CN115068602A - Surface-modified aluminum oxide composite vaccine adjuvant, preparation method and application - Google Patents
Surface-modified aluminum oxide composite vaccine adjuvant, preparation method and application Download PDFInfo
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Abstract
The application relates to the technical field of alumina vaccine adjuvants, in particular to a composite vaccine adjuvant of surface modified alumina, a preparation method and application. The preparation method comprises the following steps: and reacting alumina with polysaccharide in an alcohol solution containing at least one of benzyltriethylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium hydrogen sulfate, trioctylmethylammonium chloride, dodecyltrimethylammonium chloride and tetradecyltrimethylammonium chloride to obtain the surface-modified alumina composite vaccine adjuvant. The composite vaccine adjuvant can mediate vaccine adjuvant for obtaining immune response of Th1 and/or Th2, so as to be suitable for vaccination for preventing diseases such as intracellular infection, virus infection or cancer.
Description
Technical Field
The application relates to the technical field of alumina vaccine adjuvants, in particular to a composite vaccine adjuvant of surface modified alumina, a preparation method and application.
Background
Vaccine adjuvants are substances that, when administered in combination with a vaccine, can enhance the immune response of the body to the vaccine or alter the type of immunity. Thanks to the development of biotechnology, novel vaccines such as subunit vaccines, DNA vaccines, mRNA vaccines, etc. have been developed, which have low risk, strong specificity, and can provide lasting cellular and humoral immunity, compared to conventional vaccines. However, it has been found that these vaccines are easily immune-tolerant, and this requires the assistance of vaccine adjuvants to improve their immunogenicity.
Aluminum adjuvants are widely approved by the FDA for use in humans because of their safety and effectiveness. However, aluminium adjuvants generally induce a typical antibody-mediated (Th2) response, rather than a cell-mediated (Th1) immunity, and are therefore unsuitable for vaccination against diseases such as intracellular infections, viral infections or cancer.
Disclosure of Invention
In view of the above, the present application aims to obtain a vaccine adjuvant capable of mediating an immune response against Th1 and/or Th2, which is suitable for vaccination against diseases such as intracellular infection, viral infection, or cancer.
In a first aspect, the embodiment of the application discloses a preparation method of a surface-modified aluminum oxide composite vaccine adjuvant, which comprises the following steps: and reacting alumina with polysaccharide in an alcohol solution containing at least one of benzyltriethylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium hydrogen sulfate, trioctylmethylammonium chloride, dodecyltrimethylammonium chloride and tetradecyltrimethylammonium chloride to obtain the surface-modified alumina composite vaccine adjuvant.
In the examples herein, the sugar is selected from fructose, xylose, sucrose, trehalose or lactose.
In the embodiment of the application, the preparation method comprises the steps of dissolving fructose or sucrose in 1, 2-propylene glycol, adding nano aluminum oxide, benzyltriethylammonium chloride and anhydrous potassium carbonate, and reacting at 90-95 ℃ for 5 hours;
adding distilled water, and stirring at 90-95 ℃ for 1-2 hours;
and (2) distilling at 110-120 ℃ under reduced pressure to remove the solvent, filtering, taking a solid, directly washing the product by using a 30% NaCL solution, standing overnight, washing for 3 times by using a 5% NaCL solution, centrifugally separating at 4000r/min, washing for a plurality of times by using absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified alumina composite vaccine adjuvant.
In the embodiment of the application, the preparation method comprises the steps of dissolving lactose in n-butyl alcohol, adding nano aluminum oxide, benzyltriethylammonium chloride and anhydrous potassium carbonate, and reacting at 90-95 ℃ for 5 hours;
adding distilled water, and stirring at 90-95 ℃ for 1-2 hours;
and (2) distilling at 110-120 ℃ under reduced pressure to remove the solvent, filtering, taking a solid, directly washing the product by using a 30% NaCL solution, standing overnight, washing for 3 times by using a 5% NaCL solution, centrifugally separating at 4000r/min, washing for a plurality of times by using absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified alumina composite vaccine adjuvant.
In the embodiment of the application, the preparation method comprises the steps of dissolving trehalose in n-butanol, adding nano aluminum oxide, benzyltriethylammonium chloride and anhydrous potassium carbonate, and reacting at 90-95 ℃ for 5 hours;
adding distilled water, and stirring at 90-95 ℃ for 1-2 hours;
and (2) distilling at 110-120 ℃ under reduced pressure to remove the solvent, filtering, taking a solid, directly washing the product by using a 30% NaCL solution, standing overnight, washing for 3 times by using a 5% NaCL solution, centrifugally separating at 4000r/min, washing for a plurality of times by using absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified alumina composite vaccine adjuvant.
In the embodiment of the application, the preparation method comprises the steps of dissolving trehalose in n-butanol, adding nano alumina, tetrabutylammonium hydrogen sulfate, trioctylmethylammonium chloride and anhydrous potassium carbonate, and reacting at 90-95 ℃ for 5 hours;
adding distilled water, and stirring at 90-95 ℃ for 1-2 hours;
and (2) distilling at 110-120 ℃ under reduced pressure to remove the solvent, filtering, taking a solid, directly washing the product by using a 30% NaCL solution, standing overnight, washing for 3 times by using a 5% NaCL solution, centrifugally separating at 4000r/min, washing for a plurality of times by using absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified alumina composite vaccine adjuvant.
In a second aspect, the present application discloses a composite vaccine adjuvant prepared by the preparation method of the first aspect, which has an alumina substrate and a sugar group attached to the surface of the alumina substrate, wherein the sugar group is selected from fructosyl, xylosyl, sucrose, trehalose, or lactose.
