Cpg oligodeoxynucleotides, adjuvants comprising same, vaccines and uses

Abstract

The application discloses a CpG oligodeoxynucleotide, which has a nucleotide sequence as shown in any one of SEQ ID NO. 1-8; or a nucleotide sequence with one or more nucleotides substituted or added to the nucleotide sequence as shown in SEQ ID NO. 8 and with the same function. The application has the beneficial effect that animal experiments prove that a vaccine containing the CpG oligodeoxynucleotide has a higher IgG binding antibody level in the serum GMT detection result of mice 14 days after the second immunization, is obviously better than the negative control and the positive control, and the effect is related to the specific base sequence of the CpG oligodeoxynucleotide. The above results prove that the CpG oligodeoxynucleotide can obviously enhance the immunogenicity of the vaccine, reduce the number of immunization needles, reduce the antigen usage, and has a better vaccine adjuvant effect when applied to the preparation of the vaccine.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of adjuvants, and particularly relates to a CpG oligodeoxynucleotide, an adjuvant containing the same, a vaccine and application. BACKGROUND

[0002] CpG oligodeoxynucleotide (CpG ODN) is a kind of non-methylated deoxyoligonucleotide containing CpG dinucleotide motif artificially synthesized, which is an agonist of intracellular pattern recognition receptor TLR9 (Toll-like receptor 9). After CpG ODN binds to TLR9, it activates the downstream signal cascade reaction, promotes the activation of B cells, plasmacytoid dendritic cells (pDC) T cells and other cells, and therefore has the potential of vaccine adjuvant.

[0003] CN112156183A discloses a CpG composite adjuvant, which comprises polylactic acid nanomicrosphere and an immunostimulant, the immunostimulant comprises CPG ODN, and the CPG ODN is coupled to the surface of the polylactic acid nanomicrosphere. The CpG composite adjuvant can effectively enhance the cellular immune and humoral immune response induced by the antigen as a novel coronavirus vaccine adjuvant; CN113304257A discloses a double-adjuvant novel coronavirus inactivated vaccine, the novel coronavirus inactivated stock solution is prepared according to a specified protein content or antigen content, and then the CpG adjuvant and the aluminum adjuvant are added to obtain the vaccine. The vaccine can effectively improve the serum titer and neutralizing antibody level of the current aluminum adjuvant novel coronavirus inactivated vaccine, effectively reduce the antigen dosage, reduce the production cost, and improve the vaccine yield; CN115845042A discloses a recombinant novel coronavirus S protein trimer vaccine composition and application thereof. The vaccine composition contains a recombinant novel coronavirus S protein trimer and an immunoadjuvant (CpG adjuvant and / or aluminum adjuvant), and can effectively induce cellular immunity and humoral immunity of the body. CN114569714B discloses a composite adjuvant for a novel coronavirus inactivated vaccine, which comprises a nano-aluminum preparation and further comprises CpG ODN, and can significantly improve the immunogenicity of the novel coronavirus inactivated vaccine. CN115252772A discloses a composition comprising CpG ODN and one or more other adjuvants that work together with the immunomodulatory CpG ODN, such as aluminum adjuvant. The CpG adjuvant and the aluminum adjuvant produce a synergistic effect when used together, and the double-adjuvant vaccine shows better immunogenicity than the single-adjuvant vaccine.

[0004] As a new and efficient immune activator, CpG ODN has become a hot spot in the research of new coronavirus vaccine in recent years. However, CpG ODN shows different immune stimulation effects on different species, cells and antigen components, and its immune stimulation effect is closely related to its sequence structure. Therefore, it is necessary to improve and design CpG ODN to adapt to different application scenarios and antigen components. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a CpG oligodeoxynucleotide, an adjuvant comprising the same, a vaccine and applications thereof.

[0006] In one aspect of the present application, a CpG oligodeoxynucleotide is provided, which has a nucleotide sequence as shown in any one of SEQ ID NO. 1-8; or a nucleotide sequence having one or more nucleotides substituted or added to the nucleotide sequence as shown in SEQ ID NO: 8 and having the same function. Preferably, it has a nucleotide sequence as shown in any one of SEQ ID NO. 1-10. Preferably, one or more nucleotides of the CpG oligodeoxynucleotide are sulfur-modified; more preferably, all nucleotides of the CpG oligodeoxynucleotide are sulfur-modified, which can improve the resistance of CpG ODN to nucleases.

[0007] In another aspect of the present application, an adjuvant is provided, which comprises the aforementioned CpG oligodeoxynucleotide. Preferably, the adjuvant further comprises an aluminum salt, preferably aluminum hydroxide.

