy agar plate (TSA/NAD plate for short) containing 5 percent of newborn bovine serum and 0.005 percent of nicotinamide adenine dinucleotide NAD (American BBI company), and cultured for 24-48 hours at 37 ℃, and more than 5 typical colonies are respectively selected and used as first-class seeds after pure inspection is qualified. And (2) selecting a single colony from the primary seed, inoculating tryptic soy broth (TSB/NAD liquid culture medium for short) containing 5% of newborn bovine serum and 0.005% of NAD, shaking and culturing for 12 hours at 37 ℃ by 180 r/min in a shaking way, sampling gram staining, observing the uniform shape of bacteria under a microscope, conforming to the morphological characteristics of haemophilus parasuis, and taking the bacteria as the secondary seed without any mixed bacteria growth.
Wherein, the preparation method of the TSA/NAD plate containing 5% serum comprises the following steps: adding 945ml of distilled water into 40 g of Tryptic Soy Agar (TSA, BD Difico product), fully shaking, heating to fully dissolve, sterilizing for 15 minutes by high-pressure steam at 121 ℃, cooling to about 60 ℃, adding 50ml of filter-sterilized bovine serum and 5ml of filter-sterilized 1% NAD, fully shaking, and pouring into a flat dish; the preparation method of the TSB/NAD liquid culture medium containing 5% newborn calf serum comprises the following steps: adding 940ml distilled water into 30g Tryptic Soy Broth (TSB, BD Difico product), shaking thoroughly, heating to dissolve thoroughly, sterilizing with high pressure steam at 121 deg.C for 15 min, adding 50ml filter sterilized bovine serum, and 10ml filter sterilized 0.01% NAD.
And respectively inoculating the qualified secondary seeds of the JS strain 4 and the ZJ strain 5 of the haemophilus parasuis in a TSB/NAD liquid culture medium according to the ratio of 1:100V/V, and culturing the secondary seeds in a shaker at 37 ℃ and 200 rpm. Culturing for 12-16 h, and collecting bacterial liquid.
The harvested bacterial liquid (JS strain 4 type and ZJ strain 5 type of haemophilus parasuis) is respectively centrifuged by a continuous centrifuge (10000 r/min), then re-dissolved by PBS solution to the volume before centrifugation, and then concentrated by 5 times V/V by a Millipore membrane (molecular cut-off is 300 Kda).
Sampling bacterial liquid, diluting 10 times in series according to method known by skilled person, and taking 10 times-6、10-72 dilutions were inoculated into the aforementioned TSA/NAD solid medium, 0.1ml of each plate, and after 24 hours of incubation at 37 ℃, plates with colony counts between 30 and 300 were selected for colony counting. The counting result shows that: the content of the Haemophilus parasuis type 4 JS strain and the 5 ZJ strain are both 5 multiplied by 109CFU/ml。
1.3.2 inactivation and identification of bacterial solutions of Haemophilus parasuis type 4 JS strain and type 5 ZJ strain
Adding formaldehyde solution into the harvested bacterium solution to enable the final concentration of the formaldehyde solution to be 0.3% V/V, inactivating the formaldehyde solution for 24 hours at 37 ℃, stirring the solution for 1 time every 4 hours and 10 minutes every time, and storing the inactivated bacterium solution at 2-8 ℃ after inactivation is finished.
And (3) performing inactivation inspection on the inactivated bacterial liquid, preparing 6 plates of TSA/NAD solid culture medium, dropwise adding 1 drop of the inactivated bacterial liquid on 3 TSA/NAD solid culture medium plates in an aseptic operation, drawing lines by using an inoculating ring, culturing in a common incubator at 37 ℃, and simultaneously setting 3 non-inoculated TSA/NAD solid culture medium as a control. After 24 hours no bacterial growth was observed in the plates, while the control, 2 media without inoculation, should also be free from bacterial growth, and the observation continued until no bacterial growth occurred in 6 plates at 48 hours.