In the embodiment of the application, the composite vaccine adjuvant is in a nanometer shape and has a length of 816cm -1 、748cm -1 、555cm -1 At 3452cm -1 、1630cm -1 、3387cm -1 And 1735cm -1 Characteristic absorption peak of infrared.
In a third aspect, the present application discloses a vaccine comprising the composite vaccine adjuvant of the second aspect.
In a fourth aspect, the examples of the present application disclose the use of the composite vaccine adjuvant prepared by the preparation method of the first aspect, or the composite vaccine adjuvant of the second aspect in the preparation of an antiviral vaccine.
Compared with the prior art, the application has at least the following beneficial effects:
the application discloses a preparation method of a surface modified alumina composite vaccine adjuvant, which comprises the step of reacting alumina with polysaccharide to prepare the surface modified alumina composite vaccine adjuvant. The surface modified alumina is coupled with glycosyl to obtain a novel nano particle. The nano-particles not only have excellent dispersibility, low hydromechanical editing and low Zeta potential (mV)V Sedimentation It also has low endotoxin content and has long-term structural stability.
In vitro experiments prove that the novel surface modified aluminum oxide has a stimulation effect on mouse spleen T cells and an immune activation effect on mouse DC2.4 cells. Further, in vivo experiments prove that the novel surface modified aluminum oxide can be used as a composite vaccine adjuvant, can promote the immunogenicity of novel coronavirus RBD, stimulate an organism to generate a higher titer antibody level, and promote a mouse organism to generate Thl humoral immune response so as to be matched with single Th2 humoral immune response, thereby enriching the immune protection effect of the vaccine.
Drawings
FIG. 1 is an infrared spectrum of the composite vaccine adjuvants provided in examples 1-5 and comparative examples 1-2 of the present application.
Fig. 2 is a graph of staining of tissue sections HE of mice liver (example 4 (panel a), example 5 (panel B), comparative example 1 (panel C), comparative example 2 (panel D)), spleen (example 4 (panel E), example 5 (panel F), comparative example 1 (panel G), comparative example 2 (panel H), and kidney (example 4 (panel I), example 5 (panel J), comparative example 1 (panel K), comparative example 2 (panel L)) after RBD vaccination of mice formulated with a composite vaccine adjuvant as provided herein.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
Preparation of surface modified alumina composite vaccine adjuvant
1. Preparation method
The embodiment of the application discloses a preparation method of a surface modified aluminum oxide composite vaccine adjuvant, which comprises the step of reacting aluminum oxide with polysaccharide in an alcohol solution containing at least one of benzyltriethylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium hydrogen sulfate, trioctylmethylammonium chloride, dodecyltrimethylammonium chloride and tetradecyltrimethylammonium chloride. In some embodiments, the sugar is selected from fructose, xylose, sucrose, trehalose, and lactose.
In one example 1, 14.4g fructose was dissolved in 100mL1, 2-propanediol, 8.16g nano alumina (R003866, 99.99% crystal form. gamma., 20nm, Rone reagent) was added, 0.228g benzyltriethylammonium chloride (CAS: 56-37-1, purity 99%, St. Tokyo chemical technology Co., Ltd.) and 1.6g anhydrous K were added 2 CO 3 Reacting for 5 hours in a 250mL four-neck flask under stirring at the temperature of 90-95 ℃; 10mL of the solution was addedHeating and stirring distilled water at 90-95 ℃ for 1-2 h; and (2) distilling under reduced pressure at 110-120 ℃ to remove the solvent, filtering, taking a solid, directly washing the product by using a 30% NaCL solution, standing overnight, washing for 3 times by using a 5% NaCL solution, centrifugally separating at 4000r/min, washing for a plurality of times by using absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified aluminum oxide composite vaccine adjuvant.
In one example 2, 28.8g sucrose was dissolved in 100mL1, 2-propanediol, 8.16g nano alumina (R003866, 99.99% form γ, 20nm, Ron reagent) was added, 0.228g benzyltriethylammonium chloride (CAS: 56-37-1, purity 99%, St. Tokyo & Chemicals, Inc.) and 1.6g anhydrous K 2 CO 3 Reacting for 5 hours in a 250mL four-neck flask under stirring at the temperature of 90-95 ℃; then adding 10mL of distilled water, heating and stirring at 90-95 ℃ for 1-2 h; and (2) distilling under reduced pressure at 110-120 ℃ to remove the solvent, filtering, taking a solid, directly washing the product by using a 30% NaCL solution, standing overnight, washing for 3 times by using a 5% NaCL solution, centrifugally separating at 4000r/min, washing for a plurality of times by using absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified aluminum oxide composite vaccine adjuvant.
In one example 3, 27.4g lactose was dissolved in 100mL n-butanol, 8.16g nano alumina (R003866, 99.99% form. gamma., 20nm, Ron reagent) was added to 0.228g benzyltriethylammonium chloride (CAS: 56-37-1, 99% purity, Nissan Dry Toyol chemical technology Co., Ltd.) and 1.6g anhydrous K 2 CO 3 Reacting for 5 hours in a 250mL four-neck flask under stirring at the temperature of 90-95 ℃; then adding 10mL of distilled water, heating and stirring at 90-95 ℃ for 1-2 h; and (2) distilling under reduced pressure at 110-120 ℃ to remove the solvent, directly washing the product with 30% NaCL solution, standing overnight, washing for 3 times with 5% NaCL solution, centrifugally separating at 4000r/min, washing for a plurality of times with absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified aluminum oxide composite vaccine adjuvant.
