CN106421775A - Adjuvant for vaccine, vaccine composition containing adjuvant and application of vaccine composition - Google Patents
Adjuvant for vaccine, vaccine composition containing adjuvant and application of vaccine composition Download PDFInfo
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Abstract
The invention provides an adjuvant for a vaccine. The adjuvant is prepared from alumina gel, chitosan and lecithin. The invention further discloses vaccine composition containing the adjuvant and an application of the vaccine composition. The adjuvant can make an organism produce effective humoral immunity when used for the inactivated vaccine, cannot cause body temperature rise and side effects and can have better immune response during lower antigen content and single immunity.
Description
Technical field
The invention belongs to veterinary biologicses technical field is and in particular to be used for enhancement antigen
The adjuvant of immunne response.
Background technology
Antibacterial, virus and parasitic infection are distributed widely in humans and animals, by these infection
The disease usual combating microorganisms medical therapy that agent causes has resistance, from without effective
Therapeutic Method.Therefore, the more generally used vaccinology in this area method, to control infection, has
Body is by the pathogen with living, the pathogen through inactivation or its product, or through gene work
Pathogen protein subunit is prepared into vaccine by journey means, carries out vaccination to induce spy
Specific immunological.However, when individually giving some antigen it is impossible to produce to immune system
Enough stimulations.Therefore, it is necessary to add make exempting from of immune response increase
Epidemic disease adjuvant is obtaining the protection antibody of q.s.
Immunological adjuvant is the requisite ingredient of vaccine, can not only affect body to epidemic disease
The intensity of Seedling immunne response, and can answer for the maximally effective immunity of special pathogen induction
Answer type (Mbow ML, De Gregorio E, Valiante NM, et al.New
adjuvants for human vaccines.Current opinion in immunology,2010,
22(3):411-416).At present, the immunological adjuvant type of research is a lot, such as aluminium glue, oil
Adjuvant, Freund adjuvant, microorganism and its metabolite, nucleic acid and the like, cell
(Dey AK, the Srivastava IK.Novel adjuvants and such as the factor, liposome
delivery systems for enhancing immune responses induced by
immunogens.Expert review of vaccines,2011,10:227-251), but by
In there are some inevitable defects such as different degrees of toxic and side effects or potential safety hazard and
It is difficult to practical application (Mbow ML, De Gregorio E, Valiante NM, Rappuoli
R.New adjuvants for human vaccines.Current opinion in
immunology,2010,22(3):411-416；Batista-Duharte A,Lindblad EB,
Oviedo-Orta E.Progress in understanding adjuvant immunotoxicity
mechanisms.Toxicology letters,2011,203(2):97-105), these are led to
Adjuvant far can not meet the demand of new generation vaccine development.Especially, substantial amounts of clinic is exempted from
Epidemic disease test thinks that aluminium glue adjuvant has some problems：(1) body fluid of Th2 mediation is exempted from
Epidemic disease reaction has very strong excitation, and the work of the cell immune response to Th1 mediation
With weaker, be only applicable to antibody be main protective immunity vaccine (Exley C,
Siesjo P,Eriksson H.The immunobiology of aluminum adjuvants:
how do they really work？Trends in immunology,2010,31:
103-109)；(2) IgE antibody can be promoted to produce, easily induction body produces allergy
Reaction；(3) there is local response, granuloma can be formed, local extremely can be occurred individually aseptic
Property abscess；(4) because of its physicochemical property, vaccine containing aluminium glue is afraid of to freeze, and after jelly, aluminium glue vaccine holds
Changeableness；(5) people, animal nervous system may be had an impact (see Chinese patent
CN101402666A).Thus, the immunological adjuvant found safely, effectively, new is
One focus (Schijins VEJC, Lavelle EC.Teends of current vaccines research field
in vaccine adjuvants.Expert review of vaccines,2011,10(4):
539-550).And, immunological adjuvant research has been listed in the pioneer field of vaccine research
(Harandi AM,Medaglini D,Shattock RJ.Vaccine adjuvants:A
priority for vaccine research.Vaccine,2010,28(12):2363-2366；
Harand AM,Brewe J,Schijn V.Conference scene:recent
advancements in immunopotentiators for modern vaccines.
Immunotherapy,2011,3(11):1297-1301).
In addition, the vaccine of current commercialization is (as bloodthirsty in Mycoplasma hyopneumoniae bacterin, secondary pig
Bacillus vaccine) be by the culture medium containing serum grow gained organism preparation and
Become, thus serum component present in immune material is (as immune complex or nonimmune former
Property specific proteinses) can induce side reaction occur (see Chinese patent CN104334186A).
And, body temperature occurs after finding existing commercialized vaccine immunity pig during clinical practice
The phenomenon raising, the vaccine involved by other prior arts also has similar report such as China specially
Sharp CN103194413A, CN104312964A.
Content of the invention
In order to solve the deficiencies in the prior art, the invention provides a kind of vaccine adjuvant and
Vaccine combination containing this adjuvant and its application.
A first aspect of the present invention is to provide a kind of vaccine adjuvant, and described adjuvant includes
Aluminium glue, shitosan.
The invention still further relates to a kind of vaccine adjuvant, described adjuvant include aluminium glue, shitosan and
Phospholipid.
Vaccine adjuvant of the present invention has with low cost, easy to use, immunocompetence
Strong the advantages of, the vaccine combination containing this adjuvant can not only make animal body generation body fluid exempt from
Epidemic disease, cellular immunization, also do not result in body temperature rising, side reaction occurs.
It is another aspect of the invention to provide a kind of vaccine combination, described vaccine combination
Thing comprises the antigen of vaccine adjuvant of the present invention and immune effective dose.