Example 2 preparation of adjuvants
Preparing carbomer mother solution: dissolving carbomer 971P in distilled water, standing overnight at room temperature to fully swell it to obtain 20mg/ml carbomer solution, adding 3M sodium hydroxide solution to adjust pH to 7.2, adding appropriate amount of distilled water to adjust carbomer solution to obtain 15mg/ml carbomer mother liquor.
Preparing a sodium carboxymethylcellulose mother liquor: mixing 1g of sodium carboxymethylcellulose with 100ml of distilled water, heating and boiling until the sodium carboxymethylcellulose is completely dissolved, and then cooling to room temperature to prepare sodium carboxymethylcellulose mother liquor with the concentration of 10 mg/ml.
Preparation of ISCOM matrix mother liquor: adding 12mg/ml of lipid mixture stock solution into PBS solution, adding 30mg/ml of QuilA stock solution to make the final concentration of QuilA be 15mg/ml, phosphatidylcholine be 3mg/ml and cholesterol be 3mg/ml, fully mixing them uniformly, making ice-bath ultrasonic treatment, standing at room temperature for 1.5 hr, transferring into dialysis bag, using PBS solution as external liquor, dialyzing at room temperature for 3 days, using 0.22 micrometer filter membrane to make sterilization, and storing at 4 deg.C to obtain ISCOM matrix mother liquor with 15mg/ml of QuilA final concentration.
The mother liquor is sterilized under high pressure at 121 ℃ for 30 minutes for standby.
EXAMPLE 3 preparation of inactivated vaccine composition and animal experiments
3.1 preparation of inactivated vaccine composition
The antigen prepared in example 1 and the mother solution of each component of the adjuvant prepared in example 2 were diluted with PBS solution, so that the final concentrations of the diluted antigen solutions and the adjuvant contained in the inactivated vaccine composition in Table 1 were mixed, stirred at a rotation speed of 500-.
TABLE 1 inactivated vaccine compositions containing the components
3.2 efficacy testing of porcine circovirus antigens in inactivated vaccine compositions
Vaccines 1-1, 1-2, 1-3, 1-6, 1-7, 1-9, 2-1, 2-2, 2-3, 2-6, 2-7, 2-9 prepared in example 3.1 and a reference vaccine control group (PCV2 vaccine, produced by procheco bioengineering gmbh) were injected intramuscularly to the neck of piglets 14-21 days old (excluding porcine reproductive and respiratory syndrome, porcine circovirus type 2 and swine fever), 5 piglets/group and 2 ml/head, while a blank control group was set, and 14 groups (70 piglets in total) were set. Each pig was bled 21, 35, 60, and 90 days after immunization, and sera were collected and stored at-20 ℃ until testing.
Evaluating the immune effect of the porcine circovirus antigen part according to a serological method, which comprises the following steps:
(1) ELISA antibody detection with Escherichia coli expression of PCV2ORF2 protein (according to Chinese patent CN101101296A preparation) as antigen, by matrix titration test to determine the antigen optimal coating concentration. Diluting the antigen to the optimal coating concentration, coating an enzyme label plate with 100 mul/hole, and then coating overnight at 4 ℃; washing for 3 times, each time for 3-5 minutes; adding 200 μ l of 0.15% M/V BSA blocking solution to each well, and allowing the mixture to act at 37 ℃ for 2 hours; washing; diluting the serum to be detected by PBS (phosphate buffer solution) in a multiple ratio, adding 100 mu 1 to each hole of each sample in a line, and acting at 37 ℃ for 1 h; washing; then adding enzyme-labeled SPA (diluted by 1:10000 times), 100 mu l/hole, and acting at 37 ℃ for 1 h; washing; adding substrate solution TMB for color development, and finally using 2mol/L H2SO4The reaction was terminated. And (4) judging a result: OD of serum to be detected450Value/negative serum OD450The value is more than or equal to 2.1, and the positive result is obtained.