In one example 4, 27.4g trehalose was dissolved in 100mL n-butanol, 8.16g nano alumina (R003866, 99.99% form. gamma., 20nm, Ron reagent) was added, 0.228g benzyl triethyl chloride was addedAmmonium chloride (CAS: 56-37-1, purity 99%, Wuxi dry-mass chemical technology Co., Ltd.) and 1.6g of anhydrous K 2 CO 3 Reacting for 5 hours in a 250mL four-neck flask under stirring at the temperature of 90-95 ℃; then adding 10mL of distilled water, heating and stirring at 90-95 ℃ for 1-2 h; and (2) distilling under reduced pressure at 110-120 ℃ to remove the solvent, directly washing the product with 30% NaCL solution, standing overnight, washing for 3 times with 5% NaCL solution, centrifugally separating at 4000r/min, washing for a plurality of times with absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified aluminum oxide composite vaccine adjuvant.
In one example 5, 27.4g trehalose was dissolved in 100mL n-butanol, 8.16g nano alumina (R003866, 99.99% form. gamma., 20nm, Rone reagent) was added, 0.114g tetrabutylammonium hydrogen sulfate (CAS: 56-37-1, purity 99%, Wuxi Dry Toyobo chemical Co., Ltd.), 0.114g trioctylmethylammonium chloride (CAS: 5137-55-3 purity: 99% HPLC, Hubei Ushu Shun Biotech Co., Ltd.) and 1.6g anhydrous K 2 CO 3 Reacting for 5 hours in a 250mL four-neck flask at the temperature of 90-95 ℃ under stirring; then adding 10mL of distilled water, heating and stirring at 90-95 ℃ for 1-2 h; and (2) distilling under reduced pressure at 110-120 ℃ to remove the solvent, directly washing the product with 30% NaCL solution, standing overnight, washing for 3 times with 5% NaCL solution, centrifugally separating at 4000r/min, washing for a plurality of times with absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified aluminum oxide composite vaccine adjuvant.
In a comparative example 1, 14.75g of methyl stearate is dissolved in 100mL of chloroform solution and put into a three-neck flask, 20g of nano-alumina (R003866, 99.99% crystal form gamma, 20nm, Rone reagent) is added, mechanical stirring is carried out for 2h at room temperature, suction filtration is carried out, absolute ethyl alcohol is used for washing for a plurality of times, drying is carried out in a drying oven at 120 ℃, and grinding is carried out, thus obtaining the surface modified alumina composite vaccine adjuvant.
In a comparative example 2, nano-alumina was used as a vaccine adjuvant.
2. Performance analysis of composite vaccine adjuvants
And (3) diluting the adjuvants respectively prepared in the examples 1-5 and the comparative examples 1-2 to 50 microgram/mL by using ultrapure water, and analyzing the appearance of the composite vaccine adjuvant by using a transmission electron microscope.
The adjuvant nanoparticles prepared in examples 1 to 5 and comparative examples 1 to 2 were diluted to 500 ng/mL with deionized water, and subjected to ultrasonic treatment to uniformly disperse the material, and further subjected to respective tests on the hydraulic radius and Zeta potential of the nanoparticles with a nanoparticle sizer (Nicomp Z3000 nanoparticle size and Zeta potential analyzer, us PSS).
Drying and fully grinding the nano adjuvants respectively prepared in the examples 1-5 and the comparative examples 1-2 to be tested, mixing the nano adjuvants with potassium bromide powder according to the ratio of 20:1, firstly scanning the miscellaneous peak of the air by using a Fourier transform infrared spectrometer (Henchld, model LIDA-20), then scanning and analyzing the nano vaccine adjuvants respectively prepared in the examples 1-5 and the comparative examples 1-2 and deducting the miscellaneous peak to obtain a sample surface functional group peak diagram, wherein the measured wavelength range is 4000-40cm -l 。
Weighing 1.000g of the nano composite vaccine adjuvants respectively prepared in the examples 1-5 and the comparative examples 1-2 in a measuring cylinder filled with 10mL of liquid paraffin, carrying out ultrasonic treatment for 30 min, fully shaking up, standing for 12h until the volume does not change, reading the stabilized volume, and calculating the sedimentation volume according to the following formula:V sedimentation =V 1 /mIn the formulaV 1 Volume of sediment, mL;mis the nano adjuvant mass, g.
To further verify the stability of the nanocomposite vaccine formulations provided in examples 1 to 5 and comparative examples 1 to 2 of the present application, the nanocomposite vaccine formulations provided in examples 1 to 5 and comparative examples 1 to 2 were prepared in 0.67% physiological saline at 4 ℃, stored for 4h, 72h, 96 h, and 2 months in sequence, the supernatant of the composite adjuvant was collected by centrifugation at corresponding times, the above kits were used to sequentially test the contents of the dissociated sugars and organic acid esters in the supernatant, the sugar content was determined by a high performance ion chromatography-pulsed amperometric detection method (refer to "journal of free sugar content in commercially available beverages in China," china food sanitation journal, 2021 st year 1 "), the organic acid ester content was determined by a reverse phase high performance liquid chromatography (RP-HPLC) (refer to" high performance liquid chromatography for determination of methyl linoleate content [ J ] in china brewing, year 2014, stage 11 ").
In order to meet the research of the vaccine on endotoxin, the method for detecting the endotoxin content in the vaccine adjuvants respectively provided in examples 1-5 and comparative examples 1-2 by using a limulus reagent further comprises the following steps: (1) dissolving a bottle of endotoxin working standard (10 EU/bottle) with 1mL of inspection water, performing ultrasonic treatment for 15 min, performing vortex oscillation for sufficient and uniform, and performing gradient dilution on the standard with the inspection water to obtain endotoxin standard solutions with the concentration of 0.5, 0.25, 0.125 and 0 EU/mL in sequence; (2) taking the dried composite adjuvant, and diluting the dried composite adjuvant to 250 mug/mL by using inspection water; (3) adding water for endotoxin examination, endotoxin standard solution and adjuvant to be detected into a pyrogen-free 96-well plate, adding 25 SHAN of reagent solution into the 96-well plate with a discharging gun, slightly moving and uniformly mixing, and incubating at 37 deg.C for 14 min; (4) taking out the 96-well plate, adding 50 muL of chromogenic matrix solution by using a row gun, uniformly mixing, and then continuously incubating at 37 ℃ for 6 min; (5) after the incubation is finished, adding 25 muL of reaction terminator by using a row gun, slightly and uniformly mixing, and reading a light absorption value at a wavelength of 405nm by using an enzyme labeling instrument; (6) and (5) drawing a standard curve equation according to the standard solution to obtain the bacterial endotoxin level of the composite adjuvant.
