CN109432416B - Oligosaccharide vaccine for preventing invasive fungal infection - Google Patents
Oligosaccharide vaccine for preventing invasive fungal infection Download PDFInfo
- Publication number
- CN109432416B CN109432416B CN201811479983.6A CN201811479983A CN109432416B CN 109432416 B CN109432416 B CN 109432416B CN 201811479983 A CN201811479983 A CN 201811479983A CN 109432416 B CN109432416 B CN 109432416B
- Authority
- CN
- China
- Prior art keywords
- oligosaccharide
- chitosan oligosaccharide
- solution
- vaccine
- carrier protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/385—Haptens or antigens, bound to carriers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Communicable Diseases (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention provides an oligosaccharide vaccine for preventing invasive fungal infection, which comprises chitosan oligosaccharide coupled with an immunogenic carrier protein, wherein the coupling mass ratio of the immunogenic carrier protein to the chitosan oligosaccharide is 0.7-210:1, the deacetylation degree of the chitosan oligosaccharide is 0.05-98%, the average relative molecular weight is less than 5000Da, and the immunogenic carrier protein is non-human protein. The oligosaccharide vaccine provided by the invention effectively activates Th17 and Th1 cell immunity through proper deacetylation degree, thereby preventing broad-spectrum fungal infection. The oligosaccharide vaccine can treat or prevent invasive fungi such as Candida albicans, Aspergillus, Cryptococcus and Yersinia pneumocystis.
Description
Technical Field
The invention belongs to the field of vaccine biomedicine, and particularly relates to an oligosaccharide vaccine for preventing invasive fungal infection.
Background
With the increasing incidence of new immunosuppressive therapy, organ transplantation and AIDS, the incidence of invasive drug-resistant fungal infections and mortality has increased dramatically worldwide. According to the standard of international medical community, fungal infections are classified into superficial, subcutaneous and invasive (or systemic) fungal infections of 3 types, the latter two also known as deep fungal infections. Common pathogenic strains are candida, aspergillus, cryptococcus and yersinia pneumocystis, accounting for about 70% of fungal infections, affecting internal organs of the human body or forming mycosis. Clinical deep fungal infections have become a significant cause of death for nosocomial infections, and the number of deaths worldwide due to fungal infections is as high as 150 million people each year. U.S. studies have shown that white minds are in hospital infectious sepsis 4 th, disease mortality first, and the cost of a single treatment is $ 20-40 million/year in the united states alone. The morbidity of invasive mycosis of different populations in hospitals in China is 4.1-41.2%, and the mortality is 9.8-60%. The antifungal medicines are only ten or more, and the new medicines are slowly developed. The clinical medicine has few selectable varieties, long treatment period (> 1-6 months), and unsatisfactory curative effect, and makes the anti-invasive fungi become a difficult clinical problem.
The research of the fungal vaccine starts late and has little progress, and no vaccine is on the market at present. In recent years, 2 vaccines entering clinical use are designed aiming at adhesion factors and virulence factors of a particular species of candida albicans strain, exert a neutralization effect through humoral immunity, and are expected to be applied to gynecological candida albicans infection of specific pathogenic strains rather than fatal invasive fungal infection.
Fungal cell walls generally contain mannose, β -glucan and chitin polysaccharides. Previous researches find that the immunogenicity of sugar is low and the immune effect of the body is difficult to stimulate, and the research on fungal vaccines represented by beta-glucan can improve the immune response of the body by coupling carrier protein. The understanding of the immune mechanism of the body against fungal infection is gradually deepened, and the cell immunity and the innate immunity which are mainly Th17 are found to play a leading role. However, the research on the beta-glucan-KLH vaccine mainly evaluates the efficacy by using the antibody titer, namely humoral immunity, so that the research on the efficacy of the beta-glucan-KLH vaccine is still deficient. Meanwhile, because the tested saccharide is chemically synthesized completely, the reaction is limited by protecting groups one by one and synthesizing sugar chains with limited polymerization degrees one by one, the reaction is complex, the cost is high, and generally only a plurality of oligosaccharides with polymerization degrees of about 2-5 are synthesized for testing, so that the screening effect cannot be achieved. However, no vaccine targeting fungal cell wall chitin has been reported, and the mechanisms of recognition by the immune system are still unclear. Moreover, only chitosan coupled with protein has been reported as an immune adjuvant.
Disclosure of Invention
Aiming at the problem of the lack of the existing vaccine for preventing invasive fungal infection, the invention provides the oligosaccharide vaccine for preventing invasive fungal infection, which has low cost and broad-spectrum antifungal effect.
The invention also aims to provide a preparation method of the oligosaccharide vaccine for preventing invasive fungal infection, which has the advantages of easily obtained raw materials and simple preparation process.
In order to achieve the purpose, the invention adopts the following technical scheme.
An oligosaccharide vaccine for the prevention of invasive fungal infections comprising chitosan oligosaccharides conjugated to an immunogenic carrier protein. The coupling mass ratio of the immunogenic carrier protein to the chitosan oligosaccharide is 0.7-210:1, and preferably 1-101: 1.
The chitosan oligosaccharide has deacetylation degree of 0.05-98% and average relative molecular weight<5000 Da. Preferably, the degree of deacetylation is from 0.1 to 20%. Preferably, the chitosan oligosaccharide has a relative average molecular weight of 1000-3000 Da. Preferably, A is a 2% aqueous solution of the unconjugated chitosan oligosaccharide 420 <0.1; the resulting solution was centrifuged at 13000rpm for 2min, and no precipitate was formed. A is described 420 Refers to the absorbance value of the solution to 420nm light. The chitosan oligosaccharide may be purchased commercially or prepared by itself by degrading chitosan. Generally, a chitosan oligosaccharide is called a chitosan oligosaccharide when the degree of deacetylation is less than 15%, but it is also called a chitosan oligosaccharide in the present invention.
The immunogenic carrier protein is a non-human protein; including but not limited to diphtheria toxin non-toxic variants, KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin), OVA (chicken egg albumin), Blue carrier TM (highly soluble hemocyanin of mollusc origin), tetanus toxin/toxoid, high molecular weight protein (HMP) isolated from non-acquired haemophilus influenzae, detoxified pseudomonas aeruginosa toxin a, cholera toxin/toxoid, pertussis toxin/toxoid, clostridium perfringens exotoxin/toxoid, hepatitis b surface antigen, hepatitis b core antigen, rotavirus VP7 protein, diphtheria toxin mutants CRM, CRM191, CRM3201, respiratory syncytial virus F and G protein. KLH, BSA, OVA are preferred.
The attachment of the above-described chitosan oligosaccharide to the immunogenic carrier protein may be by conventional immunological means, such as coupling with a coupling agent. Preferably, the preparation method of the oligosaccharide vaccine comprises the following steps:
(1) dissolving the immunogenic carrier protein, the chitosan oligosaccharide and the coupling agent in a coupling solution, and incubating;
(2) adding a terminator for incubation;
(3) purifying to obtain the oligosaccharide vaccine.
In step (1), the coupling agent is selected from but not limited to CHO (CH) 2 ) m CHO，BS 3 （bis[sulfosuccinimidyl]suberate, bis (sulfo) succinate), DSS (disuccinimidyl suberate) and bs (peg) n. The larger the value of m or n is, the longer the arm of the coupling agent as a bridge is, the more favorable the reduction of the binding steric hindrance in the immune stage is, but the preparation purity of the overlong coupling agent is reduced. Preferably, m = 3-10; n = 2-15.