Vaccine combination containing adjuvant of the present invention is antigenic content is relatively low, single immunization when
Animal can be made to obtain preferable immunne response level.
The invention still further relates to the antigen containing mycoplasma hyopneumoniae of present invention preparation and secondary pig are bloodthirsty
The vaccine combination of bacteroides antigen and vaccine adjuvant is in preparation prevention and treatment hyopneumoniae
Mycoplasma and haemophilus parasuises infect the application in the medicine of relevant disease.
Immunne response level after the vaccine adjuvant of the present invention and antigen co-immunization animal
It is on close level or even higher with the immunne response of existing commercial vaccine, the antibody persistent period
Also longer, no body temperature raises, no side reaction occurs, effect stability；And in antigenic content
Also animal can be made when relatively low to obtain preferable immune effect, immune time also can make pig less
Stress reduces even disappearance.
Specific embodiment
Hereinafter, embodiments of the present invention are illustrated.
A first aspect of the present invention is to provide a kind of vaccine adjuvant, and described adjuvant includes
Aluminium glue, shitosan.
Term " adjuvant " refers to, when with antigen together with casting, make experimenter anti-to this
The enhanced compound of former immunoreation.
Term " aluminium glue ", also known as aluminium salt or aluminium adjuvant, is well-known in the art,
And as in Harlow E. and D.Lane (Antibodies:a laboratory manual gold
Spring harbor laboratory.1988) and Nicklas W. (Aluminum salts,
research in immunology.1992,143:Described in 489-493), can extensively answer
For vaccine preparation.Aluminium glue includes but is not limited to aluminium hydroxide (Aluminium hydroxide
Or Amphogel, also known as the alumina hydrate, hydrate of aluminum, aluminium trihydrate, amorphous
Aluminium oxide, including monohydrate alumina, Alumina hydrate ATH), aluminum phosphate, sulfur
One or more of sour aluminum, ammonia-alum, potassium alum or aluminium silicate, preferably aluminium hydroxide
Or aluminum phosphate.Described aluminium glue can also include following arbitrary product such asHPA
(General Chemical,LLC)、M9400SP(J.M.Huber
Corporation)、(Merck)、(Brenntag
Biosector)、Aluminum hydroxide(Sigma-aldrich、Watson、Bisely
＆Company Pty Ltd., Noah thchnologies corporation or CellMark
USA, LLC), gel aluminum hydroxide (Inner Mongol Xin Hong bio tech ltd),
20% aluminium hydroxide gel normal saline (Zhongmu Industry Co., Ltd).
Term " shitosan " (Chitosan) include natural chitosan, modification of chitosan,
At least one in shitosan salt, chitosan derivatives, can obtain from multiple manufacturers
, described manufacturer includes KitoZyme SA, Fluka chemine AG, the
NovaMatrix unit of FMC Biopolymer AS, Heppe Medical and
Sigma-Aldrich Co..Shitosan is by the cationic polymerization of glucamine monomer composition
Thing, can have multiple number-average molecular weights, such as from about 400-2000kDa, about 10-500kDa or
About 10-100kDa, or as the below about Ultra-low molecular weight material of 50kDa number-average molecular weight,
The low molecular weight material of about 50-200kDa number-average molecular weight, the equal molecule of about 200-500kDa number
The high molecular weight material of the middle-molecular-weihydroxyethyl material of amount, greater than about 500kDa number-average molecular weight,
Also can be de- through chitin (poly-n-acetyl base-D-Glucose amine) by hydrolysis
Acetyl effect and eliminate N- Acetyl Groups to synthesize.Wherein, described natural chitosan is such as
The natural polysaccharide extracting from the marine product garbage such as Crusta Penaeus seu Panulirus, Carapax Eriocheir sinensis, be by glycosamine and
The polysaccharide of N- acetyl glucosamine (N-acetyl-D-glucosamine) copolymer composition,
It is the product of chitin (Chitin) deacetylation；Described modification of chitosan is through chemistry
Modified as acylated, esterification, alkylation, or through composite modified as graft copolymerization, crosslinking,
Chelating, the shitosan of blending gained；Described shitosan salt includes but is not limited to citric acid
One of salt, nitrate, lactate, phosphate, chloride and glutamate, Glu or many
Kind；Its derivant described is one or more hydroxyls or amino has been modified for changing
The dissolubility of derivant or mucosa blocking characteristics, including sulphuric acid alcoholization chitosan derivatives and
Non-sulfuric acid refines chitosan derivatives, such as carboxymethyl, acetylation, alkylation or sulfonation shell
Polysaccharide (such as O- alkyl ether, O- acyl ester, cationization N-trimethyl chitosan TMC and use poly- second
The shitosan of glycol modification), can obtain from multiple sources, such as sulphuric acid alcoholization shitosan can
From ThioMatrix Forschungs Beratungs company and Mucobiomer
Biotechnologische forschungs-und entwicklungs company obtains, or passes through
Shitosan and suitable thiolating reagent react preparation such as application number PCT
WO03/020771A1 or Roldo et al. (Mucoadhesive thiolated chitosans as
platforms for oral controlled drug delivery:synthesis and in vitro
evaluation.European Journal of Pharmaceutic and Biopharmaceutics,
2004,57:115-121), Krauland et al. (Viscoelastic properties of a new
in situ gelling thiolated chitosan conjugate.Drug Development And
Industrial Pharmacy.2005,31:885-893), Bernkop-schn ü rch et al.