(2) The serum neutralization test employs a fixed virus dilution serum method. Heating the serum to be detected at 56 ℃ for 30 minutes, centrifuging at 10000 rpm for 5 minutes, carefully sucking out the supernatant, diluting at 1:2, and then diluting by multiple times; the cells were mixed with an equal amount of PCV2 virus solution, and inoculated into 96-well plates containing a monolayer of PK15 cells at 37 ℃ for 1h, 100 mu 1/well, 4 wells for each dilution, and cell control and virus control wells were set. Culturing at 37 deg.C for 12h, treating with 300mmol/L D-glucosamine, culturing at 37 deg.C for 48h, fixing cells with 80% acetone, and determining the number of wells containing fluorescence at each dilution by indirect immunofluorescence. The maximum dilution of serum in the cell well that can suppress the specific fluorescent cell count by 50% was taken as the neutralization titer of the serum to be tested, and the average value per group was calculated.
The results of the serological evaluation are shown in Table 2.
TABLE 2 efficacy test results for porcine circovirus antigens of inactivated vaccine compositions
Note: the PCV2 vaccine used in the reference vaccine control group was produced by Poleco bioengineering GmbH, and had an antigen content of 107.0TCID50Perml, twice immunizations were performed according to the manufacturer's instructions.
As can be seen from Table 2: when the groups A1-1 to A1-3 and A2-1 to A2-3 contain the same amount of PCV2 antigen (single vaccine) and have different adjuvant contents, the generated antibodies after immunization slightly increase along with the increase of the adjuvant content, are basically consistent with the antibodies generated by the PCV2 reference single vaccine with high antigen content and can last for at least 60 days; the antibody produced after the PCV2 combined vaccine containing the same amount of adjuvant is basically consistent with the antibody produced by a PCV2 reference single vaccine and can last for at least 60 days, which shows that the vaccines 1-1, 1-2, 1-3, 1-6, 1-7, 1-9, 2-1, 2-2, 2-3, 2-6, 2-7 and 2-9 can stimulate the organism to produce PCV2 specific resistance after respectively immunizing animals, and the effect is reliable, and other antigens of the vaccines 1-6, 1-7, 1-9, 2-6, 2-7 and 2-9 have no influence on the immune effect of the PCV2 antigen; and the adjuvant in the vaccine composition contains carbomer, ISCOM and sodium carboxymethylcellulose, and has better immune effect than the adjuvant containing carbomer and sodium carboxymethylcellulose.
3.3 efficacy testing of Mycoplasma hyopneumoniae antigens in inactivated vaccine compositions
The vaccine compositions 1-4, 1-6, 1-8, 1-9, 2-4, 2-6, 2-8 and 2-9 prepared in example 3.1 were injected intramuscularly to piglets of 14-21 days old (excluding porcine reproductive and respiratory syndrome, porcine circovirus type 2 and swine fever), 5 piglets/group and 2 ml/head, respectively, at the same timeA reference vaccine control group and a challenge control group are arranged, and 10 groups (50 piglets in total) are arranged. Observing body temperature and side reaction to 28 days after immunization, and injecting Mycoplasma hyopneumoniae Jinan virulent strain CVCC354 (with a deposit number of CVCC354 and a preservation unit of national veterinary microorganism strain preservation and management center) 100MID into all pig tracheas at 28 days50First, dissect after 28 days of observation and observe lung lesions, which were scored according to 28 points. Before and after the challenge, the piglets in the test group were weighed, and the average daily gain was calculated.
After about 10 days of the challenge, 4 pigs in the challenge control group showed symptoms of cough, asthma and the like successively, and the skin and hair were not smooth; 1 pig in the reference vaccine control group showed cough and had side effects; pigs in the other immunised groups did not show similar symptoms of asthma. Before and after the challenge, the average score of pneumonia lesions and the average daily gain result of pigs in each test group are shown in table 3.
TABLE 3 efficacy test results for Mycoplasma hyopneumoniae antigens of inactivated vaccine compositions
Note 1: the vaccine selected by the reference vaccine control group is a swine mycoplasma pneumonia inactivated vaccine (strain J) in the existing product, namely, Anbach (M + PAC), and the immunization is carried out according to the manufacturer's instructions.
Note 2: the body temperature is "none" indicating that a transient body temperature rise does not occur, and "some" indicating that a transient body temperature rise occurs, wherein the transient body temperature rise means that the body temperature briefly assumes 40.5 degrees on days 1-2 after the second immunization and returns to normal body temperature on day 3.