In the step (1), the coupling solution is selected from water or 0.01-0.3M buffer solution with pH of 6.9-9 and without amine group. Preferably, the pH of the coupling solution is 7.2-8, and the concentration is 0.05-0.2M.
In step (1), the concentration of the immunogenic carrier protein is 0.5-100 mg/mL, preferably 10-90 mg/mL.
In the step (1), the mass ratio of the immunogenic carrier protein to the chitosan oligosaccharide is 0.5-100:1, preferably 10-90: 1.
In the step (1), the mass ratio of the coupling agent to the chitosan oligosaccharide is 0.5-10:1, preferably 1-5: 1.
In the step (1), the incubation temperature of the immunogenic carrier protein, the chitosan oligosaccharide and the coupling agent is 0-35 ℃, and the incubation time is 10-150 min, preferably 4-25 ℃ and 20-120 min.
In the step (1), the coupling agent and the chitosan oligosaccharide can be added after being dissolved in a proper solution or directly added in a solid form.
In the step (2), the mass ratio of the terminating agent to the coupling agent is 5-85:1, preferably 10-50: 1.
In the step (2), the terminator is selected from Tris-HCl, glycine, lysine or NaBH 4 . Preferably, the terminating agent is1-4M Tris-HCl buffer at pH 7-8. More preferably, the terminator is Tris-HCl buffer at a concentration of 1M at pH 7.5.
In the step (2), the incubation temperature after adding the terminator is 0-35 ℃, and the temperature is kept for 5-75min, preferably 4-25 ℃, and 10-60 min.
In the step (3), the purification method can be desalting column or dialysis method to remove low molecular impurities, so as to obtain the immunogenic carrier protein coupled chitosan oligosaccharide. Wherein the buffer solution or water for desalting or dialysis is a solution for dissolving the immunogenic carrier protein.
Preferably, the packing types for the Desalting column are G10-G50, Bio-gel P2-P10, Bio-gel P2-P6DG, Thermo Scientific cassettes Zeba cassettes Spin desaling Columns, preferably G25 and Thermo Scientific cassettes Zeba cassette Spin desaling Columns. The Dialysis membrane has a pore size of 1-10kDa, and is selected from, but not limited to Thermo Scientific Slide-A-Lyzer-systems Cassettes; preferably, the Thermo Scientific Slide-A-Lyzer-system Cassettes have a pore size of 3 kDa.
Preferably, step (3) is followed by a step of making the oligosaccharide vaccine solution into a solid. Conventional procedures such as vacuum freeze drying or spray drying may be employed.
The application of the oligosaccharide vaccine as a medicament for treating or preventing invasive fungi; the fungi include, but are not limited to, Candida albicans, Aspergillus, Cryptococcus, and Yersinia pneumocystis.
The invention has the following beneficial effects:
the oligosaccharide vaccine provided by the invention adopts deacetylated chitosan oligosaccharide, and utilizes-NH exposed by deacetylation 2 The chitosan oligosaccharide coupling protein is prepared as the coupling site of the chitosan oligosaccharide and the carrier protein, so that the immunogenicity of the chitosan oligosaccharide is increased. However, the number of couplable sites is too small due to too low deacetylation degree, the difference between chitosan oligosaccharide molecules and chitin molecules of cell walls of pathogenic bacteria is too large due to too high deacetylation degree, and the Th17 and Th1 cell immunity is effectively activated through proper deacetylation degree so as to prevent broad-spectrum fungal infection. The oligosaccharide vaccine can be used for treating or preventing invasive fungi, such as Candida albicansFungi, Aspergillus, Cryptococcus, and Yersinia pneumocystis.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.
EXAMPLE 1 preparation of Chitosan oligosaccharide
1.1
Dissolving 15g of chitosan with the deacetylation degree of 1% by 300mL of 0.3M acetic acid, adjusting the pH value to 5.0 after complete dissolution, and adding water to reach the constant volume of 500 mL. Adding 1.5g (130U/g) of chitosanase, and stirring in a water bath at 25-35 ℃ for 8 h. Filtering the chitosan enzymolysis liquid by a plate frame, and then performing ultrafiltration and nanofiltration by using filter membranes with different pore diameters (400-<5000Da、<4000 Da、<3000 Da、<2000 Da、<1000Da、<800Da、<600Da、<400 Da). Concentrating by rotary evaporation for 5 times, and spray drying to collect chitosan oligosaccharide. A of 2% aqueous solution of the resulting Chitosan oligosaccharide 420 <0.1; the resulting solution was centrifuged at 13000rpm for 2min, and no precipitate was formed.
1.2
Dissolving 18g of chitosan with the deacetylation degree of 95% by 300mL of 0.2M hydrochloric acid, adjusting the pH value to 6.5 after complete dissolution, and adding water to reach the constant volume of 500 mL. Adding 1.5g (200U/g) of chitosanase, and stirring in a water bath at 25-35 ℃ for 16 h. Filtering the chitosan enzymolysis liquid by a plate frame, and then performing ultrafiltration by using filter membranes with different apertures to intercept chitosan oligosaccharide (1000-. Concentrating by rotary evaporation for 10 times, and spray drying to collect chitosan oligosaccharide. A of 2% aqueous solution of the resulting Chitosan oligosaccharide 420 <0.1; the resulting solution was centrifuged at 13000rpm for 2min, and no precipitate was formed.
1.3
Dissolving 10g of chitosan with the deacetylation degree of 10% by 300mL of 0.3M acetic acid, adjusting the pH value to 5.5 after complete dissolution, and adding water to reach the constant volume of 500 mL. Adding 1.5g (100U/g) of chitosanase, and stirring in a water bath at 25-35 ℃ for 8 h. Filtering the chitosan enzymolysis liquid by a plate frame, and then performing ultrafiltration and interception on chitosan oligosaccharide (1000-2000 Da, 1000-3000Da, 2000-3000Da, 3000-4000 Da and 4000-5000 Da) with corresponding molecular weight sections by using filter membranes with different apertures. Concentrating by rotary evaporation for 15 times, and freeze drying to collect chitosan oligosaccharide. A of 2% aqueous solution of the resulting Chitosan oligosaccharide 420 <0.1; 2% aqueous solution 13000rpm centrifugation 2min, no precipitate.
1.4
Dissolving 15g of chitosan with the deacetylation degree of 20% by 300mL of 0.3M acetic acid, adjusting the pH value to 5.5 after complete dissolution, and adding water to reach the constant volume of 500 mL. Adding 1.5g (130U/g) of chitosanase, and stirring in a water bath at 25-35 ℃ for 8 h. Filtering the chitosan enzymolysis liquid by a plate frame, and then using filter membranes with different pore diameters to carry out ultrafiltration and nanofiltration on chitosan oligosaccharide(s) with corresponding molecular weight sections<5000Da、<4000 Da、<3000 Da、<2000 Da、<1000Da、<800Da、<600Da、<400 Da). Concentrating by rotary evaporation for 5 times, and spray drying to collect chitosan oligosaccharide. A of 2% aqueous solution of the resulting Chitosan oligosaccharide 420 <0.1; the resulting solution was centrifuged at 13000rpm for 2min, and no precipitate was formed.