(Bernkop-Schnürch,Thiomers:A new generation of mucoadhesive
polymers.Advanced Drug Delivery Reviews.2005,57:1569-1582；
Thiomers:Preparation and in vitro evaluation of a mucoadhesive
nanoparticulate drug delivery system.International journal of
Pharmaceutics.2006,317:Prepared by method described in document 76-81).
As one embodiment of the present invention, the described vaccine adjuvant of the present invention includes
Aluminium glue, shitosan and phospholipid.
Term " phospholipid " (phospholipids/phosphatides) is also known as phospholipid, phosphorus
Lipid, is the lipid containing phosphoric acid, and including but not limited to lecithin, cephalin are phospholipid
Acyl sphingomylin acyl glycerol, phosphatidylinositols, phosphatidyl serine, phosphatidic acid, sheath
One or more of phospholipid, cuorin, are isolated from natural origin or are conventionally synthesized.
Term " lecithin (Lecithin) is also known as phosphatidylcholine
It is mixed with fatty acid and phosphoric acid that (phosphatidylcholine, PC) is glycerol and choline
Close esters and its derivant, including but not limited to natural phosphatidyl choline, synthesis lecithin, big
Bean lecithin, Egg Yolk Lecithin (PC-98T), the DPPC of synthesis, partly or completely perhydro
The lecithin changed and its one or more of mixture, be selected from plant origin such as Semen sojae atricolor,
Semen Maydiss, Semen arachidis hypogaeae, Semen Helianthi, Semen Brassicae campestriss, animal origin such as egg yolk and animal brain,
Or the Phospholipon of various manufacturer such as American lecithin Company supply
90G.
As one embodiment of the present invention, in the described vaccine adjuvant of the present invention,
Aluminium glue includes aluminium hydroxide, aluminum phosphate, aluminum sulfate, ammonia-alum, potassium alum, aluminium silicate
In at least one.
As a kind of preferred implementation of the present invention, the described vaccine adjuvant of the present invention
In, aluminium glue includes aluminium hydroxide, at least one in aluminum phosphate.
As a kind of most preferred embodiment of the present invention, the described vaccine assistant of the present invention
In agent, aluminium glue includingHPA、M9400SP、Aluminum hydroxide, gel aluminum hydroxide,
At least one in 20% aluminium hydroxide gel normal saline.
As one embodiment of the present invention, in the described vaccine adjuvant of the present invention,
Shitosan include natural chitosan, modification of chitosan, shitosan its esters or shitosan its
At least one in derivant.
As one embodiment of the present invention, in the described vaccine adjuvant of the present invention,
Phospholipid includes lecithin, cephalin, phosphatidyl glycerol, phosphatidylinositols, phosphatidyl silk
At least one in amino acid, phosphatidic acid, sphingomyelins, cardiolipin.
As a kind of preferred implementation of the present invention, the described vaccine adjuvant of the present invention
In, described phospholipid is lecithin.
As a kind of most preferred embodiment of the present invention, the described vaccine assistant of the present invention
In agent, lecithin includes natural phosphatidyl choline, synthesis lecithin, soybean lecithin, egg yolk
Lecithin, the DPPC of synthesis, partly or completely in the lecithin of perhydrogenating
At least one.
It is another aspect of the invention to provide a kind of vaccine combination it is characterised in that
Described vaccine combination comprises the antigen of adjuvant of the present invention and immune effective dose.
Term " immune effective dose " also known as immunoprotection amount or produces the effective of immunne response
Amount, for can in receiver's body effective inducing immunogenic response amount.Described immunity should
Answer and may be enough to be used in diagnostic purpose or other test, or be likely to be suitable for prevention disease
Sign or symptom, including the unfavorable health knot caused by the infection being caused by pathogen
Fruit or its complication.Humoral immunity or by both cell-mediated immunity or this
It is induced.Animal can be by for example measuring antibody to the immunne response of immunogenic composition
Potency, lymphocyte proliferation assays and indirect assessment, or after being attacked with wild type strains
Directly assessed by monitoring sign or symptom, and the protective immunity that should be provided by vaccine
Power can by measure such as experimenter the clinical symptom such as minimizing of mortality rate, sickness rate,
Temperature numerical, experimenter's general physiological state and general health and performance are assessing.Described
Immunne response may include but be not limited to inducing cell and/or humoral immunity.
Term " antigen ", also known as immunogen, refers to any material stimulating immunne response,
It is a kind of molecule that can be selectively bound by the antibody additionally it is possible to induction produces B- and/or T- cell
Humoral immunoresponse(HI) and/or cellullar immunologic response, also there are one or more epi-position (B-
With T- epi-position).Described antigen includes antibacterial killing, inactivateing, or its cultured cells
Preparation, supernatant.Wherein, described antibacterial that kill, inactivation refers to containing no longer
Enough infectious organisms replicating or growing or pathogen, pathogen can be by various methods
Including freeze thawing, chemical treatment (as processed with thimerosal or formalin), sonication,
Irradiate, hot or any other common method enough to stop organism from replicating or to grow is come in fact
Now inactivate and maintain its immunogenicity.
As one embodiment of the present invention, institute in the described vaccine combination of the present invention
State the antigen that antigen is inactivation.
As one embodiment of the present invention, in the described vaccine combination of the present invention,
Aluminum content is 5%-30%V/V, and chitosan concentration is 0.1-10mg/ml.
As a kind of preferred implementation of the present invention, the described vaccine combination of the present invention
In, aluminum content is 5%-30%V/V, and chitosan concentration is 0.1-10mg/ml, lecithin
Lipid concentration is 0.2-8mg/ml.
As one embodiment of the present invention, in the described vaccine combination of the present invention,
Aluminum content is 8%-20%V/V, and chitosan concentration is 1-5mg/ml.