Note 3: the side reaction indicates that no side reaction occurs, and the side reaction indicates that the side reaction occurs, wherein the side reaction refers to the occurrence of at least one of transient mental depression, anorexia and anaphylaxis.
Note 4: in statistical analysis of differences, when compared among groups, the ones with the same letter indicate no significant difference, and the ones with different letters indicate significant difference (p < 0.05).
As can be seen from Table 3: groups B1-1 to B1-4 (namely, vaccines 1-4, 1-6, 1-8 and 1-9 respectively) and groups B2-1 to B2-4 (namely, vaccines 2-4, 2-6, 2-8 and 2-9 respectively) are immunized by using single vaccine or combined vaccine containing mycoplasma hyopneumoniae antigen and adjuvant with the same content, and then are subjected to challenge treatment, so that the immunized pigs have no excessive temperature rise and no side reaction, the reduction rate of pneumonia lesions is respectively more than 73 percent and more than 84 percent, the average daily gain is respectively 0.0412-0.0415kg and 0.0445-0.0453kg, and the groups B1 and B2 have no significant difference (p is more than 0.05); compared with a reference vaccine control group, the differences are all significant (p is less than 0.05); compared with the challenge control group, the differences are obvious (p is less than 0.05); and the adjuvant in the vaccine composition contains carbomer, ISCOM and sodium carboxymethylcellulose, and the immune effect is remarkably superior to that of the adjuvant containing carbomer and sodium carboxymethylcellulose (p is less than 0.05).
3.4 efficacy testing of Haemophilus parasuis antigens in inactivated vaccine compositions
Respectively injecting the vaccine compositions 1-5, 1-7, 1-8, 1-9, 2-5, 2-7, 2-8 and 2-9 prepared in the example 3.1 into piglets of 14-21 days of age (excluding porcine reproductive and respiratory syndrome, porcine circovirus type 2 and swine fever), 5 piglets/group and 2 ml/head through neck muscle injection, and simultaneously arranging a reference vaccine control group and a challenge control group for 10 groups (named as C1-1, C1-2, C1-3, C1-4, C2-1, C2-2, C2-3, C2-4, reference vaccine control group 1 and challenge control group 1); at the same time, 10 groups (named as C1-5, C1-6, C1-7, C1-8, C2-5, C2-6, C2-7, C2-8, reference vaccine control group 2 and challenge control group 2) were tested in duplicate, and the details are shown in Table 4.
The vaccine is administered 28 days after immunization, and the first 10 groups are administered 3ml of Haemophilus parasuis type 4 intraperitoneal injection at a dose of 9.0 × 109CFU/head; the last 10 groups were administered 3ml intraperitoneal injection of Haemophilus parasuis type 5 with a dose of 6.0 × 109CFU/head, after attacking, the clinical manifestations of each group of pigs were observed, and after 14 days of observation, pathological observation was performed by killing. Before and after attacking toxinThe test piglets were weighed to calculate the average daily gain.
TABLE 4 efficacy test results for Haemophilus parasuis antigens of inactivated vaccine compositions
Note 1: the inactivated vaccine of Haemophilus parasuis disease used in reference vaccine control group is produced by Chengdu pharmaceutical machinery factory of Zhongmu practical industry, and has antigen content of 4.0 × 109CFU/head, twice immunizations were performed according to the manufacturer's instructions.
Note 2: in statistical analysis of differences, when compared among groups, the ones with the same letter indicate no significant difference, and the ones with different letters indicate significant difference (p < 0.05).