3. Results Table 1
As shown in Table 1, the hydraulic radius (nm), Zeta potential (mV), and the values of Zeta potential (mV) of the surface-modified aluminum oxide composite vaccine adjuvants obtained in examples 1 to 5 and comparative examples 1 to 2, respectivelyV Sedimentation (mL/g). Multiple comparisons and significant difference labeling were also performed on each column of data in table 1. As can be seen from table 1, the hydraulic radius of the composite vaccine adjuvant of surface-modified alumina provided in examples 1 to 5 is slightly higher than that of comparative example 2, but the Zeta potential is significantly lower than that of comparative example 2, and the sedimentation volume is significantly lower than that of comparative example 2. Thus, the nano composite vaccine adjuvant obtained by modifying the alumina in the manner provided in examples 1 to 5 has a slightly increased hydraulic radius and a significantly decreased sedimentation volume, and the hydraulic radius does not cause sedimentation, so that the dispersibility of the nano composite vaccine adjuvant in the system can be maintainedIt is advantageous for its general application properties as a vaccine adjuvant, which may be related to its surface modified sugar group. In addition, Table 1 also shows that the endotoxin content in the vaccine adjuvants provided in examples 1-5 and comparative examples 1-2 is less than 0.5EU/mL, which meets the relevant standards as a vaccine adjuvant.
In order to verify that the modification processes of alumina are respectively completed in examples 1 to 5 and comparative example 1, infrared spectrogram analysis is also performed in the application, and the result is shown in fig. 1. In FIG. 1, the infrared curve of the nano-alumina provided in comparative example 2 is at 816cm -1 、748cm -1 、555cm -1 A characteristic absorption peak appears at and is 3452cm -1 And 1630cm -1 The absorption peaks correspond to the stretching vibration absorption peak of O-H and the bending vibration absorption peak of O-H on the surface of the nano alumina respectively; comparative example 1 provides a modified alumina IR curve at 2924cm relative to comparative example 2 -1 And 2854 cm -1 2 absorption peaks appear corresponding to the stretching vibration absorption peaks of methyl and methylene respectively and at 1555cm -1 And 1464 cm -1 Has obvious absorption peaks corresponding to COO - The asymmetric and symmetric stretching vibration absorption peaks of (a) indicate that the organic acid is successfully connected to the surface of the alumina; the adjuvant of the composite vaccine provided in examples 1-5 is at 3387cm -1 And 1735cm -1 And a glycosyl-OH peak and a C = O peak are correspondingly generated respectively, which indicates that glycosyl is successfully connected on the surface of the alumina.
For this reason, the present application further investigated the stability of the composite vaccine adjuvant in which organic acid and glycosyl group are successfully attached to the surface of nano-alumina, and the result is shown in table 2, wherein in table 2, "-" indicates that the detection item is absent or not detected. As can be seen from table 2, the compound vaccine adjuvants provided in examples 1 to 5 have trace amount of free sugar precipitated after 2 months, which indicates that the compound vaccine adjuvants provided in the present application have high stability.
TABLE 2
In vitro assay
1. Stimulation of mouse T cells
The vaccine adjuvants provided in examples 1-5 and comparative examples 1-2 of the present application are used to treat T cells of mice respectively, and the stimulation effect of the vaccine adjuvants on the T cells and the T cells is detected by a flow cytometer. The method comprises the following specific steps:
the PBS solutions of the vaccine adjuvants provided in examples 1-5 and comparative examples 1-2 were prepared accurately at a concentration of 5mg/mL (test solution).
Taking 20mg of BALB/c mouse (Unico organism) spleen tissue under aseptic condition, adding 5mLRPMI 1640 basic culture medium into a 200-mesh cell sieve, grinding, placing into a grinding fluid in a 15mL centrifuge tube, centrifuging at 1500rpm for 5min, discarding supernatant, adding 2mL of 1 Xerythrocyte lysate into the centrifuge tube, blowing uniformly, and standing for 5 min; adding 8mL of PBS to terminate the reaction, centrifuging at 1500rpm for 10min, and removing the supernatant; adding 5mL of RPMI 1640 basic culture medium, and uniformly blowing; centrifuging at 1500rpm for 5min, and discarding the supernatant; adding 1mL of RPMI 1640 complete culture medium, and uniformly blowing; adding 990 muL serum-free RPMI 1640 into a 1.5mL EP tube, adding 10 muL cell suspension, uniformly mixing, counting (cells are diluted by 100 times), supplementing the RPMI 1640, completely culturing, and enabling the final number of the cells to be 1 x 10 in a centrifugal tube 7 one/mL.
The MTS method is used for detecting the stimulation effect of the vaccine adjuvant on mouse spleen T cells:
adding BALB/c mouse spleen cell suspension into a 96-well plate at 100 muL/well, and dividing the suspension into a stimulation group, a positive control group, a negative control group and a blank group. Adding 100 mu L of test article into each small hole of the stimulation group; adding 100 muL of RPMI 1640 complete culture solution to each hole of the negative control group; 200 μ L of RPMI 1640 complete medium without spleen cell suspension was added to each well of the blank group of wells, and the plates were placed at 37 ℃ in 5% CO 2 Culturing for 68h in a cell culture box, adding 40 muL/well MTS solution (Shangbao organism), culturing for 4h in the culture box, measuring OD490 absorbance, thereby evaluating the cell proliferation level, and calculating a stimulation index SI = (stimulation OD 490-blank OD 490)/(negative control OD 490-blank OD 490). And ELISA kit (mouse gamma interferon (IFN-gamma) ELISA kit, product code: E-EL-M0048 c; Small)A mouse interleukin 10(IL-10) enzyme-linked immunosorbent assay kit, the product code of which is E-EL-M0046c, Wuhanyilai organisms) detects the INF-gamma and IL-10 contents in the culture solution of each group of cells cultured for 68 h.