As a kind of preferred implementation of the present invention, the described vaccine combination of the present invention
In, aluminium glue is 8%-20%V/V, and shitosan is 1-5mg/ml, and lecithin lipid concentration is
0.5-5mg/ml.
As one embodiment of the present invention, in the described vaccine combination of the present invention,
Described antigen includes mycoplasma hyopneumoniae antigen, haemophilus parasuises antigen, kills Bath more
Moral bacteroides antigen, Streptococcus suis antigen, Staphylococcus hyicus antigen, bronchitis Boulder are special
Bacterium antigen, Actinobacillus pleuropneumoniae antigen, bacillus coli antigen, bronchus deteriorated blood ripple
Family name's bacteroides antigen and toxigenic P.multocida antigen, Salmonella choleraesuls antigen, intestinal
Scorching Salmonella antigen, pig erysipelothrix rhusiopathiae antigen, mycoplasma hyorhinis antigens, pig are slided
One or more of liquid mycoplasma antigen, pathogenic leptospire antigen.
In a preferred embodiment of the present invention, described antigen includes mycoplasma hyopneumoniae
Antigen, haemophilus parasuises antigen, Pasteurella multocida antigen, Streptococcus suis antigen,
Staphylococcus hyicus antigen, bronchitis Boulder spy's bacterium antigen, Actinobacillus pleuropneumoniae
Antigen, bacillus coli antigen, atrophic rhinitiss antigen, Salmonella choleraesuls antigen,
Salmonella enteritidis antigen, pig erysipelothrix rhusiopathiae antigen, mycoplasma hyorhinis antigens, pig
One or more of Mycoplasma synoviae antigen, pig Leptospira bacteria antigen.
As a kind of preferred implementation of the present invention, the described vaccine combination of the present invention
In, described antigen is mycoplasma hyopneumoniae antigen and/or haemophilus parasuises antigen.Term
" mycoplasma hyopneumoniae antigen " refers to containing at least one mycoplasma hyopneumoniae
Any combinations thing of (Mycoplasma hyopneumoniae, Mhp) antigen forms,
Described mycoplasma hyopneumoniae antigen can induce, stimulate or strengthen opposing mycoplasma hyopneumoniae sense
The immunne response of dye, described antigen forms include but is not limited to inactivation or subunit anti-
Former, or its culture supernatant.Described mycoplasma hyopneumoniae antigen includes HN0613 strain (to be protected
Hide number be CCTCC No.M2012230, referring to Chinese patent CN103083655A), J
Strain (purchased from American type culture collection ATCC, it is 25934 that ATCC numbers),
(preserving number is CCTCC No.M2012286, referring to Chinese patent for NJ strain
CN103585622A), HDZK-Mhp57 strain (preserving number be CGMCC No.8096,
Referring to Chinese patent CN103484414A), AN306 strain (preserving number be CCTCC No.
M2012431, referring to Chinese patent CN103740625A), DJ-166 strain (preserving number
For CGMCC No.4545, referring to Chinese patent CN103184171A) and
(preserving number is NM04/41259 strain, referring to Chinese patent for NM04/41259 strain
CN102458462A one or more), can also comprise the arbitrary of following compositionss
Auspicious times of suitable Respisure of kind of antigen such as company of Harbin Pharmaceutical Group and auspicious times fit-and prosperous
RespisureOne, peace hectogram M+PAC of Intervet company, Boehringer Ingelheim company
'sM.hyo, the MycoGard of Portec Inc. of the U.S., Pfizer Inc.
RespiFend MH, large rise (Shanghai) animal health-care product company limited Rui Fute-
Prosperous Suvaxyn MH-one, the pig gram of Cimmeria animal health company breathe heavily SprintVac,
The happiness Yifeng Mypravac Suis of Hai Bolai company.
Term " haemophilus parasuises antigen " refers to containing at least one haemophilus parasuises
Any combinations thing of (Haemophilus parasuis, HPS) antigen forms, described pair
Haemophilus suis antigen inoculation pig can induce, stimulate or strengthen opposing haemophilus parasuises sense
The immunne response of dye, described antigen forms include but is not limited to inactivation or subunit anti-
Former, or its culture supernatant.Described haemophilus parasuises antigen includes people in the art
The street strain being clinically separated known to member, comprise at present 1-15 serotype of identification or
The haemophilus parasuises of serotype cannot be measured, the mixing comprising at least two serotypes resists
It is former that as 4 types, (such as JS strain, preserving number is CCTCC No.M2011172, referring to China
Patent CN103083655A) and 5 types (such as ZJ strain, preserving number be CCTCC
No.M2011173, referring to Chinese patent CN103083655A) hybrid antigen one
Plant or multiple, any one antigen such as Boehringer Ingelheim of following compositionss can also be comprised
The Hiprasuis-Glasser of Ingelvac HP-1, Hai Bolai company Hipra of company, in
Herd Haemophilus parasuis inactivated vaccine and the force of Chengdu medical instruments factory of Industry Co., Ltd
Section Funing (the 4 type MD0322 strains+5 of Han Keqian animal biological product Co., Ltd
Type SHO165 strain).
As a kind of preferred implementation of the present invention, the described vaccine combination of the present invention
In, described mycoplasma hyopneumoniae antigen include HN0613 strain, J strain, NJ strain,
HDZK-Mhp57 strain, AN306 strain, DJ-166 strain, NM04/41259 strain antigen,
Or auspicious times of suitable Respisure and auspicious times suitable-prosperous RespisureOne, peace hectogram M+PAC,M.hyo, MycoGard, RespiFend MH, Rui Fute-prosperous Suvaxyn
MH-one, pig gram breathe heavily SprintVac, happiness Yifeng Mypravac Suis；And
Described haemophilus parasuises antigen includes 4 type JS strains and the mixing of 5 type ZJ strains resists
Former, or Ingelvac HP-1, Hiprasuis-Glasser, section Funing, in herd industry share
One of Haemophilus parasuis inactivated vaccine of Chengdu medical instruments factory of company limited or many
Kind.