1.5
Dissolving 15g of chitosan with deacetylation degree of 50% in 300mL of 0.3M acetic acid, adjusting pH to 6.0 after complete dissolution, and adding water to reach a constant volume of 500 mL. Adding 1.5g (130U/g) of chitosanase, and stirring in a water bath at 25-35 ℃ for 8 h. Filtering the chitosan enzymolysis liquid by a plate frame, and then using filter membranes with different apertures to carry out ultrafiltration and interception on chitosan oligosaccharide with corresponding molecular weight sections (2000-. Concentrating by rotary evaporation for 5 times, and freeze drying to collect chitosan oligosaccharide. A of 2% aqueous solution of the resulting Chitosan oligosaccharide 420 <0.1; the resulting solution was centrifuged at 13000rpm for 2min, and no precipitate was formed.
1.6
Dissolving 15g of chitosan with the deacetylation degree of 0.05% in 300mL of 0.3M acetic acid, adjusting the pH value to 5.0 after complete dissolution, and adding water to reach the constant volume of 500 mL. Adding 1.5g (130U/g) of chitosanase, and stirring in a water bath at 25-35 ℃ for 8 h. Filtering the chitosan enzymolysis liquid by a plate frame, and then using filter membranes with different pore diameters to carry out ultrafiltration and nanofiltration on chitosan oligosaccharide(s) with corresponding molecular weight sections<5000Da、<4000 Da、<3000 Da、<2000 Da、<1000Da、<800Da、<600Da、<400 Da). Concentrating by rotary evaporation for 5 times, and spray drying to collect chitosan oligosaccharide. A of the resulting 2% aqueous solution of chitooligo-2 saccharide 420 <0.1; the resulting solution was centrifuged at 13000rpm for 2min, and no precipitate was formed.
1.7
Dissolving 15g of chitosan with the deacetylation degree of 0.1% by using 300mL of 0.3M acetic acid, adjusting the pH value to 5.0 after complete dissolution, and adding water to reach the constant volume of 500 mL. Adding 1.5g (130U/g) of chitosanase, and stirring in a water bath at 25-35 ℃ for 8 h. Plate frameFiltering the chitosan enzymolysis liquid, ultrafiltering with filter membrane with different pore diameters, and nanofiltering to intercept chitosan oligosaccharide with corresponding molecular weight segment<5000 Da、<4000 Da、<3000 Da、<2000 Da、<1000 Da、<800 Da、<600 Da、<400 Da). Concentrating by rotary evaporation for 5 times, and spray drying to collect chitosan oligosaccharide. A of 2% aqueous solution of the resulting Chitosan oligosaccharide 420 <0.1; the resulting solution was centrifuged at 13000rpm for 2min, and no precipitate was formed.
1.8
Dissolving 15g of chitosan with the deacetylation degree of 98% by 300mL of 0.3M acetic acid, adjusting the pH value to 5.0 after complete dissolution, and adding water to reach the constant volume of 500 mL. Adding 1.5g (130U/g) of chitosanase, and stirring in a water bath at 25-35 ℃ for 8 h. Filtering the chitosan enzymolysis liquid by a plate frame, and then using filter membranes with different pore diameters to carry out ultrafiltration and nanofiltration on chitosan oligosaccharide(s) with corresponding molecular weight sections<5000 Da、<4000 Da、<3000 Da、<2000 Da、<1000Da、<800Da、<600Da、<400 Da). Concentrating by rotary evaporation for 5 times, and spray drying to collect chitosan oligosaccharide. A of 2% aqueous solution of the resulting Chitosan oligosaccharide 420 <0.1; the resulting solution was centrifuged at 13000rpm for 2min, and no precipitate was formed.
EXAMPLE 2 preparation of oligosaccharide vaccine
50mg of KLH was dissolved in 1ml of 0.1M sodium phosphate buffer pH7.2 containing 0.15M NaCl. 1mg of chitosan oligosaccharide with deacetylation degree of 10% and molecular weight of 1000-3000Da is added, and the mixture is dissolved and mixed evenly. Taking glutaraldehyde (CHO (CH) 2 ) 3 CHO) 50% aqueous solution 2. mu.L, add substrate solution, mix well, maintain 25 ℃ and shake gently for 60 min. Adding glycine solid 50mg, mixing, and keeping at 4 deg.C for 30 min. Using 0.1M sodium phosphate buffer solution containing 0.15M NaCl as mobile phase, removing low molecular impurities through Thermo Scientific field protein solution, obtaining carrier protein solution of coupling chitosan oligosaccharide, placing the carrier protein solution in a 3000Da dialysis bag for pure water dialysis for 24h, and obtaining the oligosaccharide vaccine after freeze-drying.
EXAMPLE 3 preparation of oligosaccharide vaccine
50mg of KLH was dissolved in 1ml of 0.1M sodium phosphate buffer pH7.2 containing 0.15M NaCl. 1mg of chitosan oligosaccharide with deacetylation degree of 95% and molecular weight of 1000-3000Da is added, and the mixture is dissolved and mixed evenly. Taking glutaraldehyde (CHO (CH) 2 ) 3 CHO) 50% aqueous solution 11. mu.L, adding substrate solution, and mixingThe temperature was kept at 25 ℃ and the vibration was gently carried out for 60 min. Adding glycine solid 250mg, mixing, and keeping at 4 deg.C for 30 min. Using 0.1M sodium phosphate buffer solution containing 0.15M NaCl as mobile phase, removing low molecular impurities through Thermo Scientific field protein solution, obtaining carrier protein solution of coupled chitosan oligosaccharide, placing in 2000Da dialysis bag for pure water dialysis for 24h, and obtaining oligosaccharide vaccine after freeze-drying.
EXAMPLE 4 preparation of oligosaccharide vaccine
50mg of BSA was dissolved in 1ml of 0.1M sodium phosphate buffer pH7.2 containing 0.15M NaCl. 1mg of chitosan oligosaccharide with deacetylation degree of 10% and molecular weight of 1000-3000Da is added, and the mixture is dissolved and mixed evenly. Taking glutaraldehyde (CHO (CH) 2 ) 3 CHO) 50% aqueous solution 2. mu.L, add substrate solution, mix well, maintain 25 ℃ and shake gently for 60 min. Adding glycine solid 50mg, mixing, and keeping at 4 deg.C for 30 min. Using 0.1M sodium phosphate buffer solution containing 0.15M NaCl as mobile phase, removing low molecular impurities through Thermo Scientific regions Zeba's protein desaling Columns to obtain carrier protein solution coupled with chitosan oligosaccharide, placing in 1000Da dialysis bag for pure water dialysis for 24h, and freeze-drying to obtain oligosaccharide vaccine.
EXAMPLE 5 preparation of oligosaccharide vaccine
50mg of BSA was dissolved in 1ml of 0.1M sodium phosphate buffer pH7.2 containing 0.15M NaCl. 1mg of chitosan oligosaccharide with deacetylation degree of 95% and molecular weight of 1000-3000Da is added, and the mixture is dissolved and mixed evenly. Taking glutaraldehyde (CHO (CH) 2 ) 3 CHO) 50% aqueous solution 11. mu.L, substrate solution was added, mixed well and gently shaken for 60min at 25 ℃. Adding glycine solid 250mg, mixing, and keeping at 4 deg.C for 30 min. Using 0.1M sodium phosphate buffer solution containing 0.15M NaCl as mobile phase, removing low molecular impurities through Thermo Scientific field protein solution, obtaining carrier protein solution of coupling chitosan oligosaccharide, placing the carrier protein solution in a 3000Da dialysis bag for pure water dialysis for 24h, and obtaining the oligosaccharide vaccine after freeze-drying.