[0008] In another aspect of the present application, a vaccine is provided, which comprises an antigen and the aforementioned adjuvant. The antigen is a microbial antigen or a self-antigen; the microbial antigen is selected from any one of a bacterial antigen, a viral antigen and a parasitic antigen, preferably a viral antigen; the viral antigen is preferably a recombinant protein antigen of a new coronavirus, more preferably an Omicron BA.4 / 5-Delta strain recombinant new coronavirus protein. When the vaccine comprises an Omicron BA.4 / 5-Delta strain recombinant new coronavirus protein, a CpG oligodeoxynucleotide and an aluminum adjuvant, the final concentration of the protein is 45-55 μg / ml, the aluminum concentration is 0.4-0.6 mg / ml, and the concentration of the CpG oligodeoxynucleotide is 45-55 μg / ml; preferably, the final concentration of the protein is 50 μg / ml, the aluminum concentration is 0.5 mg / ml, and the concentration of the CpG oligodeoxynucleotide is 50 μg / ml.

[0009] In another aspect of the present application, the aforementioned CpG oligodeoxynucleotide and adjuvant are used for preparing a vaccine.

[0010] The present application has the following beneficial effects:

[0011] The animal experiment proves that the vaccine containing the CpG oligodeoxynucleotide of the application has a higher IgG binding antibody level, which is obviously better than the negative control and the positive control, and the effect is related to the specific base sequence of the CpG oligodeoxynucleotide. The above results prove that the CpG oligodeoxynucleotide of the application applied to the preparation of the vaccine can obviously enhance the immunogenicity of the vaccine, reduce the number of immunization needles, reduce the amount of antigen used, and has a good vaccine adjuvant effect. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1A Figure 1 is the serum GMT detection result of the mice 14 days after the second immunization;

[0013] Figure 1B Figure 2 is the serum GMT detection result of the mice 14 days after the second immunization-2;

[0014] Figure 2 Figure 3 is the ELIspot IL-2 detection result of the spleen cells of the mice 14 days after the second immunization;

[0015] Figure 3 Figure 4 is the ELIspot IL-5 detection result of the spleen cells of the mice 14 days after the second immunization. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the embodiments of the application clearer, the technical scheme of the embodiments of the application will be clearly and completely described below in conjunction with the drawings of the embodiments of the application. Obviously, the described embodiments are some of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0017] The sequences of the CpG oligodeoxynucleotides in the following examples and comparative examples are shown in Table 1:

[0018] Table 1 CpG oligodeoxynucleotide and sequence

[0019]

[0020] Example 1

[0021] ZF-CpG1 was added dropwise to the aluminum hydroxide adjuvant, and then the Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly (10 min), the mixture was slowly added dropwise into the diluent, in which the final protein concentration was 50 μg / ml, the aluminum concentration was 0.5 mg / ml, the ZF-CpG1 concentration was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of the dropwise addition, adsorption was performed for 30 min to obtain the vaccine semi-finished product.

[0022] Example 2

[0023] ZF-CpG2 was added dropwise to the aluminum hydroxide adjuvant, and then the Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly (10 min), the mixture was slowly added dropwise into the diluent, in which the final protein concentration was 50 μg / ml, the aluminum concentration was 0.5 mg / ml, the ZF-CpG2 concentration was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of the dropwise addition, adsorption was performed for 30 min to obtain the vaccine semi-finished product.

[0024] Example 3

[0025] ZF-CpG3 was added dropwise to the aluminum hydroxide adjuvant, and then the Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly (10 min), the mixture was slowly added dropwise into the diluent, in which the final protein concentration was 50 μg / ml, the aluminum concentration was 0.5 mg / ml, the ZF-CpG3 concentration was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of the dropwise addition, adsorption was performed for 30 min to obtain the vaccine semi-finished product.

[0026] Example 4

[0027] ZF-CpG4 was added dropwise to the aluminum hydroxide adjuvant, and then the Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly (10 min), the mixture was slowly added dropwise into the diluent, in which the final protein concentration was 50 μg / ml, the aluminum concentration was 0.5 mg / ml, the ZF-CpG4 concentration was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of the dropwise addition, adsorption was performed for 30 min to obtain the vaccine semi-finished product.

[0028] Example 5

[0029] ZF-CpG5 was added dropwise into aluminum hydroxide adjuvant, and then Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly (10 min), the mixture was slowly added dropwise into the diluent, in which the final concentration of the protein was 50 μg / ml, the concentration of aluminum was 0.5 mg / ml, the concentration of ZF-CpG5 was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of dropwise addition, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0030] Example 6

[0031] ZF-CpG6 was added dropwise into aluminum hydroxide adjuvant, and then Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly (10 min), the mixture was slowly added dropwise into the diluent, in which the final concentration of the protein was 50 μg / ml, the concentration of aluminum was 0.5 mg / ml, the concentration of ZF-CpG6 was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of dropwise addition, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0032] Example 7