As a kind of preferred implementation of the present invention, the described vaccine combination of the present invention
Including 1 × 108The mycoplasma hyopneumoniae HN0613 strain antigen of the inactivation of CCU/ head part, 1
×109The haemophilus parasuises 4 type JS strain antigen of the inactivation of CFU/ head part and 1 ×
109The haemophilus parasuises 5 type ZJ strain antigen of the inactivation of CFU/ head part；And 10%V/V
Aluminium glue, 4mg/ml shitosan and 2mg/ml lecithin.
Term " pig " refers to any animal belonging to Suidae (Suidae) member, including
The member of the Suidaes such as pigletss, sow, gilt, boar.
Third aspect present invention be to provide described vaccine combination in preparation prevention and/or
Treatment mycoplasma hyopneumoniae and haemophilus parasuises infect relevant disease medicine in should
With.
Term " prevention and/or treatment " refers to when being related to bacterium infection suppress answering of antibacterial
Make, suppress the propagation of antibacterial or prevent antibacterial from settling down in its host, and mitigate thin
Bacterium infection disease or disease symptom.If bacterial load amount reduces, infection symptoms subtract
Light and/or food ration and/or growth increase, then just it is considered that described treatment reaches
Therapeutic effect.
To further describe the present invention with reference to specific embodiment, advantages of the present invention and
Feature will be with describing apparent.But these embodiments are only exemplary, not
Any restriction is constituted to the scope of the present invention.It will be understood by those skilled in the art that
Lower without departing from the spirit and scope of the present invention can to the details of technical solution of the present invention and
Form is modified or is replaced, but these modifications and replacement each fall within the protection model of the present invention
In enclosing.
The mycoplasma hyopneumoniae to inactivate for the mycoplasma hyopneumoniae antigen in the embodiment of the present invention
HN0613 strain, haemophilus parasuises antigen with the 4 type haemophilus parasuises JS strains that inactivate and
It is illustrated as a example 5 type haemophilus parasuises ZJ strains, but no matter this embodiment is in any feelings
Limitation of the invention is not all constituted under condition.
In the embodiment of the present invention, pneumonopathy varying index is passed judgment on using 28 point-scores.Described 28
Point-score is to observe pathological changes according to the typical pulmonary of mycoplasmal pneumonia of swine, and occurring degree is carried out
Quantify to judge, the substantive sample pathological changes occurring including apex pulmonis leaf, lobus cardiacus, lobus diaphragmaticus, middle leaf,
As " carnification ", " change of pancreas sample ".Pneumonia pathological changes standards of grading are：2 sharp leaves, 2
Individual lobus cardiacus, 2 lobus diaphragmaticus, 1 middle leaf amount to 7 lobes of the lung, and each lobe of the lung full marks is 4
Point, 28 points altogether.Ratio according to this leaf shared by lobe of the lung area that substantive pathological changes occur,
Respectively each lobe of the lung is given a mark.No pneumonia pathological changes are designated as 0 point；Lesion area ratio
For 1%-25%, it is designated as 1 point；Lesion area ratio 26%-50%, is designated as 2 points；Disease
Variable area ratio is 51%-75%, is designated as 3 points；Lesion area ratio is 76%-100%,
It is designated as 4 points.As lobe of the lung tow sides all have pathological changes, carried out with the big one side of lesion area
Score.The pneumonia disease that each lobe of the lung marking summation is this morbidity strain becomes score.Right respectively
After immune group pig and matched group pig are scored, according to below equation Computation immunity group pig
Lobe of the lung pathological changes slip：
Embodiment of the present invention statistical analysis technique is：The pneumonopathy varying index of 7 lobes of the lung of statistics,
Determine lesion degree.Carry out ANOVA analysis with SPSS computer software, relatively more each
Group difference, determines the effectiveness of pathological changes difference.
PH7.4PBS solution formula used in the present invention is：Add in 800ml distilled water
Enter NaCl 8g, KCl 0.2g, Na2HPO41.42g、KH2PO40.27g, adjusts pH value
To 7.4, it is settled to 1000ml, through using after 121 DEG C of autoclavings 30 minutes, but should
No matter embodiment does not all constitute limitation of the invention under any circumstance.
In the present invention used chemical reagent be analysis pure, purchased from Chinese medicines group, but
No matter this embodiment does not all constitute limitation of the invention under any circumstance.
For making the present invention easier to understand, with reference to specific embodiment, explain further
State the present invention.It should be understood that these embodiments are only used for the present invention rather than limit this
Bright scope.Experimental technique of the present invention, if no specified otherwise, is routine side
Method；Described biomaterial, if no specified otherwise, all commercially obtains.
The preparation of embodiment 1 antigen
The preparation of 1.1 mycoplasma hyopneumoniae antigens
It is prepared mycoplasma hyopneumoniae according to Chinese patent CN103083655A
HN0613 strain bacterium solution and the content measuring bacterium solution, result：Mycoplasma hyopneumoniae HN0613
The content of strain is 1 × 109CCU/ml；And bacterium solution inactivated and checks, result：Training
Foster base coloring does not change, and Medium's PH Value does not reduce, and shows mycoplasma hyopneumoniae
HN0613 strain bacterium solution inactivates successfully.