In table 3, multiple comparisons and significant difference labeling were performed for each column of data. As shown in Table 3, the vaccine adjuvants provided in examples 1-5 have stronger stimulation effect on mouse spleen T cells than comparative examples 1-2, and have higher contents of cytokines INF-gamma and IL-10 for stimulating the secretion of T cells than comparative examples 1-2, and have stronger immunogenicity (and the best results of example 5).
TABLE 3
2. Effect on DC2.4 cells
The vaccine adjuvants provided in examples 1-5 and comparative examples 1-2 of the present application were used to treat mouse DC2.4 cells, and the stimulation effect of the vaccine adjuvants on the two was detected by a flow cytometer. The method comprises the following specific steps:
PBS solutions of the vaccine adjuvants provided in examples 1 to 5 and comparative examples 1 to 2 were prepared accurately at a concentration of 5mg/mL (test solution), and 1 mg/mL LPS was dissolved in sterile double distilled water as a control.
Serum-free DMEM and RPMI 1640 culture media are respectively diluted to prepare LPS serum-free DMEM culture media containing 6 mug/mL, and PBS solutions of the vaccine adjuvants provided in the above examples 1-5 and comparative examples 1-2 are respectively prepared to test solution containing 20 mug/mL by using the serum-free DMEM culture media. At 5% CO 2 Culturing DC2.4 (cambium) in a 37 ℃ cell culture box until logarithmic phase, adjusting the cell number to 10 by using a culture medium 6 and/mL, preparing DC2.4 cell suspension, respectively paving in a 96-well plate, culturing each well by 100 muL, changing a novel culture solution to an RPMI 1640 culture medium containing 10% fetal calf serum after culturing again to a logarithmic phase, adding 20 muL of a sample solution as a test group, adding 20 muL of 6 mug/mL LPS as a positive control group, and adding 20 muL of a DMEM culture medium containing 10% fetal calf serum as a negative control group. Each group of cells was placed in a medium containing 5% CO 2 37 ℃ cell culture boxCulturing for 48H, mixing with cell suspension by adding 20 μ L of flow-type antibodies PE anti-mouse H-2Kb (Biolegend, MHC-I), FITC anti-mouse I-A/I-E (BioLegend, MHC-II), PE anti-mouse CD80 (Emmetie technologies Co., Ltd.) and PE anti-mouse CD86 (Emmetie technologies Co., Ltd.), shading and staining for 30 min at 4 ℃, washing for 1 time with PBS, discarding supernatant, adding 1mL PBS to resuspend cells, collecting in a 1.5mL centrifuge tube, gently blowing with a pipette, and detecting with a flow cytometer according to the sequence of 4 mass concentration gradients of each antibody negative control group, positive control group and test group.
TABLE 4
Table 4 shows the expression levels (percentages) of MHC-I, MHC-II, CD80 and CD86 on the cell surface after treating DC2.4 cells and RAW264.7 cells with the composite vaccine adjuvants provided in examples 1-5 and comparative examples 1-2, respectively, in vitro, and each column of data is subjected to multiple comparisons and marked for significant differences in Table 4. As shown in Table 4, compared with the negative control, the expression levels of MHC-I, MHC-II, CD80 and CD86 on the cell surface of the compound vaccine adjuvant provided in examples 1-5 are significantly increased after the compound vaccine adjuvant acts on DC2.4 cells; after the composite vaccine adjuvant provided by the comparative examples 1-2 acts on DC2.4 cells, the expression level of MHC-I, MHC-II, CD80 and CD86 on the cell surfaces is increased, but the activation effect of the expression level is inferior to that of the examples 1-5. Thus, the composite vaccine adjuvants provided in examples 1-5 of the present application have a strong immune activation effect on DC2.4 cells in vitro (and example 5 is the best); APC plays an important role in immune recognition, immune response and immune regulation of the body as the most basic immune response cell of the body, and APC cell plays a role by expressing important surface proteins, CD80, CD8, MHC-I, MHC-II, and secreting important cytokines IL-12, TNF-alpha and the like with anti-tumor effects. Therefore, the composite vaccine adjuvants provided in embodiments 1 to 5 of the present application have an immune activation effect in vitro.
In vivo assay
The adjuvant of the novel coronavirus vaccine is used as an important component of the novel coronavirus vaccine, and the currently commonly used adjuvant comprises aluminum salt, emulsion and the like. Therefore, in the experiment, the composite vaccine adjuvants respectively prepared in the above examples 1-5 and comparative examples 1-2 and the novel coronavirus S-RBD protein are used as immunogens to prepare vaccines so as to further evaluate the adjuvant activation efficacy.
1. Vaccine formulation and immunization
The vaccines were formulated as test samples according to the vaccine formulation shown in table 5.
A6-8-week female Balb/C mouse (synbiotics) is selected, a clean-grade mouse food and sterile water are used for feeding the female mouse in the period, and the female mouse is divided into an experimental group, a control group and a blank group. In the experimental group, vaccines prepared in the following table 4 are administered in examples 1-5 and comparative examples 1-2 by intramuscular injection of the outer thigh of a mouse, and the immunization program is that 1 injection is performed on the 0 th and 21 st days, and the injection dose is 50 muL/mouse; the control group was immunized with 50 μ L of 3 μ g/μ L of new coronavirus RBD protein (cat # Z03479, Gensript) following the same immunization protocol, and the blank group was immunized with the same amount of saline. Wherein, the preparation process of the adjuvant and the antigen is carried out in a clean bench. In Table 4, the adjuvants are the nano-modified aluminas provided in examples 1 to 5 and comparative examples 1 to 2, respectively.