EXAMPLE 6 preparation of oligosaccharide vaccine
50mg of OVA was dissolved in 1ml of 0.01M sodium phosphate buffer solution (pH 7.2). 1mg of chitosan oligosaccharide with deacetylation degree of 10% and molecular weight of 1000-3000Da is added, and the mixture is dissolved and mixed evenly. Taking glutaraldehyde (CHO (CH) 2 ) 3 CHO) 50% aqueous solution 2. mu.L, add bottomMixing the solution, and keeping at 25 deg.C and slightly vibrating for 60 min. Adding glycine solid 50mg, mixing, and keeping at 4 deg.C for 30 min. Using 0.01M sodium phosphate buffer solution with pH7.2 as mobile phase, removing low molecular impurities through Thermo Scientific field protein solution to obtain carrier protein solution coupled with chitosan oligosaccharide, placing in 3000Da dialysis bag for pure water dialysis for 24h, and lyophilizing to obtain oligosaccharide vaccine.
EXAMPLE 7 preparation of oligosaccharide vaccine
50mg of OVA was dissolved in 1ml of 0.01M sodium phosphate buffer solution (pH 7.2). 1mg of chitosan oligosaccharide with deacetylation degree of 95% and molecular weight of 1000-3000Da is added, and the mixture is dissolved and mixed evenly. Taking glutaraldehyde (CHO (CH) 2 ) 3 CHO) 50% aqueous solution 11. mu.L, substrate solution was added, mixed well and gently shaken for 60min at 25 ℃. Adding glycine solid 250mg, mixing, and keeping at 4 deg.C for 30 min. Using 0.01M sodium phosphate buffer solution with pH7.2 as mobile phase, removing low molecular impurities through Thermo Scientific field protein solution to obtain carrier protein solution coupled with chitosan oligosaccharide, placing in 3000Da dialysis bag for pure water dialysis for 24h, and lyophilizing to obtain oligosaccharide vaccine.
EXAMPLE 8 preparation of oligosaccharide vaccine
100mg of BSA was dissolved in 1ml of 0.1M sodium phosphate buffer (containing 0.15M NaCl) at pH 6.9. Adding 1mg of a polymer with a deacetylation degree of 0.05% and a molecular weight<Dissolving chitosan oligosaccharide of 400Da, and mixing. 50% glutaraldehyde (CHO (CH) 2 ) 3 CHO) 1. mu.L of aqueous solution was added and mixed, and gently shaken at 0 ℃ for 150 min. Adding NaBH 4 The solid 42.5mg was mixed well and kept at 0 ℃ for 75 min. Taking 0.1M sodium phosphate buffer solution (containing 0.15M NaCl) as a mobile phase, removing low molecular impurities through a desalting prepacked column G10 to obtain a carrier protein solution coupled with chitosan oligosaccharide, placing the carrier protein solution in a 3000Da dialysis bag for pure water dialysis for 24h, and freeze-drying to obtain the oligosaccharide vaccine.
EXAMPLE 9 preparation of oligosaccharide vaccine
90mg of KLH was dissolved in 1ml of 0.1M sodium phosphate buffer pH7. Adding 1mg of a polymer with a deacetylation degree of 0.05% and a molecular weight<600Da chitosan oligosaccharide, and dissolving and mixing uniformly. 50% of hexanedial (CHO (CH) 2 ) 4 CHO) 1.4. mu.L of aqueous solution was added and mixed, and gently shaken at 2 ℃ for 140 min. Adding 1M pH7.4Tris-HCl buffer 463. mu. Lmg was mixed well and kept at 2 ℃ for 70 min. Removing low molecular impurities by desalting pre-packed column G15 with 0.1M sodium phosphate buffer solution as mobile phase to obtain carrier protein solution coupled with chitosan oligosaccharide, placing in 3000Da dialysis bag, dialyzing with pure water for 24 hr, and lyophilizing to obtain oligosaccharide vaccine.
EXAMPLE 10 preparation of oligosaccharide vaccine
80mg of OVA was dissolved in 1ml of 0.01M sodium phosphate buffer solution (pH 7.2). Adding 1mg of a polymer having a degree of deacetylation of 0.1% and a molecular weight<Dissolving chitosan oligosaccharide of 800Da, and mixing. 50% heptanedial (CHO (CH) 2 ) 5 CHO) 1.8. mu.L of aqueous solution was added and mixed, and gently shaken at 4 ℃ for 120 min. Adding glycine 67.5mg, mixing, and keeping at 4 deg.C for 60 min. Taking 0.01M sodium phosphate buffer solution (pH7.2) as mobile phase, removing low molecular impurities through desalting pre-packed column G25 to obtain carrier protein solution coupled with chitosan oligosaccharide, placing in 3000Da dialysis bag, dialyzing with pure water for 24 hr, and lyophilizing to obtain oligosaccharide vaccine.
EXAMPLE 11 preparation of oligosaccharide vaccine
70mg of blue carrier was taken TM Dissolved in 1ml of 0.1M carbonate buffer, pH 7.1. Adding 1mg of a polymer having a degree of deacetylation of 0.1% and a molecular weight<Dissolving 1000Da chitosan oligosaccharide, and mixing. 50% suberaldehyde (CHO (CH) was taken 2 ) 6 CHO) 2. mu.L of aqueous solution was added and mixed, and gently shaken for 110min at 8 ℃. Adding lysine 70mg, mixing, and maintaining at 8 deg.C for 55 min. Using 0.1M carbonate buffer solution as a mobile phase, and removing low molecular impurities through a desalting prepacked column G50 to obtain a carrier protein solution coupled with the chitosan oligosaccharide.
EXAMPLE 12 preparation of oligosaccharide vaccine
65mg of tetanus toxin was dissolved in 1ml of 0.02M HEPES buffer solution at pH 7.3. 1mg of a polymer having a degree of deacetylation of 1% and a molecular weight<2000Da chitosan oligosaccharide, and dissolving and mixing uniformly. Taking 50% nonane dialdehyde (CHO (CH) 2 ) 7 CHO) aqueous solution 3. mu.L was added and mixed, and gently shaken for 90min at 10 ℃. Adding 2M Tris-HCl buffer solution 403. mu. Lmg with pH8.0, mixing, and keeping at 10 deg.C for 45 min. Taking 0.02M HEPES buffer solution as a mobile phase, and removing low molecular impurities through a desalting column Bio-gel P2 to obtain a carrier protein solution coupled with the chitosan oligosaccharide.
EXAMPLE 13 preparation of oligosaccharide vaccine
60mg of HMP was dissolved in 1ml of 0.05M borate buffer pH 7.4. 1mg of a polymer having a degree of deacetylation of 1% and a molecular weight<3000Da chitosan oligosaccharide, and uniformly dissolving. 50% of decanedial (CHO (CH) was taken 2 ) 8 CHO) aqueous solution 4. mu.L was added and mixed, and gently shaken for 80min at 14 ℃. Adding glycine 120mg, mixing, and maintaining at 14 deg.C for 40 min. And removing low-molecular impurities by using 0.05M borate buffer solution as a mobile phase through a desalting column Bio-gel P4 to obtain a chitosan oligosaccharide-coupled carrier protein solution.