The preparation of 1.2 haemophilus parasuises antigens
Prepare haemophilus parasuises 4 type JS according to Chinese patent CN103083655A respectively
Strain and 5 type ZJ strain bacterium solution and mensure bacterium solution content, result：Haemophilus parasuises 4 type
The content of JS strain and 5 type ZJ strains is 5 × 109CFU/ml；And bacterium solution is inactivated
And inspection, result：No any bacterial growth in plate, shows haemophilus parasuises 4 type
JS strain and the inactivation of 5 type ZJ strain bacterium solution are all successful.
The preparation of embodiment 2 adjuvant
Aluminium glue：Purchased from U.S. General Chemical, LLC'sHPA helps
Agent.
Shitosan mother solution：Shitosan purchased from Qingdao Hai Hui biological engineering company limited (is divided
Son is measured as 145kD, and deacetylation is 50%) it is dissolved completely in aqueous acetic acid containing 1%V/V
In be configured to concentration be 15mg/ml chitosan-acetic acid solution, with NaOH adjust solution to
PH7.4, and carry out filtration sterilization, obtain final product 15mg/ml shitosan mother solution.
Lecithin mother solution：By the 1g lecithin purchased from Beijing source magnificent phospholipid Science and Technology Ltd.
With ethanol dissolving, carry out vacuum distillation after adding PBS solution mix homogeneously, treat that ethanol is waved
It is placed in tissue mashing machine after sending out to the greatest extent to stir 10 minutes, in 30 points of 115-125 DEG C of steam sterilization
Clock, stirs twice with ultrasonic echography, and 10 minutes/time add PBS solution to 100ml,
And carry out filtration sterilization, obtain final product 10mg/ml lecithin mother solution.
The preparation of embodiment 3 inactivated vaccine composition and animal experiment
The preparation of 3.1 inactivated vaccine compositions
Adjuvant prepared by antigen prepared by embodiment 1, embodiment 2 uses PBS solution dilute
Release, make each antigenic solution after dilution and adjuvant final concentration by the vaccine combination inactivateing in table 1
Component mixing contained by thing, stirs 10-15 minute with 500-800 rev/min of rotating speed, at end
The thimerosal of 1%V/V is added (purchased from the limited public affairs of Chinese medicines group chemical reagent before only stirring
Department) solution so as to final concentration be less than ten thousand/, fully vibration mix, subpackage,
After subpackage, 2-8 DEG C saves backup.
Component contained by table 1 inactivated vaccine composition
The animal experiment of the mycoplasma hyopneumoniae antigen of 3.2 inactivated vaccine compositions
By the vaccine combination A1 prepared by embodiment 3.1, A2, A3, A5, B1,
B2, B3, B5, (exclusion pig is numerous for difference musculi colli injecting immune 14-21 age in days piglet
Grow and respiration syndrome, pig annulus 2 type and swine fever), 5/group, 2ml/ head, simultaneously
Setting is with reference to vaccine control group, counteracting toxic substances matched group, totally 10 groups (totally 50 piglets).
Measure body temperature after immunity inoculation, observe side reaction to 28 days, and on 28th according to Pulmonis Sus domestica
Scorching mycoplasma antibody assay kit (purchased from IDEXX) description carries out antibody test,
The results are shown in Table 2；Simultaneously to all pig tracheas injection mycoplasma hyopneumoniae Jinan system poison by force
(purchased from National Veterinary Microbiological Culture Collection administrative center, preserving number is for CVCC354 strain
CVCC354), 100MID50/ head, dissects after observing 28 days, and observes pulmonary lesion,
According to 28 point-scores, pulmonary lesion is scored.Before and after counteracting toxic substances, respectively to test group piglet
Weighed, and calculated average daily gain.
About 10 days about after counteracting toxic substances, wherein 5 pigs of counteracting toxic substances matched group successively occur cough,
The symptoms such as asthma, fur is rough；2 pigs are had to occur in that cough with reference to vaccine control group
Symptom and there occurs side reaction；The similar of asthma in the pig of other immune group
Symptom.Before and after counteracting toxic substances, the pneumonia disease of each test group pig becomes average and average daily gain
The results are shown in Table 2.
The animal test results of the mycoplasma hyopneumoniae antigen of table 2 inactivated vaccine composition
Note 1：Vaccine with reference to selected by vaccine control group is the porcine mycoplasmal in existing product
Pneumonia inactivated vaccine (J strain), that is, peace hectogram (M+PAC), is entered by shop instruction
Row immunity.
Note 2：Body temperature "None" shows not occurring transient body temperature to raise, and " having " shows to send out
The transient body temperature of life raises, and wherein transient body temperature raises and refers to 1-2 after two immunity
Its body temperature of short duration can assume 40.5 degree, and recovered normal body temperature in the 3rd day.
Note 3：Side reaction "None" shows side reaction, and " having " shows to occur
Side reaction, wherein side reaction refer to occur that transience spirit is depressed, loss of appetite, allergy
At least one in reaction.
Note 4：In diversity statistical analysiss, compare between group, alphabetical identical person represents difference not
Significantly, alphabetical difference person represents significant difference (p ＜ 0.05).