As can be seen from Table 4: groups C1-1 to C1-4, groups C1-5 to C1-8 (i.e. immunization vaccines 1-5, 1-7, 1-8 and 1-9 respectively), groups C2-1 to C2-4 and groups C2-5 to C2-8 (i.e. immunization vaccines 2-5, 2-7, 2-8 and 2-9 respectively) are immunized by using single vaccine or combined vaccine containing haemophilus parasuis antigen and adjuvant with the same content, and then challenge, the result shows that the immunized pigs are completely protected, the average daily gain is 0.0311-0.0317kg and 0.0388-0.0344kg respectively, and the differences among groups C1 and C2 are not significant (p is more than 0.05); compared with a reference vaccine control group, the low antigen content can also ensure high immune protection efficiency and obvious average daily gain difference (p is less than 0.05); compared with the challenge control group, the average daily gain difference is obvious (p is less than 0.05); and the adjuvant in the vaccine composition contains carbomer, ISCOM and sodium carboxymethylcellulose, and the immune effect is remarkably superior to that of the adjuvant containing carbomer and sodium carboxymethylcellulose (p is less than 0.05).
EXAMPLE 4 preparation of live vaccine compositions and animal testing
4.1 preparation of live vaccine of Mycoplasma hyopneumoniae vaccine (strain 168)
The live vaccine composition was prepared as shown in table 5 using the mycoplasma hyopneumoniae live vaccine (168 strain) prepared in example 1.2 as an antigen and the adjuvant prepared in example 2 diluted with PBS as a live vaccine diluent, and 1 part of the mycoplasma hyopneumoniae live vaccine (168 strain) was dissolved in 2ml of the live vaccine diluent.
TABLE 5 dilution of the Mycoplasma hyopneumoniae live vaccine containing the components and final concentration
4.2 animal test of live vaccine against Mycoplasma hyopneumoniae (strain 168)
The vaccine compositions prepared in example 4.1 were used for immunizing 45 piglets (excluding porcine reproductive and respiratory syndrome, porcine circovirus type 2 and swine fever) of 14-21 days old, 5 piglets/group and 2ml piglets/head by neck intramuscular injection according to table 5, and a reference vaccine control group, a prior art control group and a challenge control group are simultaneously arranged for 9 groups.
Observing body temperature and side reaction to 28 days before and after immunization, and injecting Mycoplasma hyopneumoniae and south China virulent strain CVCC354 (with a deposit number of CVCC354 and a preservation unit of national veterinary microorganism strain preservation and management center) 100MID into trachea of all pigs at 28 days50First, dissect after 28 days of observation and observe lung lesions, which were scored according to 28 points. Before and after the challenge, the piglets in the test group were weighed, and the average daily gain was calculated.
Safety test results: the body temperatures of the immune groups I-VI have no obvious change, and no obvious pathological change is found when lung lesions are observed after killing.
The result of the challenge test is as follows: after about 10 days of toxin attack, the pigs (4 of them) in the toxin attack control group have symptoms of cough, asthma and the like, and the skin and hair are not smooth; one pig in each of group VII and VIII showed cough; no pigs in the other groups showed similar symptoms of asthma. Before and after the challenge, the average score of pneumonia lesions and the average daily gain result of each test group of pigs are shown in table 6.
TABLE 6 porcine mycoplasma live vaccine immunization post observation and efficacy test results
Note 1: the reference vaccine control group is zhibitung produced by Nanjing Tianbang Biotechnology Co., Ltd, and intrapulmonary injection is carried out according to the manufacturer's instructions; the control group in the prior art is a mycoplasma hyopneumoniae live vaccine (168 strain) prepared according to Chinese patent CN103071151B, contains a special diluent for the vaccine mentioned in the patent, and is immunized in a neck intramuscular injection mode according to the patent at intervals of 2 weeks for 2 times.
Note 2: the body temperature is "none" indicating that a transient body temperature rise does not occur, and "some" indicating that a transient body temperature rise occurs, wherein the transient body temperature rise means that the body temperature briefly assumes 40.5 degrees on days 1-2 after the second immunization and returns to normal body temperature on day 3.
Note 3: the side reaction indicates that no side reaction occurs, and the side reaction indicates that the side reaction occurs, wherein the side reaction refers to the occurrence of at least one of transient mental depression, anorexia and anaphylaxis.
Note 4: in statistical analysis of differences, when compared among groups, the ones with the same letter indicate no significant difference, and the ones with different letters indicate significant difference (p < 0.05).