EXAMPLE 14 preparation of oligosaccharide vaccine
55mg of Pseudomonas aeruginosa toxin A was dissolved in 1mL of pure water at pH 7.5. 1mg of chitosan oligosaccharide with deacetylation degree of 10% and molecular weight of 1000-2000 Da is added, and the mixture is dissolved and mixed evenly. Taking 50% CHO (CH) 2 ) 10 Adding 5 μ L of CHO aqueous solution, mixing, and slightly vibrating at 17 deg.C for 70 min. Adding lysine 137.5mg, mixing, and keeping at 17 deg.C for 35 min. Pure water is used as a mobile phase, and low molecular impurities are removed through a desalting column Bio-gel P6 to obtain a carrier protein solution coupled with the chitosan oligosaccharide.
EXAMPLE 15 preparation of oligosaccharide vaccine
50mg of cholera toxin was dissolved in 1ml of 0.01M sodium phosphate buffer pH 7.6. 1mg of chitosan oligosaccharide with deacetylation degree of 10% and molecular weight of 2000-3000Da is added, and the mixture is dissolved and mixed evenly. 4mgBS3 was dissolved in 80. mu.L of starting sodium phosphate buffer, 60. mu.L was added to the substrate solution, mixed well and kept at 20 ℃ with gentle shaking for 60 min. Adding 3M Tris-HCl buffer 413 mu Lmg with pH7.0, mixing, and keeping at 20 deg.C for 30 min. And removing low-molecular impurities by using 0.01M sodium phosphate buffer solution as a mobile phase through a desalting column Bio-gel P10 to obtain a carrier protein solution of the coupled chitosan oligosaccharide.
EXAMPLE 16 preparation of oligosaccharide vaccine
40mg of pertussis toxin was dissolved in 1ml of 0.1M sodium phosphate buffer, pH 7.7. 1mg of chitosan oligosaccharide with deacetylation degree of 10% and molecular weight of 3000-4000 Da is added, and the mixture is dissolved and mixed evenly. 4mgDSS was dissolved in 120. mu.L DMSO, 105. mu.L of the DMSO solution was added to the substrate solution and mixed well, and the mixture was gently shaken for 50min at 22 ℃. Adding glycine 157.5mg, mixing, and maintaining at 22 deg.C for 25 min. And removing low-molecular impurities by using 0.1M sodium phosphate buffer solution as a mobile phase through a desalting prepacked column Bio-gel P6DG to obtain a carrier protein solution of the coupled chitosan oligosaccharide.
EXAMPLE 17 preparation of oligosaccharide vaccine
30mg of Clostridium perfringens exotoxin was dissolved in 1ml of 0.1M sodium phosphate buffer (containing 0.15M NaCl) at pH 7.8. Adding 1mg of a polymer having a degree of deacetylation of 20% and a molecular weight<Dissolving and mixing chitosan oligosaccharide of 4000 Da. Mixing 100mg (100. mu.L) of BS (PEG) 2 0.9mL of DMSO was added to prepare a 250mM reagent, 40. mu.L of the reagent was added to the substrate solution and mixed well, and the mixture was gently shaken at 24 ℃ for 40 min. Adding lysine 160mg, mixing, and maintaining at 24 deg.C for 20 min. Low-molecular impurities were removed by Thermo Scientific "Zeba" cells using 0.1M sodium phosphate buffer (containing 0.15M NaCl) as the mobile phase to obtain a solution of chitosan oligosaccharide-conjugated carrier protein.
EXAMPLE 18 preparation of oligosaccharide vaccine
20mg of hepatitis B surface antigen was dissolved in 1ml of 0.1M carbonate buffer pH 7.9. Adding 1mg of a polymer having a degree of deacetylation of 20% and a molecular weight<5000Da chitosan oligosaccharide, and dissolving and mixing uniformly. Mixing 100mg (100. mu.L) of BS (PEG) 4 The reagent was prepared at 252mM in 720. mu.L DMSO, 36.9. mu.L was added to the substrate solution and mixed well, and the mixture was gently shaken at room temperature for 20 min. Then, the mixture was mixed with 434. mu. Lmg parts of 3M Tris-HCl buffer solution (pH 7.5) and kept at room temperature for 10 min. Using 0.1M carbonate buffer solution as mobile phase, removing low molecular impurities by Thermo Scientific Slide-A-Lyzer analysis Cassettes to obtain carrier protein solution coupled with chitosan oligosaccharide.
EXAMPLE 19 preparation of oligosaccharide vaccine
15mg of hepatitis B core antigen was dissolved in 1ml of 0.02M HEPES buffer pH8. Adding 1mg of a polymer having a degree of deacetylation of 20% and a molecular weight<3000Da chitosan oligosaccharide, and uniformly dissolving. Mixing 100mg (100. mu.L) of BS (PEG) 5 Add 650. mu.L of DMSO to make up 250mM reagent, add 37.5. mu.L of substrate solution and mix well, shake gently for 30min at room temperature. Adding glycine 150mg, mixing, and standing at room temperature for 15 min. Taking 0.02M HEPES buffer solution as a mobile phase, and removing low molecular impurities through a dialysis bag with the aperture of 10kDa to obtain a carrier protein solution of the coupled chitosan oligosaccharide.
EXAMPLE 20 preparation of oligosaccharide vaccine
10mg of rotavirus VP7 protein was dissolved in 1ml of 0.05M borate buffer solution (pH 8.1). Adding 1mg of chitosan oligosaccharide with deacetylation degree of 50% and molecular weight of 2000-4000Da, dissolving and mixing uniformly. Mixing 100mg (100. mu.L) of BS (PEG) 6 251mM reagent was prepared by adding 594. mu.L of DMSO, 38.17. mu.L of the reagent was added to the substrate solution and mixed well, and the mixture was gently shaken for 40min at room temperature. Adding lysine 137.5mg, mixing, and standing at room temperature for 20 min. And (3) removing low-molecular impurities by using 0.05M borate buffer solution as a mobile phase through a dialysis bag with the aperture of 8kDa to obtain a carrier protein solution coupled with the chitosan oligosaccharide.
EXAMPLE 21 preparation of oligosaccharide vaccine
5mg of the diphtheria toxin mutant CRM was dissolved in 1mL of purified water at pH 8.2. Adding 1mg of chitosan oligosaccharide with deacetylation degree of 50% and molecular weight of 2000-4000Da, dissolving and mixing uniformly. Taking 38.7 mu L of BS (PEG) 7 Adding substrate solution, mixing, and slightly vibrating at 25 deg.C for 20 min. Add 3M Tris-HCl buffer 331. mu. Lmg of pH7.2 and mix well, keep at 25 ℃ for 10 min. And (3) removing low-molecular impurities by using pure water as a mobile phase through a dialysis bag with the aperture of 6kDa to obtain a carrier protein solution of the coupled chitosan oligosaccharide.
EXAMPLE 22 preparation of oligosaccharide vaccine
3mg of diphtheria toxin mutant CRM191 was dissolved in 1mL of 0.01M sodium phosphate buffer, pH 8.3. 1mg of chitosan oligosaccharide with deacetylation degree of 95% and molecular weight of 1000-3000Da is added, and the mixture is dissolved and mixed evenly. 42.14 μ L of BS (PEG) 8 The substrate solution was added and mixed well and kept at 28 ℃ with gentle shaking for 18 min. Adding 105mg glycine, mixing, and keeping at 28 deg.C for 9 min. And (3) removing low-molecular impurities by using 0.01M sodium phosphate buffer solution as a mobile phase through a dialysis bag with the aperture of 5kDa to obtain a carrier protein solution of the coupled chitosan oligosaccharide.