As shown in Table 2：1-4 group, 5-8 group are respectively using same content mycoplasma hyopneumoniae
Antigen and different content, single Seedling of composition adjuvant or connection Seedling carry out immunity, the blood after immunity
Clear antibody show containing aluminium glue, the vaccine combination of shitosan adjuvant and containing aluminium glue, shitosan,
The antibody horizontal of the vaccine combination of lecithin adjuvant is above the antibody water with reference to vaccine
Flat, and the antibody horizontal containing aluminium glue, shitosan, the vaccine combination of lecithin adjuvant is high
In the antibody horizontal containing aluminium glue, the vaccine combination of shitosan adjuvant；Carry out counteracting toxic substances afterwards,
Result display 1-4 group, 5-8 group immune swine all do not occur transient body temperature to raise, and do not go out yet
Existing side reaction, and pneumonia pathological changes slip equal ＞ 67%, ＞ 80% respectively, average day
Weightening is respectively 0.0399-0.04016kg, 0.0412-0.0414kg, though 1-4 Zu Ge group,
Between each group of 5-8, difference is not notable (p ＞ 0.05), but 1-4 group is compared with 5-8 group
Difference is all notable (p ＜ 0.05)；With compared with vaccine control group, difference is also all notable
(p ＜ 0.05)；Compared with counteracting toxic substances matched group, difference is also all notable (p ＜ 0.05).
Show that in vaccine combination, the immune effect containing aluminium glue, shitosan, lecithin for the adjuvant is notable
Better than containing aluminium glue, shitosan.
The animal experiment of the haemophilus parasuises antigen of 3.3 inactivated vaccine compositions
By vaccine combination A4, A5, B4, B5 prepared by embodiment 3.1, difference
Musculi colli injecting immune 14-21 age in days piglet (exclusion Porcine reproductive and respiratory syndrome,
Pig annulus 2 type and swine fever), 5/group, 2ml/ head, setting simultaneously is with reference to vaccine comparison
Group, counteracting toxic substances matched group, totally 6 groups (be named as 1 successively, 2,3,4, with reference to vaccine pair
According to group 1, counteracting toxic substances matched group 1)；Do simultaneously and repeat to test, totally 6 groups (be named as 11,
12nd, 13,14, with reference to vaccine control group 2, counteracting toxic substances matched group 2), refer to table 3.
After immunity inoculation, measurement body temperature, observation side reaction, to 28 days, the results are shown in Table 3；And
(it is purchased from according to haemophilus parasuises antibody assay kit within 28 days after immunity inoculation
Biovet) carry out antibody test, the results are shown in Table 3.Carry out counteracting toxic substances simultaneously, use pair for first 6 groups
Haemophilus suis 4 type JS strain lumbar injection 3ml carries out counteracting toxic substances, counteracting toxic substances dosage be 9.0 ×
109CFU/ head；6 groups are carried out with haemophilus parasuises 5 type ZJ strain lumbar injection 3ml afterwards
Counteracting toxic substances, counteracting toxic substances dosage is 6.0 × 109CFU/ head, observes each group pig clinical manifestation after counteracting toxic substances,
Cut open after observing 14 days and enter row pathological observation.Respectively test group piglet is carried out before and after counteracting toxic substances
Weigh, to calculate average daily gain, the results are shown in Table 3.
The animal test results of the haemophilus parasuises antigen of table 3 inactivated vaccine composition
Note 1：Haemophilus parasuis inactivated vaccine with reference to used by vaccine control group herds reality in being
Chengdu medical instruments factory of industry limited company produces, and antigenic content is 4.0 × 109CFU/ head part,
Carry out immunity twice according to shop instruction.
Note 2：Body temperature "None" shows that pig does not all occur transient body temperature to raise, " having "
Show that at least 1 pig transient body temperature and raises, wherein transient body temperature raises and refers to
Occur after two immunity 1-3 days body temperature can of short duration present 40.5 degree and more than, and in the 4th day
Recover normal body temperature.
Note 3：Side reaction "None" shows that pig did not all occur side reaction, and " having " shows
There is side reaction at least 1 pig, wherein side reaction refer to occur transience lethargy,
Cough, dyspnea, loss of appetite, at least one in anaphylaxiss.
Note 4：In diversity statistical analysiss, compare between group, alphabetical identical person represents difference not
Significantly, alphabetical difference person represents significant difference (p ＜ 0.05).
As shown in Table 3：1-2,3-4,5-6,7-8 group is respectively using containing the bloodthirsty bar of secondary pig
Single Seedling of bacterium antigen and same content adjuvant or connection Seedling carry out immunity, all do not occur after immunity
Body temperature raises, also side reaction does not occur, and produced serum antibody shows containing aluminium glue, shell
Adjuvant self-control vaccine and contain aluminium glue, shitosan, the vaccine combination of lecithin adjuvant
The antibody horizontal of thing is higher than the antibody horizontal with reference to vaccine, and contains aluminium glue, shitosan, ovum
The antibody horizontal of the vaccine combination of phospholipid adjuvant is higher than the epidemic disease containing aluminium glue, shitosan adjuvant
The antibody horizontal of Seedling compositionss；Carry out counteracting toxic substances afterwards, result display 1-2,3-4,5-6,
7-8 group immunity pig all obtains to be protected completely, and average daily gain is respectively
0.0324-0.0325kg、0.0336-0.0337kg、0.0324-0.0327kg、0.0338
-0.0339kg.Though 1-2 Zu Ge group, each group of 3-4,5-6 Zu Ge group, 7-8 group
Between each group, difference is not notable (p ＞ 0.05), but 1-2 group is all aobvious with 3-4 group difference
Write (p ＜ 0.05), 5-6 group and 7-8 group difference all notable (p ＜ 0.05)；With reference
Vaccine control group is compared, and when antigenic content is low, immunoprotection efficiency is also higher, and average day
Weightening difference is all notable (p ＜ 0.05)；Compared with counteracting toxic substances matched group, average daily gain is poor
Different all notable (p ＜ 0.05).Show containing aluminium glue, shitosan, lecithin adjuvant vaccine
The immune effect of compositionss is significantly better than containing aluminium glue, the vaccine combination of shitosan adjuvant
Immune effect.