As can be seen from Table 6:
(1) groups I-III (i.e., immunization live vaccines 1-3) were immunized by intramuscular injection of Mycoplasma hyopneumoniae live vaccine (168 strain) diluted with an adjuvant containing carbomer and sodium carboxymethylcellulose, followed by challenge, and the results showed that: the immune effect (pneumonia lesion reduction rate and average daily gain) is increased along with the increase of the content of each component in the adjuvant, the immune pig does not generate a transient temperature increase and side reaction, and the difference of the immune effect among all groups is obvious (p is less than 0.05); compared with the VII group (namely the reference vaccine control group, the immune component is the zhibituning), the heterodynia is obvious (p is less than 0.05) except the I group, and the intramuscular injection is simpler and more convenient than the intrapulmonary injection and has higher acceptability; compared with VIII group (i.e. the prior art control group), the heterodynia is obvious (p is less than 0.05) except the I group, the immunization frequency is low, the immunization cost is reduced, the immunization program is saved, and the method is economical and reliable; compared with the challenge control group, the differences are all significant (p is less than 0.05).
(2) Groups IV-VI (i.e., immunization live vaccines 4-6) were immunized by intramuscular injection of mycoplasma hyopneumoniae live vaccine (168 strain) in the neck using an adjuvant comprising carbomer, ISCOM, sodium carboxymethylcellulose, followed by challenge, and the results showed that: the immune effect (pneumonia lesion reduction rate and average daily gain) is increased along with the increase of the content of each component in the adjuvant, the immune pig does not generate a transient temperature increase and side reaction, and the difference among the groups is obvious (p is less than 0.05); compared with groups I-III, the immune effect of the adjuvant diluted live vaccine with the same content of components except ISCOM is better, and the difference is obvious (p is less than 0.05); compared with the VII group (namely the reference vaccine control group, the immune component is the zhibituoning), the differences are obvious (p is less than 0.05), and the intramuscular injection is simpler and more convenient to operate and has higher acceptability than the intrapulmonary injection; compared with the VIII group (namely the prior art control group), the difference is obvious (p is less than 0.05), the immunization frequency is low, the immunization cost is reduced, the immunization program is saved, and the method is economical and reliable; compared with the challenge control group, the differences are all significant (p is less than 0.05).
EXAMPLE 5 use of protein stabilizing solutions
5.1RHDV VP60 protein stability Studies
An immunogenic VP60 protein of Rabbit Hemorrhagic Disease Virus (RHDV) is prepared according to the method described in Chinese patent CN103555766A, the expressed VP60 protein is identified and confirmed to be purified undoubtedly, and the agglutination value of human O-type red blood cells is detected, and the detection result shows that the agglutination value HA of the VP60 protein is 1:217。
The mother solutions of the adjuvant components prepared in example 2, i.e. carbomer mother solution, ISOCM matrix mother solution and sodium carboxymethylcellulose mother solution, were used to prepare VP60 protein stabilizing solution, and RHDV VP60 protein solution (solutions 1-2) was prepared by diluting with PBS so that the final concentrations of the components were: VP60HA is 1:214Carbomer at 1mg/ml and sodium carboxymethylcellulose at 0.1 mg/ml. Meanwhile, solutions (solutions 3 to 4) were prepared so that the final concentrations of the respective components were: VP60HA is 1:214Carbomer 1mg/ml, saponin 0.1mg/ml, carboxymethyl celluloseThe content of vitamin sodium is 0.1 mg/ml.
2 portions of the same dose of RHDV VP60 protein solution were stored at 2-8 ℃ for 6, 12, 18, 24, 30, 36 months, respectively, and 2 portions of VP60 protein solution diluted with PBS were used as a control to perform hemagglutination assay on the protein solutions under different storage conditions, and the results are shown in Table 7.
TABLE 7 RHDV VP60 protein stability test results
As can be seen from Table 7: the solution containing carbomer and sodium carboxymethylcellulose serves as an RHDV VP60 protein stabilizing solution to stabilize VP60 protein for at least 24 months, the solution containing carbomer, ISCOM and sodium carboxymethylcellulose serves as an RHDV VP60 protein stabilizing solution to stabilize VP60 protein for at least 30 months, and HA of a control group taking PBS as a storage solution is gradually reduced along with the prolonging of time, which indicates that the protein is degraded to a certain extent or irreversibly changed.