EXAMPLE 23 preparation of oligosaccharide vaccine
1mg of diphtheria toxin mutant CRM3201 was dissolved in 1mL0.2M sodium phosphate buffer (pH8.5). 1mg of chitosan oligosaccharide with deacetylation degree of 95% and molecular weight of 3000-500Da is added, and the mixture is dissolved and mixed evenly. Taking 45.2 mu L of BS (PEG) 9 Adding substrate solution, mixing, and slightly vibrating at 30 deg.C for 15 min. Adding lysine 80mg, mixing, and maintaining at 30 deg.C for 7.5 min. Using 0.2M sodium phosphate buffer as mobile phase, passing through a pore size of 4kDaAnd removing low molecular impurities by a dialysis bag to obtain a carrier protein solution of the coupled chitosan oligosaccharide.
EXAMPLE 24 preparation of oligosaccharide vaccine
0.8mg of respiratory syncytial virus F protein was dissolved in 1ml of 0.1M sodium phosphate buffer (containing 0.15M NaCl) at pH 8.8. Adding 1mg of chitosan oligosaccharide with deacetylation degree of 98% and molecular weight of 4000-5000Da, dissolving and mixing uniformly. 42.84 μ L of BS (PEG) 12 The substrate solution was added, mixed well and kept at 33 ℃ with gentle shaking for 12 min. Adding 149 μ Lmg pH7.8 4M Tris-HCl buffer solution, mixing, and keeping at 33 deg.C for 6 min. Taking 0.1M sodium phosphate buffer solution (containing 0.15M NaCl) as a mobile phase, removing low molecular impurities through a dialysis bag with the aperture of 3kDa to obtain a carrier protein solution of the coupled chitosan oligosaccharide, and freeze-drying to obtain the chitosan oligosaccharide conjugate carrier protein solution.
EXAMPLE 25 preparation of oligosaccharide vaccine
0.5mg respiratory syncytial virus G protein was dissolved in 0.5mL 0.3M carbonate buffer at pH 9. 1mg of a buffer solution of 0.5mL of 0.1M carbonate having a degree of deacetylation of 98% and a molecular weight of 9 was dissolved in<1000Da chitosan oligosaccharide, and mixing with carrier protein solution. Direct addition of 41.2. mu.L of BS (PEG) 15 Adding substrate solution, mixing, and standing at 35 deg.C for 10 min. Adding glycine 50mg, mixing, and keeping at 35 deg.C for 5 min. Removing low molecular impurities by using 0.3M carbonate buffer solution as a mobile phase through a dialysis bag with the aperture of 1kDa to obtain a carrier protein solution of the coupled chitosan oligosaccharide, and performing spray drying to obtain the chitosan oligosaccharide.
Example 26 coupling ratio detection
(1) Standard curve 0.1030g of anhydrous glucose (water content 1.22%) are weighed out precisely, and the volume is adjusted to 50mL by pure water and mixed evenly. 5mL of the solution was taken in a 50mL measuring flask and water was added to 50mL to obtain 0.2035 mg/mL of the standard solution. Adding 0, 10, 20, 40, 60, 80, 100, 120, 140 and 160 mu L of water into a 1.5 mL centrifuge tube, respectively adding 200, 190, 180, 160, 140, 120, 100, 80, 60 and 40 mu L of water, and uniformly mixing to obtain a series of concentrations of 0-0.1628 mg/mL.
(2) Weighing oligosaccharide vaccine to prepare aqueous solution with mass concentration of 1 mg/mL, and taking carrier protein with the same concentration unconjugated chitosan oligosaccharide as blank control. 400 μ L of oligosaccharide vaccine solution was placed in a 2 mL hydrolysis tube, 80 μ L of concentrated sulfuric acid was added, the cap was tightened, and the temperature was maintained at 105 ℃ for 1.5 h. And (5) replenishing water to dilute the hydrolysate to a scale of 0.5mL, and blowing and uniformly mixing by using a pipettor to obtain a sample solution to be detected. The carrier protein blank solution was treated in the same way. If the detected value exceeds the range, diluting with water in proper amount.
(3) Sample determination A series of standard solutions and 200 μ L of sample solution to be determined are put into a 1.5 mL centrifuge tube, 600 μ L of 0.2% (w/v) anthrone sulfuric acid solution is added, the mixture is evenly mixed and covered, a boiling water bath is used for heating for 15min, and the mixture is taken out and cooled for 15min in an ice water bath. After mixing, 200. mu.L of the mixture was added to a 96-well plate, and absorbance at 625nm was measured by a microplate reader. Standard curve at tested concentration range y =4.840x-0, R 2 = 0.991. And reading the absorbance of the sample solution at 625nm, deducting the blank absorption value of the corresponding carrier protein, and calculating the sugar concentration of the sample according to the standard curve. And calculating the coupling mass ratio of the carrier protein to the chitosan oligosaccharide according to the following formula:
TABLE 1 coupling Mass ratio of Carrier protein to Chitosan oligosaccharide
Example 27 antigenicity and immunity of oligosaccharide vaccines
The antigenicity and immunity of chitosan oligosaccharide coupled with the immunogenic carrier protein are evaluated by animal inoculation immunization and peripheral blood detection to detect the change of the key IL-17F cytokine level in Th17 cells, and the significance of the parameter is equivalent to the change of IgG or IgM in B cell mediated humoral immunity.
(1) Immunization protocols
Female mice of Kunming species (SPF grade) of 4-6 weeks old were treated with 6 mice each with a physiological saline blank group, KLH adjuvant group, chitosan oligosaccharide-KLH group with a degree of deacetylation of 10% in example 2, and chitosan oligosaccharide-KLH group with a degree of deacetylation of 95% in example 3. Subcutaneous multi-point injection is carried out on the neck and the back of the mice in the saline group respectively at 0, 2, 4 and 6 weeks after the start of the test, and the dosage is 0.2 mL/mouse; the test sample containing the adjuvant was injected into each of the remaining groups of mice in the same manner at 0.2 mL/mouse.
In order to activate immune response as much as possible and reduce uncontrollable nature, conventional Freund's complete and incomplete adjuvants are selected. Freund's complete adjuvant is selected for the immunization injection at the 0 th week, and Freund's incomplete adjuvant is selected for the immunization injection at the 2 nd, 4 th and 6 th weeks. KLH, chitosan oligosaccharide 95% deacetylation-KLH and chitosan oligosaccharide 1% deacetylation-KLH are prepared into 0.1 mg/mL solution by 0.02M PBS (pH 7.2-7.4), and then mixed with Freund's complete adjuvant or Freund's incomplete adjuvant in equal volume, and the mixture is emulsified uniformly by ultrasonic wave.
(2) Intracellular cytokine detection
Lymphocyte extraction and stimulation: before the 0 th week of immunization injection and after the 6 th week of immunization injection, 0.3 mL of blood was taken from the submaxillary vein of the mouse and mixed with 1.5 mL of an anticoagulation tube, 3 mL of 1 Xerythrocyte lysate was added to lyse erythrocytes for 4 min, supernatant was discarded by centrifugation at 500 g for 10min at 4 ℃, supernatant was discarded by centrifugation at 500 g for 10min after 10 mL of 0.01M PBS was added, and the precipitated lymphocytes were suspended in a round bottom 96-well plate with 200. mu.L of a stimulating solution (formulation of stimulating solution: PMA 1/1000, inomycin 1/1000, BFA (brefeldin) 1/1000, monensin1/1000 in 1640 complete medium) and incubated for 4h in a cell culture box.