TABLE 5 vaccine formulations
2. Spleen cell extraction
The mice of each group of mice immunized twice are euthanized, the spleen of each mouse is extracted, the mice are placed in a 1640 complete culture medium, sterile slides are used for grinding respectively to remove larger tissues, aggregated cell tissues are further removed through screens of 70 mu m and 40 mu m in sequence, the mice are transferred to a 15mL centrifuge tube after being subjected to membrane filtration, the centrifugation is carried out for 6 min at 500g, the supernatant is discarded, and the mice are resuspended in 3mL of 1640 complete culture medium. Adding 4 mL of erythrocyte lysate into 1mL of the cell suspension, incubating for 3 min at room temperature, adding 3mL of 1640 complete culture medium to stop red cracking, suspending in 4 mL of 1640 complete culture medium after centrifugal washing, and counting by using a trypan blue method. 1mL of the cell suspension was collected, and CD4+ T cells and CD8+ T cells were obtained by sorting them according to the method of "study on method for sorting peripheral blood cells by flow cytometry [ J ] leukemia-lymphoma, 2010, 3 rd stage".
3. Determination of serum antibody titer levels
The method comprises the steps of taking blood from eyes of a mouse immunized twice, placing the blood on ice, standing for 2 hours, centrifuging at 4 ℃ and 1300 rpm, separating serum, discarding redundant precipitates, storing the obtained serum at-80 ℃, performing operations such as unfreezing and diluting before use, avoiding repeated freezing and thawing of the serum for many times as much as possible, and detecting the content of a new crown Antibody in the serum by using an ELISA Kit (SARS-CoV-2 centrifugation Antibody ELISA Kit, E-EL-E606, Wuhan Yirerite biology). The specific detection method comprises the following steps: preparing 5 mug/mL RBD buffer solution by using a coating buffer solution, adding the RBD buffer solution into a 96-well plate according to the amount of 50 mug/well, and incubating overnight at 4 ℃; wash the plate 3 times with Wash buffer (1 × PBS +0.05% tween 20), add fast blocking solution at 50 μ L/well, and incubate at 37 ℃ for 2 hours; after washing for 3 times by using Wash buffer, diluting the serum sample by using PBS, adding the diluted serum sample into a pore plate according to the amount of 50 muL/pore, setting a negative control group, namely adding only 50 muL PBS, and incubating for 2h at 37 ℃; washing for 3 times by Wash buffer, diluting goat anti-mouse IgG-HRP, IgGi-HRP and IgGaa-HRP by ten thousand times by PBS, adding the diluted IgG-HRP, IgGi-HRP and IgGaa-HRP into a pore plate according to the amount of 50 mu L/pore, and incubating for 2 hours at 37 ℃; washing the mixture for 5 times by using a Wash buffer, adding TMB according to the amount of 50 muL/hole for color development, and incubating for 5-20 minutes at room temperature; adding stop solution (2N H) according to the amount of 25 muL/hole 2 SO 4 ) The color development was stopped and the absorbance at 450nm was read within 30 min.
The RBD composite vaccines prepared by the adjuvants provided in examples 1-5 and comparative examples 1-2 were used for a mouse vaccination test, and Table 6 shows the measurement results of the antibody titer level in the orbital blood-collecting serum of each group of mice on day 30, in Table 6, multiple comparisons and significant difference markers are performed on each line of data, and "-" indicates that no detection is performed. As shown in Table 6, the serum antibodies of the mice were observed at day 30 after the mice were vaccinated with the novel coronavirus prepared by the adjuvant combination vaccines provided in examples 1 to 5IgG and IgG 2a Titer levels were significantly higher than control (RBD) vs. 1-2, and IgG no 2a /IgG 1 The values were also significantly higher than the control (RBD) and comparative examples 1-2 (and example 5 was best). Therefore, the vaccine prepared by the compound vaccine adjuvant provided by the embodiment of the application not only can show the effect of promoting the antibody level to be obviously improved similar to the in vitro cell level activation, but also can promote a mouse body to generate Thl humoral immune response so as to be matched with single Th2 humoral immune response, thereby enriching the immune protection effect of the vaccine.
TABLE 6
4. Determination of cellular immune effects
The cell points of IL-4 and IFN-gamma secretion of the sorted CD4+ T cells and CD8+ T cells are determined by enzyme linked immunosorbent assay (Elispot), and the specific process is as follows:
primary antibody was diluted with 1 × PBS, added to sterile ELISPOT plates (Millipore) at 100 μ L/well, incubated overnight at 4 ℃, replaced with fresh 200 μ L1 × PBS and incubated under block at room temperature for 2 h; the supernatant was discarded and the above sorted CD4+ T cells and CD8+ T cells 5X 10 were added to each well 6 Adding culture solution containing 10 mug/mLRBD into each cell in an amount of 100 muL/well, and incubating for 36h at 37 ℃; discarding cell suspension, washing with deionized water for 2 times, soaking the wells for 3-5 min each time, washing with PBS solution containing 0.05% Tween-20 for 3 times, diluting the detection secondary antibody with PBS solution containing 10% FBS, adding the diluted detection secondary antibody into a pore plate according to the amount of 100 muL/well, and incubating at room temperature for 2 h. Discarding the supernatant, washing the supernatant with PBS solution containing 0.05% Tween-20 for three times, washing each time, soaking for 1-2 min, diluting HRP with PBS solution containing 10% FBS, adding the diluted HRP into a pore plate according to the amount of 100 muL/pore, and incubating for 1 h at room temperature; discarding the supernatant, washing four times with PBS (phosphate buffer solution) containing 0.05% Tween-20, soaking for 1-2 min for each washing, washing twice with PBS containing 0.05% Tween-20, adding AEC color development liquid into the pore plate according to the amount of 100 muL/pore, developing, stopping developing with deionized water, air-drying at room temperature, and storing in dark until the plate is read.