5.2PCV2Cap protein stability study
An immunogenic Cap protein of Porcine circovirus type 2 (PCV 2) is prepared according to the method described in Chinese patent CN104017813A, the expressed Cap protein is identified and confirmed undoubtedly and then purified, and the concentration of Cap protein is quantified according to the method of the BCA protein quantification kit (purchased from Merck company) specification, and the result shows that: the concentration of Cap protein was 2 mg/ml.
Mother solutions of the adjuvant components prepared in example 2, namely carbomer mother solution, ISOCM matrix mother solution and sodium carboxymethylcellulose mother solution are used for preparing Cap protein stabilizing solution, and PCV2Cap protein solution (solutions 1-2) is prepared by diluting PBS, so that the final concentration of each component is as follows: the concentration of Cap protein is 0.40mg/ml, carbomer is 1mg/ml, and sodium carboxymethylcellulose is 0.1 mg/ml. Meanwhile, solutions (solutions 3 to 4) were prepared so that the final concentrations of the respective components were: the concentration of Cap protein is 0.40mg/ml, carbomer is 1mg/ml, saponin is 0.1mg/ml, and sodium carboxymethylcellulose is 0.1 mg/ml.
2 portions of PCV2Cap protein solution with the same dosage are stored at 2-8 ℃ for 6, 12, 18, 24, 30 and 36 months respectively, and 2 portions of Cap protein solution diluted by PBS are used as a control, and the BCA measurement is carried out on the protein solution under different storage conditions respectively, and the results are shown in Table 8.
TABLE 8 PCV2Cap protein stability test results
As can be seen from Table 8: the solution containing carbomer and sodium carboxymethylcellulose is used as PCV2Cap protein stabilizing solution to stabilize Cap protein for at least 24 months, the solution containing carbomer, ISCOM matrix and sodium carboxymethylcellulose is used as PCV2Cap protein stabilizing solution to stabilize Cap protein for at least 30 months, and the concentration of Cap protein in a control group using PBS as a preservation solution is gradually reduced along with the prolongation of the preservation time, so that the protein is degraded to a certain extent or irreversibly changed.
In conclusion, the vaccine composition containing carbomer, the immunostimulating complex and the sodium carboxymethylcellulose adjuvant has the immune effect which is remarkably superior to that of the vaccine composition containing carbomer and the sodium carboxymethylcellulose, the immune response level of the vaccine composition (1) after the animal is immunized is increased along with the increase of the adjuvant content, the immune response level is basically consistent with that of the existing commercial vaccine, the immune duration is longer, and the effect is exact; (2) when the antigen content is lower, the animal can obtain better immune response level and complete protection, the immune frequency is less, the stress reaction of the pig is reduced or even eliminated, and no side reaction occurs; (3) the adjuvant can also be used for diluting the live vaccine, so that the live vaccine immunizes pigs in an intramuscular injection mode, the pigs have no transient body temperature rise and side reaction, the pneumonia disease rate of the pigs is less, the average daily gain is increased, and the like, the good immune effect is better than that of the existing live vaccine, and the operation is simple and convenient, the cost is reduced, the program is saved, and the vaccine is economical and reliable; (4) the adjuvant can be used as protein stabilizing solution to stabilize protein without degradation or irreversible change.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. An adjuvant for a vaccine containing one or more antigens of porcine circovirus antigen, mycoplasma hyopneumoniae antigen and haemophilus parasuis antigen, wherein the adjuvant is an acrylic polymer and cellulose ether; the cellulose ether is sodium carboxymethylcellulose, the acrylic acid polymer is carbomer, the carbomer is 1mg/ml, and the sodium carboxymethylcellulose is 0.1 mg/ml; or
Carbomer is 4mg/ml, and sodium carboxymethylcellulose is 4 mg/ml; or
Carbomer 10mg/ml, sodium carboxymethylcellulose 8 mg/ml.