Extracellular staining: the 96-well plate was centrifuged at 1600 rpm at 4 ℃ for 10min (same centrifugation conditions), the supernatant was discarded, 70. mu.L/well of FACS buffer containing extracellular staining antibody (containing fluorescent staining antibodies CD3e, CD4, CCR6, and 7-AAD at 1:100, 1:200, 1:100, and 1:100, respectively) was blown, and incubated at 4 ℃ in the dark for 40min for extracellular staining.
Membrane breaking: adding FACS buffer solution containing no antibody 180 μ L/well, blowing, centrifuging, discarding supernatant, adding membrane-breaking solution at 100 μ L/well, incubating at 4 deg.C in dark for 40min, and performing membrane-breaking treatment.
Intracellular staining: the cells were blown up with 180. mu.L/well of 1 Xmembrane-breaking buffer, centrifuged and the supernatant discarded, 70. mu.L/well of membrane-breaking buffer containing intracellular staining antibody (containing fluorescent staining IL-17F antibody, added in amounts of 1:100, respectively) was added, and incubated at room temperature in the dark for 40min to stain intracellular cytokines.
Flow cytometry detection: adding FACS buffer solution into 180 mu L/hole for blowing, centrifuging, removing supernatant, adding FACS buffer solution into 100 mu L/hole for blowing, and detecting the intracellular IL-17F level of Th17 on a machine to reflect the activation of the tested sample on the mouse cell immunity.
The same procedure was performed to examine the chitosan oligosaccharide-BSA group having a degree of deacetylation of 10% in example 4, the chitosan oligosaccharide-BSA group having a degree of deacetylation of 95% in example 5, the chitosan oligosaccharide-OVA group having a degree of deacetylation of 10% in example 6, and the chitosan oligosaccharide-OVA group having a degree of deacetylation of 95% in example 7. The ratio of Th17 intracellular IL to Th17 before and after immunization of mice with each oligosaccharide vaccine is shown in tables 2-4, for example.
TABLE 2 Th17 intracellular IL-17F ratio before and after KLH-coupled Chitosan-immunized mice
Statistical difference compared to saline blank and KLH adjuvant group, α = 0.05.
TABLE 3 Th17 intracellular IL-17% ratio before and after immunization of mice with BSA coupled with chitosan
# was statistically different, α =0.05, compared to the saline blank and the BSA adjuvant groups.
TABLE 4 Th17 intracellular IL-17 ratios before and after OVA-coupled Chitosan-immunized mice
+ was statistically different, α =0.05, compared to the saline blank and BSA adjuvant groups.
Therefore, compared with a 'physiological saline blank group' and a 'carrier adjuvant' group only containing a blank carrier and an adjuvant, the increase of the IL-17F level before and after immunization has statistical significance, and the chitosan oligosaccharide coupled with KLH, BSA and OVA can effectively activate Th17 cells to secrete IL-17F with antifungal efficacy, has antigenicity and immunity, has a low deacetylation degree and has a higher IL-17F promoting effect than the deacetylation degree.
EXAMPLE 28 challenge experiments with oligosaccharide vaccines
Female mice of Kunming species (SPF grade) of 4-6 weeks old were selected, and 6 mice in each group were provided with 5 groups of a physiological saline blank group, a KLH adjuvant group, a chitosan oligosaccharide-KLH group with a deacetylation degree of 10% in example 2, a chitosan oligosaccharide-KLH group with a deacetylation degree of 95% in example 3, and a fluconazole control group.
Subcutaneous multi-point injection is carried out on the neck and the back of the mice in the saline group respectively at 0, 2, 4 and 6 weeks after the start of the test, and the dosage is 0.2 mL/mouse; 0.2 mL/mouse of a test sample containing an adjuvant is injected by the same method of a KLH adjuvant group, a chitosan oligosaccharide-KLH group with deacetylation degree of 10% and a chitosan oligosaccharide-KLH group with deacetylation degree of 95%; the fluconazole group is not injected, and the mice are subjected to intragastric administration at the dose of 50 mg/kg d at the 6 th week for 7 days continuously, so that the fluconazole group is a positive control group for preventive antifungal administration before toxicity attack.
Freund's complete adjuvant is selected for the immunization injection at the 0 th week, and Freund's incomplete adjuvant is selected for the immunization injection at the 2 nd, 4 th and 6 th weeks. KLH, a chitosan oligosaccharide-KLH group with a deacetylation degree of 10% and a chitosan oligosaccharide-KLH group with a deacetylation degree of 95% were combined with 0.02M PBS (pH 7.2-7.4) to prepare a 0.5 mg/mL solution, which was then mixed with Freund's complete adjuvant or Freund's incomplete adjuvant in equal volume, and the mixture was emulsified by sonication.
At week 8, the cultured Candida albicans was suspended in 0.01M PBS at 2X 10 4 The virus is attacked by intravenous injection from the tail of the mouse at the dose of 0.1 mL. After 10 days, the mice were sacrificed, the two kidneys were transected longitudinally and transversely, respectively, half of the kidneys were fixed with glutaraldehyde, and inflammation status was observed by HE staining; and weighing the rest half kidney tissues, homogenizing, performing plate inoculation culture on an improved Shaoer culture medium, and calculating the kidney capacity CFU of the fungus.
HE staining is mainly used for observing the infiltration and fibrosis degree of multinucleated granulocytes, lymphocytes, plasma cells, macrophages and megakaryocytes, and is used for evaluating the inflammatory response degree. HE staining score was rated 0-4, (1) 0: no infiltration is carried out; (2) 1 minute: very little infiltration (10-50, 400 times under the mirror observation); (3) and 2, dividing: slight infiltration (50-200, 400 times under the mirror observation); (4) and 3, dividing: severe infiltration (200 and 500, observed under 400 times of lens); (5) and 4, dividing: severe infiltration (> 500, 400 fold under mirror).
The same procedure was performed to examine the effects of the chitosan oligosaccharide-BSA group having a deacetylation degree of 10% in example 4, the chitosan oligosaccharide-BSA group having a deacetylation degree of 95% in example 5, the chitosan oligosaccharide-OVA group having a deacetylation degree of 10% in example 6, and the chitosan oligosaccharide-OVA group having a deacetylation degree of 95% in example 7 on the renal inflammatory response after challenge with cryptococcus. The effect of each oligosaccharide vaccine on kidney inflammation in mice after fungal challenge is shown in table 5.
TABLE 5 Effect of different vaccines on Kidney HE staining score after fungal challenge
Compared with the saline blank group, the KLH adjuvant group and the fluconazole group, the statistical difference is that the alpha = 0.05;
# was statistically different from saline blank, α = 0.05;
+ was statistically different with α =0.05 compared to saline blank, KLH adjuvant, fluconazole.
As can be seen from Table 5, the chitosan oligosaccharide coupled with the carrier protein can effectively reduce the inflammatory reaction of fungus attack on the kidney of the mouse, and the inflammation with low deacetylation degree is lighter than the inflammation with high deacetylation degree.
Claims (9)
1. An oligosaccharide vaccine for the prevention of invasive fungal infection, comprising a chitosan oligosaccharide conjugated to an immunogenic carrier protein; the deacetylation degree of the chitosan oligosaccharide is 10 percent, and the relative average molecular weight is 1000-3000 Da;
the immunogenic carrier protein is selected from any one of KLH, BSA and OVA.