The cellular levels of IL-4 and IFN-y cytokines secreted by CD4+ T cells and CD8+ T cells isolated from the spleens of various mice were measured by the ELISpot method, and the results are shown in Table 7, where multiple comparisons and significant difference labeling were performed for each column of data in Table 7. Table 7 shows that after mice are vaccinated with the new coronavirus vaccine prepared by the composite vaccine adjuvant provided in examples 1-5, the IFN-gamma spot number and the IL-4 spot number of CD4+ T cells and CD8+ T cells in splenocytes of the mice are significantly higher than those of blank groups, control groups (RBDs) and comparative examples 1-2 on day 30, which indicates that the composite vaccine adjuvant disclosed in the examples of the present application is beneficial to promoting the RBD vaccine to exert a better immune effect.
TABLE 7
5. Determination of memory cell activation levels
The sorted CD3+ T memory T cells and the sorted CD4+ T cells and the sorted CD8+ T cells are respectively inoculated into a 6-well plate at a density of 1000 ten thousand per well, each well contains 200 muL of complete culture medium, 5 mug/mL RBD diluted by 200 muL of culture medium is added at the same time, the 6-well plate is placed at 37 ℃ for treatment for 60 hours, the cells are collected, the cells are subjected to centrifugal washing and then are resuspended in PBS containing 1% blocking antibody, the room temperature incubation is carried out for 10min, and the CD69 antibody, the CD107 alpha antibody, the CD178(Fasl) antibody, the CD44 antibody and the CD62L antibody are sequentially dyed on each sample, the Fas25 antibody is incubated at 4 ℃ for 30 min, the cells are washed with PBS for 2 times after the incubation, and the cells are resuspended in FACS buffer solution for flow cytometry analysis.
TABLE 8 expression ratio (%)
Flow cytometry analysis of sorted T cell ratios in splenocytes from various groups of mice is shown, for example, at 87, in table 8, "+" indicates positive expression, "-" negative expression, and multiple comparisons and marked for significant differences were performed for each column of data in table 8. Table 8 shows that, after mice were vaccinated with the novel coronavirus vaccine formulated with the composite vaccine adjuvant provided in examples 1-5, the expression levels of CD4+ CD69+, CD8+ CD69+, CD4+ CD107 a +, CD8+ CD178 a, CD3+ CD44+ CD 62L-cells were significantly higher than those of the blank, control and comparative examples 1-2 (and example 5 is the best). Therefore, the expression level of the effector memory T cells expressing CD3+ CD44+ CD 62L-is increased, and the vaccine adjuvant provided by the embodiment of the application has the function of promoting the generation of immune memory T cells so as to migrate to inflammatory tissues; and the expression level of CD4+ CD69+, CD8+ CD69+, CD4+ CD107 alpha +, CD8+ CD178 is improved, so that the vaccine adjuvant improved by the embodiment of the application can obviously enhance the expression of a main killing medium of CTL, and has obvious immunostimulation enhancement.
6. Biocompatibility analysis
Organs such as heart, liver, spleen, lung and kidney of the mice were collected and stored in a cell tissue fixing solution of 4% paraformaldehyde for HE staining and observation of histopathological sections (test entrusted to Shandong green leaf test).
Further evaluating the in vivo safety, collecting the liver, spleen and kidney tissues of each group of mice subjected to the immunization test, storing the tissues in a cell tissue fixing solution containing 4% paraformaldehyde, making paraffin sections and carrying out HE staining observation. The results are shown in fig. 2, and all the visceral organ sections H & E staining of the mice in each group are the pathological features of inflammation, thereby demonstrating that the composite vaccine adjuvant provided by the embodiment of the application has good immunogenicity control and acceptable biosafety.
In summary, the application discloses a preparation method of a surface modified alumina composite vaccine adjuvant, which comprises the step of reacting alumina with polysaccharide to obtain the surface modified alumina composite vaccine adjuvant. The surface modified alumina is coupled with glycosyl to obtain a novel nano particle. The nano-particles not only have excellent dispersibility, low hydromechanical editing and low Zeta potential (mV)V Sedimentation It also has low endotoxin content and has long-term structural stability.
In vitro experiments prove that the novel surface modified aluminum oxide has a stimulation effect on mouse spleen T cells and an immune activation effect on mouse DC2.4 cells. Further, in vivo experiments prove that the novel surface modified aluminum oxide can be used as a composite vaccine adjuvant, can promote the immunogenicity of novel coronavirus RBD, stimulate an organism to generate a higher titer antibody level, and promote a mouse organism to generate Thl humoral immune response so as to be matched with single Th2 humoral immune response, thereby enriching the immune protection effect of the vaccine.
The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.
Claims (9)
1. A preparation method of a surface modified aluminum oxide composite vaccine adjuvant is characterized by comprising the following steps: and reacting alumina with polysaccharide in an alcohol solution containing at least one of benzyltriethylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrabutylammonium hydrogen sulfate, trioctylmethylammonium chloride, dodecyltrimethylammonium chloride and tetradecyltrimethylammonium chloride to obtain the surface-modified alumina composite vaccine adjuvant.
2. The method according to claim 1, wherein the polysaccharide is selected from fructose, xylose, sucrose, trehalose, and lactose.
3. The preparation method of claim 1, wherein the preparation method comprises the steps of dissolving fructose or sucrose in 1, 2-propylene glycol, adding nano aluminum oxide, benzyltriethylammonium chloride and anhydrous potassium carbonate, and reacting at 90-95 ℃ for 5 hours;
adding distilled water, and stirring at 90-95 ℃ for 1-2 hours;
and (2) distilling at 110-120 ℃ under reduced pressure to remove the solvent, filtering, taking a solid, directly washing the product by using a 30% NaCL solution, standing overnight, washing for 3 times by using a 5% NaCL solution, centrifugally separating at 4000r/min, washing for a plurality of times by using absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified alumina composite vaccine adjuvant.
4. The preparation method of claim 2, wherein the preparation method comprises the steps of dissolving lactose in n-butanol, adding nano alumina, benzyltriethylammonium chloride and anhydrous potassium carbonate, and reacting at 90-95 ℃ for 5 hours;
adding distilled water, and stirring at 90-95 ℃ for 1-2 hours;
and (2) distilling at 110-120 ℃ under reduced pressure to remove the solvent, filtering, taking a solid, directly washing the product by using a 30% NaCL solution, standing overnight, washing for 3 times by using a 5% NaCL solution, centrifugally separating at 4000r/min, washing for a plurality of times by using absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified alumina composite vaccine adjuvant.
5. The preparation method of claim 2, which comprises dissolving trehalose in n-butanol, adding nano alumina, benzyltriethylammonium chloride and anhydrous potassium carbonate, and reacting at 90-95 ℃ for 5 h;
adding distilled water, and stirring at 90-95 ℃ for 1-2 hours;
and distilling at 110-120 ℃ under reduced pressure to remove the solvent, filtering, taking a solid, directly washing the product by using a 30% NaCL solution, standing overnight, washing for 3 times by using a 5% NaCL solution, centrifugally separating at 4000r/min, washing for several times by using absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified alumina composite vaccine adjuvant.
6. The preparation method of claim 2, wherein the preparation method comprises the steps of dissolving trehalose in n-butanol, adding nano alumina, tetrabutylammonium hydrogen sulfate, trioctylmethylammonium chloride and anhydrous potassium carbonate, and reacting at 90-95 ℃ for 5 hours;
adding distilled water, and stirring at 90-95 ℃ for 1-2 hours;
and distilling at 110-120 ℃ under reduced pressure to remove the solvent, filtering, taking a solid, directly washing the product by using a 30% NaCL solution, standing overnight, washing for 3 times by using a 5% NaCL solution, centrifugally separating at 4000r/min, washing for several times by using absolute ethyl alcohol, drying in an oven at 120 ℃, and grinding to obtain the surface modified alumina composite vaccine adjuvant.
7. A composite vaccine adjuvant prepared by the preparation method of any one of claims 1 to 6, wherein the composite vaccine adjuvant comprises alumina as a base material and glycosyl attached to the surface of the alumina, and the glycosyl is selected from fructosyl, xylosyl, sucrose, trehalose and lactose.
8. A vaccine comprising the composite vaccine adjuvant of claim 7.
9. Use of the composite vaccine adjuvant prepared by the preparation method according to any one of claims 1 to 6 or the composite vaccine adjuvant according to claim 7 in the preparation of an antiviral vaccine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115656519A (en) * | 2022-10-25 | 2023-01-31 | 江苏乐汇生物技术有限公司 | Evaluation method capable of reducing nonspecific influence on serum titer of influenza vaccine |
| CN115656519B (en) * | 2022-10-25 | 2024-05-10 | 江苏乐聚医药科技有限公司 | Evaluation method capable of reducing serum titer of influenza vaccine with nonspecific influence |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050271684A1 (en) * | 2004-04-13 | 2005-12-08 | Trautman Joseph C | Apparatus and method for transdermal delivery of multiple vaccines |
| CN1980638A (en) * | 2004-07-07 | 2007-06-13 | 国立血清研究所 | Compositions and methods for stabilizing lipid based adjuvant formulations using glycolipids |
| CN108452299A (en) * | 2018-02-11 | 2018-08-28 | 上海络祥基因科技有限公司 | A kind of preparation method of liposome and the method for making Liposome Adjuvant |
| WO2021205042A1 (en) * | 2020-04-10 | 2021-10-14 | RNAssist Limited | Composition and method for inactivating a virus |
-
2022
- 2022-08-22 CN CN202211008477.5A patent/CN115068602B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050271684A1 (en) * | 2004-04-13 | 2005-12-08 | Trautman Joseph C | Apparatus and method for transdermal delivery of multiple vaccines |
| CN1980638A (en) * | 2004-07-07 | 2007-06-13 | 国立血清研究所 | Compositions and methods for stabilizing lipid based adjuvant formulations using glycolipids |
| CN108452299A (en) * | 2018-02-11 | 2018-08-28 | 上海络祥基因科技有限公司 | A kind of preparation method of liposome and the method for making Liposome Adjuvant |
| WO2021205042A1 (en) * | 2020-04-10 | 2021-10-14 | RNAssist Limited | Composition and method for inactivating a virus |
Non-Patent Citations (2)
| Title |
|---|
| SAIDEHFAKHARZADEH等: "The new nano-complex, Hep-c, improves the immunogenicity of the hepatitis B vaccine", 《VACCINE》 * |
| 宝航: "基于羟基氧化铝表面改性的复合疫苗佐剂", 《中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115656519A (en) * | 2022-10-25 | 2023-01-31 | 江苏乐汇生物技术有限公司 | Evaluation method capable of reducing nonspecific influence on serum titer of influenza vaccine |
| CN115656519B (en) * | 2022-10-25 | 2024-05-10 | 江苏乐聚医药科技有限公司 | Evaluation method capable of reducing serum titer of influenza vaccine with nonspecific influence |
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