2. An adjuvant for a vaccine containing one or more antigens of porcine circovirus antigen, mycoplasma hyopneumoniae antigen and haemophilus parasuis antigen, wherein the adjuvant is an acrylic polymer, cellulose ether and an immunopotentiator; the cellulose ether is sodium carboxymethylcellulose, the acrylic polymer is carbomer, the immunopotentiator is ISCOM, the carbomer is 1mg/ml, the sodium carboxymethylcellulose is 0.1mg/ml, and the ISCOM is 0.1 mg/ml; or
Carbomer at 4mg/ml, sodium carboxymethylcellulose at 4mg/ml, and ISCOM at 4 mg/ml; or
Carbomer at 10mg/ml, sodium carboxymethylcellulose at 8mg/ml, and ISCOM at 10 mg/ml.
3. A vaccine composition comprising an immunizing amount of antigen and the adjuvant of any one of claims 1-2; the antigen comprises one or more of porcine circovirus antigen, mycoplasma hyopneumoniae antigen and haemophilus parasuis antigen; the porcine circovirus antigen is a porcine circovirus SH strain inactivated antigen, the porcine mycoplasma hyopneumoniae antigen is a porcine mycoplasma hyopneumoniae HN0613 strain inactivated antigen, and the haemophilus parasuis antigens are inactivated antigens of 4 type JS strains and 5 type ZJ strains of haemophilus parasuis.
4. According to claim3, wherein the content of the inactivated antigen of the porcine circovirus SH strain is 5 multiplied by 105 TCID50The content of the inactivated antigen of the mycoplasma hyopneumoniae HN0613 strain is 1 multiplied by 108CCU/head part, the content of the Haemophilus parasuis type 4 JS strain is 1 x 109The content of the inactivated antigen of the CFU/head and the Haemophilus parasuis type 5 ZJ strain is 1 multiplied by 109CFU per head.
5. An antigenic protein stabilizing agent, wherein said stabilizing agent comprises an acrylic acid-based polymer and a cellulose ether; the cellulose ether is sodium carboxymethylcellulose, the acrylic acid polymer is carbomer, the carbomer is 1mg/ml, and the sodium carboxymethylcellulose is 0.1 mg/ml.
6. An antigenic protein stabilizing agent, wherein said stabilizing agent comprises an acrylic acid-based polymer, a cellulose ether, and a saponin-containing composition; the cellulose ether is sodium carboxymethylcellulose, the acrylic acid polymer is carbomer, the saponin-containing composition is an immunostimulating complex, the carbomer is 1mg/ml, the sodium carboxymethylcellulose is 0.1mg/ml, and the immunostimulating complex is 0.1 mg/ml.
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| CN109806391A (en) * | 2017-11-22 | 2019-05-28 | 西北民族大学 | The preparation method and applications of Water Soluble Compound adjuvant are immunized in a kind of live vaccine |
| CN108245675B (en) * | 2018-02-02 | 2021-11-30 | 四川省畜牧科学研究院 | Live vaccine diluent, preparation method and application thereof, and vaccine product |
| CN110404065B (en) * | 2018-04-27 | 2023-06-16 | 洛阳赛威生物科技有限公司 | Adjuvant composition for pigs and preparation method thereof |
| CN111346224B (en) * | 2018-12-24 | 2023-12-08 | 洛阳赛威生物科技有限公司 | Immunoadjuvant composition, preparation method and application thereof |
| CN110898218B (en) * | 2019-12-30 | 2023-10-10 | 瑞普（保定）生物药业有限公司 | Duck tembusu virus inactivated vaccine and preparation method thereof |
| CN112522212B (en) * | 2020-12-16 | 2023-03-24 | 湖北省农业科学院畜牧兽医研究所 | Porcine epidemic diarrhea virus strain and combined immunization method of inactivated vaccine and interferon thereof |
| CN113244390B (en) * | 2021-05-28 | 2023-03-24 | 西南大学 | Triple egg yolk antibody freeze-dried powder and preparation method and application thereof |
| CN115444933B (en) * | 2022-09-14 | 2023-08-29 | 贵州福斯特生物科技有限公司 | Preparation method and application of water-soluble composite adjuvant for rabbits |
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