In sum, containing aluminium glue, shitosan, the exempting from of the vaccine combination of lecithin adjuvant
Epidemic disease effect is significant is better than the immune effect containing aluminium glue, the vaccine combination of shitosan adjuvant,
Immunne response level after these vaccine combinations (1) immune animal is with the increasing of adjuvant content
Plus and raised, and be on close level with the immunne response of existing commercial vaccine or even relatively
Height, the antibody persistent period is also longer, and no body temperature raises, no side reaction occurs, and effect is true
Real；(2) animal also can be made when antigenic content is relatively low to obtain preferable immune effect, exempt from
Epidemic disease number of times also can make the stress of pig reduce even disappearance less.
The above is only the preferred embodiments of the present invention, not the present invention is appointed
How pro forma restriction is although the present invention is disclosed above with preferred embodiment, but simultaneously
It is not used to limit the present invention, any those skilled in the art, without departing from this
In the range of bright technical scheme, when the technology contents of available the disclosure above make a little more
Equivalent embodiments that are dynamic or being modified to equivalent variations, as long as be without departing from the technology of the present invention side
The content of case, according to the technical spirit of the present invention above example is made any simple
Modification, equivalent variations and modification, all still fall within the range of technical solution of the present invention.
Claims (10)
1. a kind of vaccine adjuvant, described adjuvant includes aluminium glue, shitosan.
2. according to claim 1 adjuvant it is characterised in that aluminium glue includes hydroxide
At least one in aluminum, aluminum phosphate, aluminum sulfate, ammonia-alum, potassium alum, aluminium silicate；
Preferably, aluminium glue includes aluminium hydroxide, at least one in aluminum phosphate；
Most preferably, aluminium glue includesHPA、M9400SP、Aluminum hydroxide, gel aluminum hydroxide,
At least one in 20% aluminium hydroxide gel normal saline.
3. according to claim 1 adjuvant it is characterised in that shitosan include natural
At least one in shitosan, modification of chitosan, shitosan salt or chitosan derivatives.
4. according to claim 1 adjuvant it is characterised in that described adjuvant also includes
Phospholipid；
Preferably, described phospholipid includes lecithin, cephalin, phosphatidyl glycerol, phospholipid
In acyl inositol, phosphatidyl serine, phosphatidic acid, sphingomyelins, cardiolipin at least one
Kind；
Preferably, described phospholipid is lecithin；
Preferably, described lecithin includes natural phosphatidyl choline, synthesis lecithin, Semen sojae atricolor ovum
Phospholipid, Egg Yolk Lecithin (PC-98T), the DPPC of synthesis, partly or completely perhydrogenating
At least one in lecithin.
5. a kind of vaccine combination is it is characterised in that described vaccine combination will containing right
Seek the antigen of adjuvant any one of 1-4 and immune effective dose.
6. according to claim 5 vaccine combination it is characterised in that described vaccine
In compositionss, aluminum content is 5%-30%V/V, and chitosan concentration is 0.1-10mg/ml；
Preferably, in described vaccine combination, lecithin lipid concentration is 0.2-8mg/ml.
7. according to claim 5 vaccine combination it is characterised in that aluminum content
For 8%-20%V/V, chitosan concentration is 1-5mg/ml；
Preferably, in described vaccine combination, lecithin lipid concentration is 0.5-5mg/ml.
8. according to claim 5 vaccine combination it is characterised in that described vaccine
Antigen described in compositionss include mycoplasma hyopneumoniae antigen, haemophilus parasuises antigen,
Kill p pestic antigen, Streptococcus suis antigen, Staphylococcus hyicus antigen, bronchus more
Scorching Boulder spy's bacterium antigen, Actinobacillus pleuropneumoniae antigen, bacillus coli antigen,
Trachea deteriorated blood bordetella bacilli antigen and toxigenic P.multocida antigen, Salmonella choleraesuls
Antigen, Salmonella enteritidis antigen, pig erysipelothrix rhusiopathiae antigen, mycoplasma hyorhinises resist
Former, one or more of mycoplasma hyosynoviae antigen, pathogenic leptospire antigen；
Preferably, described antigen is mycoplasma hyopneumoniae antigen and/or haemophilus parasuises resist
Former.
9. according to claim 8 vaccine combination it is characterised in that described Pulmonis Sus domestica
Scorching mycoplasma antigen include HN0613 strain, J strain, NJ strain, HDZK-Mhp57 strain,
AN306 strain, DJ-166 strain, NM04/41259 strain antigen, or auspicious times of suitable Respisure
- prosperous RespisureOne suitable with auspicious times, peace hectogram M+PAC,M.hyo、
MycoGard, RespiFend MH, Rui Fute-prosperous Suvaxyn MH-one, pig gram breathe heavily
One or more of SprintVac, happiness Yifeng Mypravac Suis；And
Described haemophilus parasuises antigen includes 4 type JS strains and the mixing of 5 type ZJ strains resists
Former, or Ingelvac HP-1, Hiprasuis-Glasser, section Funing, in herd industry share
One of Haemophilus parasuis inactivated vaccine of Chengdu medical instruments factory of company limited or many
Kind.
10. vaccine combination according to claim 9 is it is characterised in that described
Vaccine combination include 1 × 108The mycoplasma hyopneumoniae of the inactivation of CCU/ head part
HN0613 strain antigen, 1 × 109Haemophilus parasuises 4 type JS of the inactivation of CFU/ head part
Strain antigen and 1 × 109The haemophilus parasuises 5 type ZJ strain antigen of the inactivation of CFU/ head part；
And the aluminium glue of 10%V/V, 4mg/ml shitosan and 2mg/ml lecithin.
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