2. A method of preparing the oligosaccharide vaccine of claim 1, comprising the steps of:
(1) dissolving the immunogenic carrier protein, the chitosan oligosaccharide and the coupling agent in a coupling solution, and incubating;
(2) adding a terminator for incubation;
(3) purifying to obtain the oligosaccharide vaccine.
3. The method according to claim 2, wherein in step (1), the coupling agent is selected from CHO (CH) 2 ) m CHO，BS 3 DSS or BS (PEG) n Any one of the above;
in the step (2), the terminator is selected from Tris-HCl, glycine, lysine or NaBH 4 。
4. The method according to claim 3, wherein the CHO (CH) is 2 ) m M =3-10 in CHO; bs (peg) n with n = 2-15.
5. The method according to claim 2, wherein in the step (1), the mass ratio of the immunogenic carrier protein to the chitosan oligosaccharide is 0.5-100: 1; the mass ratio of the coupling agent to the chitosan oligosaccharide is 0.5-10: 1; in the step (2), the mass ratio of the terminating agent to the coupling agent is 5-85: 1.
6. The method according to claim 2, wherein in the step (1), the coupling solution is selected from water or 0.01-0.3M buffer solution with pH of 6.9-9 and without amine group; the concentration of the immunogenic carrier protein is 0.5-100 mg/mL.
7. The method according to claim 6, wherein in the step (1), the coupling solution is a buffer solution with pH of 7.2-8 and concentration of 0.05-0.2M; the concentration of the immunogenic carrier protein is 10-90 mg/mL.
8. The method of claim 2, wherein step (3) is further followed by the step of making the oligosaccharide vaccine solution into a solid.
9. Use of the oligosaccharide vaccine of claim 1 in the manufacture of a medicament for the prevention of an invasive fungal infection.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2018111155606 | 2018-09-25 | ||
| CN201811115560 | 2018-09-25 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109432416A CN109432416A (en) | 2019-03-08 |
| CN109432416B true CN109432416B (en) | 2022-08-05 |
Family
ID=65556300
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811479983.6A Active CN109432416B (en) | 2018-09-25 | 2018-12-05 | Oligosaccharide vaccine for preventing invasive fungal infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109432416B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113274488B (en) * | 2021-04-30 | 2023-08-29 | 山东省药学科学院 | Oligosaccharide vaccine for specifically preventing fungal infection and preparation method thereof |
| CN113274489B (en) * | 2021-04-30 | 2023-08-29 | 山东省药学科学院 | Chitin oligosaccharide vaccine for preventing fungal infection and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106366163A (en) * | 2016-10-31 | 2017-02-01 | 中国人民解放军第二军医大学 | Beta-1,2-D-oligomeric mannose peptide-protein conjugate and preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB201218954D0 (en) * | 2012-10-22 | 2012-12-05 | Norwegian University Of Life Sciences The | Composition |
-
2018
- 2018-12-05 CN CN201811479983.6A patent/CN109432416B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106366163A (en) * | 2016-10-31 | 2017-02-01 | 中国人民解放军第二军医大学 | Beta-1,2-D-oligomeric mannose peptide-protein conjugate and preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Conjugation of chitosan oligosaccharides via a carrier protein markedly improves immunogenicity of porcine circovirus vaccine;Guiqiang Zhang等;《Glycoconjugate Journal》;20180726;第35卷;摘要 * |
| The Positive Correlation of the Enhanced Immune Response to PCV2 Subunit Vaccine by Conjugation of Chitosan Oligosaccharide with the Deacetylation Degree;Guiqiang Zhang等;《Marine Drugs》;20170726;第15卷(第8期);摘要 * |
| 潘搏.白念珠菌甘露多糖疫苗评价及白念珠菌侵入人内皮和上皮细胞动态观察.《中国优秀博士学位论文全文数据库 医药卫生科技辑》.2016,(第03期), * |
| 白念珠菌甘露多糖疫苗评价及白念珠菌侵入人内皮和上皮细胞动态观察;潘搏;《中国优秀博士学位论文全文数据库 医药卫生科技辑》;20160315(第03期);第8-10、20-21页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109432416A (en) | 2019-03-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7491511B2 (en) | Fungal antigens and process for producing the same | |
| JPH05503952A (en) | Method for producing soluble glucans | |
| US20170157235A1 (en) | Method for producing and vaccine composition of neisseria meningitidis serogroups a, c, y, and w-135 oligosaccharides conjugated to glycan-free carrier protein | |
| JPH06107702A (en) | New glucan preparation | |
| FR2682388A1 (en) | PROCESS FOR THE PREPARATION OF AN OLIGOSIDE BY DEPOLYMERIZATION OF A POLYOSIDE FROM A PATHOGENIC AGENT, OLIGOSIDE THUS OBTAINED AND ITS USE IN PARTICULAR AS A VACCINE AGENT | |
| CN109432416B (en) | Oligosaccharide vaccine for preventing invasive fungal infection | |
| US11547719B2 (en) | Acetylation of aloe polysaccharides | |
| CN114163545B (en) | Lycium barbarum polysaccharide and application thereof in reducing blood sugar | |
| CN112741901A (en) | Vaccine containing streptococcus pneumoniae capsular polysaccharide type 5 and preparation method thereof | |
| RU2378015C2 (en) | Conjugate for immunisation and vaccination and method for improving immunogenicity | |
| IE47149B1 (en) | Vaccine | |
| CN113274489B (en) | Chitin oligosaccharide vaccine for preventing fungal infection and preparation method thereof | |
| WO2012058957A2 (en) | Polysaccharide extracts of epimedium, fractions thereof and uses thereof in vaccine adjuvants | |
| WO2023036203A1 (en) | Cs-4 fermented mycelium heteropolysaccharide, preparation method therefor and use thereof | |
| RU2260053C2 (en) | Method for isolation of bioactive fraction (baf) containing s-lipopolysaccharides (s-lps) from gram-negative bacteria, baf for prevention and treatment of diseases induced by endotoxic s-lps producing gram-negative bacteria, pharmaceutical composition, methods for inducing of protective immunity and improving state in patient in need of immune status increasing | |
| US11173193B2 (en) | Immunobiological products | |
| WO2012058935A1 (en) | Total polysaccharides of radix isatidis and their fractions, and uses thereof as vaccine adjuvants | |
| IE44448B1 (en) | Gonococcal pili processes for the preparation thereof and the use thereof | |
| CN108295253A (en) | A kind of A, C group meningitis cocci-b types haemophilus influenzae/encephalitis B combined vaccine | |
| CN1158088C (en) | Water extracted and alcohol precipitated matter of subprostrate sophora and its application in medicine | |
| CN113274488B (en) | Oligosaccharide vaccine for specifically preventing fungal infection and preparation method thereof | |
| CN114316081B (en) | A sulfated polysaccharide of Botrytis longipedicularis with SARS-CoV-2 inhibiting activity, and its preparation method and application | |
| CN113461832B (en) | Bacillus calmette-guerin mannan, preparation method and application thereof | |
| JP2851556B2 (en) | Type 1 and type F fimbriae-adhesins isolated from novel Escherichia coli strains, their preparation and their use | |
| SU1750689A1 (en) | Method for preparation of polysaccharido-protein vaccine against neisseria meningitis serogroup b |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |