CN116999546A - Acinetobacter baumannii core oligosaccharide-carrier protein conjugate, and preparation method and application thereof - Google Patents
Acinetobacter baumannii core oligosaccharide-carrier protein conjugate, and preparation method and application thereof Download PDFInfo
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- CN116999546A CN116999546A CN202311222464.2A CN202311222464A CN116999546A CN 116999546 A CN116999546 A CN 116999546A CN 202311222464 A CN202311222464 A CN 202311222464A CN 116999546 A CN116999546 A CN 116999546A
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- carrier protein
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Classifications
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- 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
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- 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
- A61K39/02—Bacterial antigens
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
- A61K39/1045—Moraxella
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- 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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- 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
- A61P31/04—Antibacterial agents
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- 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
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
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- 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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application belongs to the field of chemistry, and particularly relates to a acinetobacter baumannii core oligosaccharide-carrier protein conjugate, and a preparation method and application thereof. The core oligosaccharide of the acinetobacter baumanii core oligosaccharide-protein conjugate is alpha-Kdo- (2-4) - [ alpha-Kdo- (2-5) ] -alpha-Kdo branched trisaccharide, and the carrier protein comprises tetanus toxoid, diphtheria toxoid, a nontoxic variant of diphtheria toxin, haemophilus influenzae D protein, a group B meningococcal outer membrane protein complex or Q beta phage protein particles.
Description
The present application claims priority from chinese patent application No. [ CN202210040080.8 ] filed 1/14/2022, entitled "a core oligosaccharide fragment in serotype lipopolysaccharide of acinetobacter baumannii ATCC 17904 and method of synthesizing same", and is incorporated by reference in its entirety.
Technical Field
The application belongs to the field of chemistry, and particularly relates to a acinetobacter baumannii core oligosaccharide-carrier protein conjugate, and a preparation method and application thereof.
Background
Acinetobacter baumannii (Acinetobacter baumannii), also known as Acinetobacter baumannii, belongs to gram-negative bacteria, is a conditional pathogenic bacterium of strict aerobic and non-lactose fermentation, does not have flagella, has low mobility, but has extremely strong vitality. Acinetobacter baumannii is widely distributed in hospital environments, particularly in Intensive Care Units (ICU), emergency departments and respiratory medicine wards, and can cause acute pneumonia, meningitis and septicemia of people, and serious infection of blood and soft tissues of the human body, the mortality of patients after infection can reach 7.8% -23%, and the mortality of untreated patients infected by Acinetobacter baumannii can even reach more than 50%. Acinetobacter baumannii has the ability to rapidly acquire and spread drug resistance, and the resulting infectious disease has become the most refractory and refractory hospital acquired infectious disease worldwide. In 2017, acinetobacter baumannii was listed by the World Health Organization (WHO) as the most urgent class of bacteria for which the development of novel drugs or vaccines was needed. Therefore, there is a need to develop a novel drug or vaccine that can be used for the treatment or prevention of acinetobacter baumannii.
Disclosure of Invention
In view of the above, the present application aims to provide a series of acinetobacter baumannii core oligosaccharide-carrier protein conjugates, and a preparation method and application thereof, and the specific technical scheme is as follows.
A core oligosaccharide-carrier protein conjugate of acinetobacter baumanii, wherein the core oligosaccharide of the core oligosaccharide-carrier protein conjugate of acinetobacter baumanii is alpha-Kdo- (2- & gt 4) - [ alpha-Kdo- (2- & gt 5) ] -alpha-Kdo branched trisaccharide (hereinafter referred to as oligosaccharide 1), and the protein comprises tetanus toxoid, diphtheria toxoid, a nontoxic variant (CRM 197) of diphtheria toxin, haemophilus influenzae D protein, a meningococcal outer membrane protein complex of group B, or Qbeta phage protein particles and the like. The carrier proteins are all carrier proteins capable of enhancing or aiding in enhancing an immune response and have an amino acid structure capable of binding to a core oligosaccharide intermediate.
Further, the alpha-Kdo- (2- & gt 4) - [ alpha-Kdo- (2- & gt 5) ] -alpha-Kdo branched trisaccharide is firstly reacted with thiophosgene to obtain an intermediate hapten (hereinafter referred to as hapten 2), the hapten has a thiocyanate structure, and the thiocyanate structure is then subjected to condensation reaction with amino groups of proteins to generate thiourea, so that the core oligosaccharide and the carrier proteins are coupled together.
The preparation method of the acinetobacter baumannii core oligosaccharide-carrier protein conjugate comprises the following steps:
1) Dissolving alpha-Kdo- (2- & gt 4) - [ alpha-Kdo- (2- & gt 5) ] -alpha-Kdo branched trisaccharide serving as a starting material in sodium bicarbonate water solution, adding a chloroform solution mixed with thiophosgene at room temperature, stirring at room temperature overnight to obtain a reaction mixture, diluting the reaction mixture with water, extracting with a pure chloroform solution, obtaining a water layer and a chloroform layer after extraction, removing excessive thiophosgene from the water layer (the chloroform layer), collecting and freeze-drying to obtain a crude extract of an intermediate hapten;
2) Mixing the crude extract of the hapten obtained in the step 1) with carrier protein according to a mass ratio of 2:1, mixing, and reacting in alkaline phosphate buffer overnight to obtain the core oligosaccharide-carrier protein conjugate; the carrier protein comprises tetanus toxoid, diphtheria toxoid, nontoxic variants of diphtheria toxin, haemophilus influenzae protein D, group B meningococcal outer membrane protein complex or qβ phage protein particles.
The application of the acinetobacter baumannii core oligosaccharide-carrier protein conjugate in preparing vaccines for preventing or treating infectious diseases caused by acinetobacter baumannii.
Further, the infectious diseases include one or more of skin or wound infection, urinary tract infection, mechanical ventilation-related pneumonia, septicemia, meningitis and endocarditis.
Further, the vaccine may optionally further comprise alpha-Kdo- (2→4) - [ alpha-Kdo- (2→5) ] -alpha-Kdo branched trisaccharide.
Further, the vaccine may optionally be combined with an adjuvant. Preferred adjuvant types include, but are not limited to, aluminum hydroxide, saponins, or CpG oligodeoxynucleotides, and the like.
Further, the vaccine-induced antibodies can directly bind to whole bacterial antigens of acinetobacter baumannii for inhibiting or killing bacteria.
Further, the immunization program of the vaccine is as follows: following initial vaccination on day 0, booster immunizations were performed on days 14 and 28, respectively.
Further, the vaccination regimen of the vaccine comprises subcutaneous and/or intramuscular injection.
Beneficial technical effects
The application utilizes self-synthesized acinetobacter baumanii core oligosaccharide alpha-Kdo- (2- & gt 4) - [ alpha-Kdo- (2- & gt 5) ] -alpha-Kdo branched trisaccharide as a starting material (oligosaccharide 1) to synthesize an oligosaccharide 1-carrier protein conjugate, and utilizes the oligosaccharide 1-carrier protein conjugate to carry out an immune experiment. The results prove that:
1) Oligosaccharide 1 and oligosaccharide 1-CRM 197 The conjugates are all capable of inducing mouse to produceHigh total IgG antibody concentration and antibody titer specific for Acinetobacter baumannii, in particular oligosaccharide 1-CRM 197 The conjugate group is capable of generating stronger immune response levels and higher antibody concentrations.
2) Spleen cell proliferation results showed oligosaccharide 1 and oligosaccharide 1-CRM 197 Conjugate antigens have similar ability to induce cellular immune proliferation.
3) Oligosaccharide 1 and oligosaccharide 1-CRM 197 Antibodies in conjugate group serum can be combined with whole-cell antigens of Acinetobacter baumannii, and oligosaccharide 1-CRM 197 The capacity of the conjugate group serum antibody to bind to the whole bacterial antigen is significantly higher than that of the oligosaccharide group 1 (more than 10 times of binding capacity).
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the application and that other drawings may be derived from these drawings without inventive faculty.
FIG. 1 is a schematic diagram of the process of coupling a core oligosaccharide to a carrier protein according to the application;
FIG. 2 is a SDS-PAGE of oligosaccharide 1 and CRM 197 A protein coupled product;
FIG. 3 is a graph showing total IgG antibody concentration in mouse serum at various time points after immunization;
FIG. 4 is a graph showing the titers of specific IgG antibodies in mouse serum at various time points after immunization by indirect ELISA;
FIG. 5 is an in vitro proliferation assay of splenocytes from mice analyzed by CCK-8 method;
FIG. 6 is a diagram of an experimental chart for determining serum antibody binding of different dilution factors by using whole-cell antigen ELISA of Acinetobacter baumannii;
FIG. 7 is a diagram of the reaction pathways of starting materials for the synthesis of α -Kdo- (2.fwdarw.4) - [ α -Kdo- (2.fwdarw.5) ] - α -Kdo branched trisaccharide;
FIG. 8 is a reaction scheme for an intermediate product for the synthesis of α -Kdo- (2.fwdarw.4) - [ α -Kdo- (2.fwdarw.5) ] - α -Kdo branched trisaccharide;
FIG. 9 is a diagram of the synthetic α -Kdo- (2.fwdarw.4) - [ α -Kdo- (2.fwdarw.5) ] - α -Kdo branched trisaccharide pathway.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. It will be apparent that the described embodiments are some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.
In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, a rangeThe description of (c) should be taken as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.
The chemical formula of the alpha-Kdo- (2-4) - [ alpha-Kdo- (2-5) ] -alpha-Kdo branched trisaccharide (oligosaccharide 1) is shown as follows:
the chemical formula of the intermediate hapten (hapten 2) is shown as follows:
example 1
Synthesis of hapten 2
Oligosaccharide 1 (5 mg) was dissolved in aqueous sodium bicarbonate (1 mL), a solution of thiophosgene (6.68. Mu.L, 87.2. Mu. Mol) in chloroform (1.5 mL) was added at room temperature, and after the addition was completed, the reaction was stirred at room temperature overnight. The reaction mixture was diluted with water (10 mL), extracted with a pure chloroform solution (4 mL. Times.3), and after extraction, the chloroform layer was removed to remove excess thiophosgene. The aqueous layer was collected and lyophilized to give the crude product which was used directly for the next protein coupling step.
Preparation of conjugate vaccine
Hapten 2 (1.0 mg) was reacted with 0.5mg of CRM 197 Bovine Serum Albumin (BSA) and Bovine Serum Albumin (BSA) are respectively mixed according to the mass ratio of 2:1, reacted overnight in phosphate buffer with the pH of 8.0-9.0, and then ultrafiltered by using a 10kDa ultrafilter tube to remove free unconjugated redundant hapten 2, and the SDS-PAGE of a coupling product is shown in figure 2. Results display CRM 197 The molecular weight increases after the oligosaccharide 1 was coupled, indicating successful coupling. Protein concentration was quantified by BCA method, and then the protein concentration was adjusted to 0.2mg/ml, and oligosaccharide 1-CRM was used 197 The conjugate and oligosaccharide 1 (0.1 mg/ml) are thoroughly mixed with an aluminum adjuvant (SERVA, alu-Gel-S) in a ratio of 1:1, respectively, and the aluminum adjuvant optionally comprises aluminum hydroxide, saponin, cpG oligodeoxynucleotide, etc., but is not limited thereto.
Wherein the oligosaccharide 1-BSA conjugate is used as an antigen for coating in subsequent detection.
Example 2
Immunization of mice
Selecting male BALB/C mice of 6-8 weeks old, dividing the mice into 3 groups of 8-10 mice, group A: PBS, group B: oligosaccharide 1, group C: oligosaccharide 1-CRM 197 A conjugate. All mice were immunized by subcutaneous injection on the back in clean animal rooms, each injected with an emulsion corresponding to the corresponding antigen (10 μg oligosaccharide antigen/mouse), at a dose of 100 μl, and boosted with the same dose on days 14 and 28 after primary immunization, for 5 days.
Serum collection
0.4ml of blood was collected through the orbital veins of the mice on days 0, 7, 14, 21, 28, 35 after the primary immunization. Whole blood was first allowed to stand at room temperature for 4h, centrifuged at 10,000r/min for 15min, and then the supernatant was aspirated to obtain about 0.2ml of serum, which was frozen at-70 ℃ for subsequent antibody titer determination.
Extraction of spleen lymphocytes
The whole process of splenic lymphocyte extraction was performed under aseptic super clean bench environment conditions and on ice to maintain cell viability, the specific steps are as follows:
1) Mice on day 35 after three complete immunizations were sacrificed at cervical scission and sterilized by soaking in 75% ethanol for 5min.
2) In an ultra clean bench, 1mL of RPMI-1640 medium was added to infiltrate the cell sieve, and an appropriate amount of sterile PBS was poured into a glass petri dish. The sterilized mice were fixed on foam plates with their backs facing downward and with needles, after cutting off the abdominal cavity, the spleens (located at the upper right part of the abdominal cavity) were removed and placed in 10cm glass petri dishes and washed with PBS.
3) The washed spleen was placed on a cell sieve, gently grinded with a grinding rod until the spleen was completely dispersed into individual spleen cells, and the spleen cells remaining on the cell sieve and the grinding rod were washed to a bottom centrifuge tube with 15mL of RPMI-1640 medium.
4) The prepared spleen lymphocyte suspension was centrifuged at 1000g at 4℃for 5min, and the supernatant was discarded to collect the spleen cell pellet.
5) And adding erythrocyte lysate into the spleen cell sediment, and slightly sucking and blowing to resuspend the cell sediment so as to avoid generating bubbles. After full resuspension, the mixture was left at room temperature for 4min.
6) After lysis was completed, 10mL of RPMI-1640 medium was rapidly added to terminate lysis, and after mixing, 1000g was centrifuged for 5min.
7) The cell supernatant was discarded, the cell pellet was collected and resuspended in 1mL of RPMI-1640 medium, the remaining spleen lymphocyte tissue mass was picked up, 9mL of the RPMI-1640 medium was added, and the spleen cell pellet was collected by centrifugation under the same conditions as above.
8) And adding 1mL of complete RPMI-1640 (containing 10% FBS and 1% diabody) culture medium into the cell sediment to resuspend the cell sediment, and uniformly dispersing to prepare single lymphocyte suspension which is used for spleen lymphocyte proliferation experiments.
Example 3
Indirect ELISA determination of mouse serum-specific IgG antibody titres
1) Coating: oligosaccharide 1-BSA conjugate (10. Mu.g/ml, 100. Mu.L/well) was coated overnight (12-16 hours) at 4℃and sealed with a sealing membrane to prevent evaporation (coating buffer: 50mmol/L bicarbonate buffer, pH 9.6), carefully placed in a wet box.
2) Washing the plate: the original coating liquid in the plate was discarded, washed 3 times with PBST wash solution, and then the plate was dried by pipetting.
3) Closing: incubating with 200 μl of skim milk blocking solution at 37deg.C for 2h; the ELISA plate should be used immediately after being patted dry, so that complete drying is avoided.
4) Washing the plate: the blocking solution was discarded, washed 3 times with PBST wash solution, and the mixture was patted dry.
5) An anti-binding: after the test strip is dried, firstly adding a diluent, diluting 50 mu L/well, diluting the tested serum in an ELISA plate, using the ELISA plate transversely, adding 50 mu L of the diluted tested serum into the column 1, mixing column 1 with a row gun, adding 50 μl into column 2, mixing, adding 50 μl into column 3, mixing, diluting to column 10, discarding 50 μl liquid, and incubating at 37deg.C for 2 hr.
6) Washing the plate: the sample was discarded, washed 5 times with PBST wash and patted dry.
7) And (2) secondary antibody: 100 μl of oxidase (HRP) -conjugated IgG goat anti-mouse secondary antibody (diluted 1:10000, and enzyme-labeled antibody was added in the last plate wash) and incubated for 2h at room temperature.
8) Washing the plate: the solution in the plate was discarded, and the plate was washed 7 times with PBST and dried by patting.
9) Color development: 100. Mu.L of TMB color development solution was added under a dark condition, and incubated at room temperature for 30min (note dark operation).
10 Terminating: the reaction was quenched with 50. Mu.L of 2mol/L H2SO 4.
11 Reading a plate: absorbance values at 450nm were read to evaluate antibody activity. The reaction was stopped for 30min, absorbance at 450nm was read, and 620nm wavelength was set up as background absorbance subtraction.
12 Calculation of antibody titer: note that PBS control wells should not be hemolyzed, typically 2 times the average of negative wells is set as a threshold, wells above which are judged positive, and the highest dilution of positive wells is the antibody titer of the serum sample.
Experimental results:
antibody titer is an important index for evaluating humoral immunity, and the antibody titer of serum of immunized mice was measured by indirect ELISA, and the results are shown in FIG. 4. Serum antibody titers showed an increasing trend as a whole after mice were immunized. However, the levels of antibody titers in mice were low within 14 days after primary immunization. On day 28 after priming, the experimental group was significantly higher than the PBS group, indicating that each group of antigens elicited an effective humoral immune response in mice. After three complete immunizations, the mice had significantly increased antibody titers in vivo on day 35. Oligosaccharide 1, unconjugated protein, was also able to induce specific antibodies in mice compared to PBS. But oligosaccharide 1 and CRM 197 After conjugation, the immune response in mice induced by it was stronger, yielding higher antibody titers. Indicating that oligosaccharide 1 is purified by CRM 197 Protein coupleIn combination, helps to enhance its ability to induce an immune response in vivo.
Determination of the concentration of mouse serum Total IgG antibodies
The method for determining the concentration of the total IgG antibodies in the serum of the mice is determined according to the manual method of the total IgG antibody kit of the blood of the mice. Through PBS group, oligosaccharide 1 group and oligosaccharide 1-CRM 197 The total IgG concentration in the samples was determined using a kit standard mouse IgG standard concentration curve from the serum of mice 0-7 weeks after immunization of each conjugate.
Experimental results:
the total IgG antibody concentration was an important index for evaluating the ability of the vaccine to elicit an immune response, and the total IgG antibody concentration in serum of immunized mice was measured by a commercial kit, and the results are shown in fig. 3. Serum total IgG antibody concentration showed a trend of continuously increasing as a whole after mice were immunized. Laboratory mice serum total IgG antibody concentrations were all significantly elevated compared to PBS group. Oligosaccharide 1-CRM 197 The conjugate group was more able to induce higher levels of IgG antibody concentration in mice than the oligosaccharide 1 group, indicating oligosaccharide 1 and CRM 197 After protein coupling, its immunogenicity in mice was significantly enhanced.
Example 4
Spleen cell proliferation assay
In the experiment, the CCK-8 method is adopted to detect the proliferation capacity of spleen lymphocytes under the action of in vitro antigen stimulus, and the specific operation is as follows: cell concentration of lymphocyte suspension by trypan blue counting method and adjusting cell concentration to 5×l0 with RPMI-1640 complete medium 6 cell/mL, then added to a 96-well plate, the number of cells added per well being 5X 10 5 And each. In addition, PBS, oligosaccharide 1-CRM is added into each hole 197 The conjugate (the concentration of which is adjusted to 10 mug/mL by using a complete culture medium and acid according to the mass of polysaccharide) is blown and evenly mixed, so that air bubbles are avoided. The negative control group only contains cells, and no stimulus is added; the blank control group is only added with culture medium; each sample was provided with 3 duplicate wells, the total volume of each well being 200 μl. 96-well cell culture plates were placed at 37℃with 5% CO 2 Is cultured for 72 hours in the incubator of (2). mu.L of CCK-8 solution was added to each well and gently blown with a gunMixing was continued for 4h in an incubator, and then absorbance at 450nm was measured. The proliferation intensity of splenic lymphocytes was expressed as absorbance at 450 nm.
Experimental results:
first, proliferation of mouse spleen cell population was measured by CCK-8 method, and as shown in FIG. 5, in vitro was performed by oligosaccharide 1, oligosaccharide 1-CRM 197 After conjugate stimulation, spleen cell proliferation levels were significantly higher in both groups of mice than in PBS, oligosaccharide 1 group and oligosaccharide 1-CRM 197 The differences between the conjugate groups were not significant, indicating oligosaccharide 1 and oligosaccharide 1-CRM 197 The conjugates can effectively stimulate the organism to generate similar cellular immune response.
Example 5
Experiment of mouse serum antibody combined with Bowman
Acinetobacter baumannii (Hua Xi clinical laboratory) is cultured on LB medium and 1% agarose solid medium, colonies are picked up, resuspended in LB medium, and the concentration of thallus is regulated to 1×10 8 Mu.l of Acinetobacter baumannii suspension was inoculated into each well at a volume of 100. Mu.l/ml using a 96-well plate, the supernatant medium was discarded after standing overnight at 37℃and 100. Mu.l of 5% glutaraldehyde solution (0.1 mol/L NaHCO) was added to each well 3 95ml of 25% glutaraldehyde solution 5 ml) was fixed, allowed to act at 37℃for 2 hours, and washed 3 times with distilled water; then 200 μl of blocking solution was added to each well at 4deg.C overnight or 37deg.C, and after blocking for 2 hours, the wells were washed three times. After 50 μl of seventh week mouse serum diluted with different multiples was added, three wells were multiplexed per sample, and after two hours of binding at 37 ℃, PBST was washed three times, and then after binding of secondary antibody (HRP-labeled goat anti-mouse antibody), washing, color development and termination, absorbance values were read by a microplate reader.
Experimental results:
PBS group, oligosaccharide 1-CRM 197 The results of ELISA detection experiments of whole-cell antigen coating of the conjugate group and Acinetobacter baumannii after dilution by different multiples are shown in FIG. 6, compared with PBS control group, oligosaccharide 1 group and oligosaccharide-CRM 197 The conjugate group mouse serum can be combined with the Bowman whole-cell antigen, indicating that the oligosaccharide 1 and the oligosaccharide 1-CRM 197 The conjugate antigen induced antibodies can be directly combined with BowmanThe bacteria can directly inhibit and kill bacteria. The results also show that at 10-fold, 100-fold and 1000-fold dilutions, at the same dilution, the oligosaccharides 1-CRM 197 The OD value of the conjugate group was significantly higher than that of the oligosaccharide 1 antigen alone, indicating that oligosaccharide 1-CRM 197 The conjugate group serum can be combined with Bowman antigen with higher dilution, which indicates that oligosaccharide 1-CRM 197 Higher levels of specific antibodies capable of binding directly to Bowman were produced in the conjugate group serum.
Conclusion of the experiment of the application:
1) Oligosaccharide 1 and oligosaccharide 1-CRM 197 The conjugates are capable of inducing mice to produce high levels of total IgG antibody concentration and antibody titer specific for Acinetobacter baumannii, in particular oligosaccharide 1-CRM 197 The conjugate group is capable of generating stronger immune response levels and higher antibody concentrations.
2) Spleen cell proliferation results showed oligosaccharide 1 and oligosaccharide 1-CRM 197 Conjugate antigens have similar ability to induce cellular immune proliferation.
3) Oligosaccharide 1 and oligosaccharide 1-CRM 197 Antibodies in conjugate group serum can be combined with whole-cell antigens of Acinetobacter baumannii, and oligosaccharide 1-CRM 197 The capacity of the conjugate group serum antibody to bind to the whole bacterial antigen is significantly higher than that of the oligosaccharide group 1 (more than 10 times of binding capacity).
Example 6
Synthesis example of alpha-Kdo- (2.fwdarw.4) - [ alpha-Kdo- (2.fwdarw.5) ] -alpha-Kdo branched trisaccharide (Compound 15 in this example)
The compound S-1 is subjected to TBS protecting group removal under the action of TBAF, and then the C5 benzoyl group is removed under the action of sodium methoxide, so that the compound S-2 is obtained in two steps of 85% yield. Compound S-2 was allyl over C-4OH selectivity to give receptor 2 in 87% yield.
Wherein, the chemical formulas of S-1 and S-2 are as follows:
donor 1 and C5-OH acceptor 2 under the catalysis of TBPAGlycosylation was performed to give 2, 5-linked Kdo disaccharide compound 3 in 80% yield. In one aspect, disaccharide compound 3 is at Et 3 After removal of 8'-O-TBS and 5',7'-O-DTBS protecting groups by N-3HF, 7',8'-OH was protected with isopropylidene acetal to give 5' -OH disaccharide receptor 4 in 73% yield in two steps. On the other hand, after 4-O-All was removed by the action of activated iridium complex, mercury oxide and mercury chloride, disaccharide compound 3 gave 4-OH disaccharide acceptor 5 (yield 75%) and disaccharide by-product 6 (yield 13%)
Wherein, the chemical formulas of the donor 1, the acceptor 2, the compound 3 and the acceptor 5 are respectively shown as follows:
glycosylation of 4-OH disaccharide acceptor 5 with donor 12 gave Kdo branched trisaccharide 13 in 70% yield. Kdo branched trisaccharide 13 removes 8' -O-TBS and 5',7' -O-DTBS protecting group to give compound 14 (yield 87%). Compound 14 gave the target branched trisaccharide 15 in 58% yield over three steps by removing the isopropylidene group under acidic conditions, removing all the ester protecting groups by saponification, and converting the azide group to amino group while removing the benzyl protecting group by catalytic hydrogenation reduction.
Wherein the chemical formulas of the donor 12 and the compound 14 are as follows:
the embodiments of the present application have been described above with reference to the accompanying drawings, but the present application is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present application and the scope of the claims, which are to be protected by the present application.
Claims (9)
1. A acinetobacter baumannii core oligosaccharide-carrier protein conjugate, wherein the core oligosaccharide of the acinetobacter baumannii core oligosaccharide-carrier protein conjugate is alpha-Kdo- (2-4) - [ alpha-Kdo- (2-5) ] -alpha-Kdo branched trisaccharide, and the carrier protein comprises tetanus toxoid, diphtheria toxoid, a nontoxic variant of diphtheria toxin, haemophilus influenzae D protein, group B meningococcal outer membrane protein complex, or qβ phage protein particles; the alpha-Kdo- (2-4) - [ alpha-Kdo- (2-5) ] -alpha-Kdo branched trisaccharide is reacted with thiophosgene to obtain an intermediate product hapten, the hapten has a thiocyanate structure, and the thiocyanate structure is subjected to condensation reaction with amino groups of proteins to generate thiourea, so that the core oligosaccharide and the carrier proteins are coupled together; the intermediate hapten has the following structural formula:
2. the method for preparing the acinetobacter baumannii core oligosaccharide-carrier protein conjugate according to claim 1, comprising the following steps:
1) Dissolving alpha-Kdo- (2- & gt 4) - [ alpha-Kdo- (2- & gt 5) ] -alpha-Kdo branched trisaccharide serving as a starting material in sodium bicarbonate water solution, adding chloroform solution mixed with thiophosgene at room temperature, stirring at room temperature overnight to obtain a reaction mixture, diluting the reaction mixture with water, extracting with pure chloroform solution, obtaining a water layer and a chloroform layer after extraction, removing excessive thiophosgene from the water layer, collecting and freeze-drying to obtain a crude extract of an intermediate hapten;
2) Mixing the crude extract of the hapten obtained in the step 1) with carrier protein according to a mass ratio of 2:1, mixing, and reacting in alkaline phosphate buffer overnight to obtain the core oligosaccharide-carrier protein conjugate; the carrier protein comprises tetanus toxoid, diphtheria toxoid, nontoxic variants of diphtheria toxin, haemophilus influenzae protein D, group B meningococcal outer membrane protein complex or qβ phage protein particles.
3. Use of the acinetobacter baumannii core oligosaccharide-carrier protein conjugate according to claim 1 in the preparation of a vaccine for preventing or treating infectious diseases caused by acinetobacter baumannii.
4. The use according to claim 3, wherein the infectious disease comprises one or more of skin or wound infection, urinary tract infection, mechanical ventilation-related pneumonia, sepsis, meningitis and endocarditis.
5. The use according to claim 3, wherein the vaccine comprises α -Kdo- (2→4) - [ α -Kdo- (2→5) ] - α -Kdo branched trisaccharide.
6. The use according to claim 3, wherein the vaccine is optionally combined with an aluminium adjuvant.
7. The use according to claim 3, wherein the vaccine-induced antibodies bind directly to the whole bacterial antigen of acinetobacter baumannii for inhibiting or killing bacteria.
8. The use according to claim 3, wherein the immunization program of the vaccine is: following initial vaccination on day 0, booster immunizations were performed on days 14 and 28, respectively.
9. The use according to claim 8, wherein the vaccination regimen of the vaccine comprises subcutaneous and/or intramuscular injection.
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