[0033] ZF-CpG7 was added dropwise into aluminum hydroxide adjuvant, and then Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly (10 min), the mixture was slowly added dropwise into the diluent, in which the final concentration of the protein was 50 μg / ml, the concentration of aluminum was 0.5 mg / ml, the concentration of ZF-CpG7 was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of dropwise addition, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0034] Example 8

[0035] ZF-CpG8 was added dropwise into aluminum hydroxide adjuvant, and then Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly (10 min), the mixture was slowly added dropwise into the diluent, in which the final concentration of the protein was 50 μg / ml, the concentration of aluminum was 0.5 mg / ml, the concentration of ZF-CpG8 was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of dropwise addition, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0036] Example 9

[0037] ZF-CpG0041 was added dropwise into aluminum hydroxide adjuvant, and then Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly (10 min), the mixture was slowly added dropwise into a diluent, in which the final concentration of the protein was 50 μg / ml, the concentration of aluminum was 0.5 mg / ml, the concentration of ZF-CpG0041 was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of the dropwise addition, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0038] Example 10

[0039] ZF-CpG004 was added dropwise into aluminum hydroxide adjuvant, and then Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly (10 min), the mixture was slowly added dropwise into a diluent, in which the final concentration of the protein was 50 μg / ml, the concentration of aluminum was 0.5 mg / ml, the concentration of ZF-CpG004 was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of the dropwise addition, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0040] Comparative Example 1

[0041] Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added into aluminum hydroxide adjuvant, and then the mixture was slowly added dropwise into a diluent, in which the final concentration of the protein was 50 μg / ml, the concentration of aluminum was 0.5 mg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of the dropwise addition, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0042] Comparative Example 2

[0043] CpG7909 was added dropwise into aluminum hydroxide adjuvant, and then Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly (10 min), the mixture was slowly added dropwise into a diluent, in which the final concentration of the protein was 50 μg / ml, the concentration of aluminum was 0.5 mg / ml, the concentration of CpG7909 was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of the dropwise addition, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0044] Comparative Example 3

[0045] CpG1018 was dropped into aluminum hydroxide adjuvant, and then Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After uniform mixing (10 min), the mixture was slowly dropped into the diluent, in which the final protein concentration was 50 μg / ml, the aluminum concentration was 0.5 mg / ml, the CpG1018 concentration was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of dropping, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0046] Comparative Example 4

[0047] ZF-CpG9 was dropped into aluminum hydroxide adjuvant, and then Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After uniform mixing (10 min), the mixture was slowly dropped into the diluent, in which the final protein concentration was 50 μg / ml, the aluminum concentration was 0.5 mg / ml, the ZF-CpG9 concentration was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of dropping, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0048] Comparative Example 5

[0049] ZF-CpG10 was dropped into aluminum hydroxide adjuvant, and then Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After uniform mixing (10 min), the mixture was slowly dropped into the diluent, in which the final protein concentration was 50 μg / ml, the aluminum concentration was 0.5 mg / ml, the ZF-CpG10 concentration was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of dropping, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0050] Comparative Example 6

[0051] ZF-CpG11 adjuvant was dropped into aluminum hydroxide adjuvant, and then Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After uniform mixing (10 min), the mixture was slowly dropped into the diluent, in which the final protein concentration was 50 μg / ml, the aluminum concentration was 0.5 mg / ml, the ZF-CpG11 concentration was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of dropping, adsorption was performed for 30 min to obtain a vaccine semi-finished product.

[0052] Comparative Example 7

[0053] The ZF-CpG12 adjuvant was added dropwise into the aluminum hydroxide adjuvant, and then the Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly for 10 min, the mixture was slowly added dropwise into the diluent, in which the final protein concentration was 50 μg / ml, the aluminum concentration was 0.5 mg / ml, the ZF-CpG12 concentration was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of the dropwise addition, adsorption was performed for 30 min to obtain the vaccine semi-finished product.

[0054] Comparative Example 8

[0055] The ZF-CpG13 adjuvant was added dropwise into the aluminum hydroxide adjuvant, and then the Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein stock solution was slowly added. After mixing uniformly for 10 min, the mixture was slowly added dropwise into the diluent, in which the final protein concentration was 50 μg / ml, the aluminum concentration was 0.5 mg / ml, the ZF-CpG13 concentration was 50 μg / ml, and the diluent was 10 mM His+3.5% glucose solution (pH 6.0). After the end of the dropwise addition, adsorption was performed for 30 min to obtain the vaccine semi-finished product.

[0056] Test Example

[0057] The vaccine semi-finished products prepared in the above examples and comparative examples were subjected to quality attribute detection, and the detection methods and results are as follows:

[0058] 1. Appearance detection: visual detection was used, and the appearance should be a milky white suspension liquid, which could be stratified due to precipitation, was easy to shake and disperse, and should not have lumps that could not be shaken apart.

[0059] 2. pH value detection: according to the General Rules 0631 in Volume III of the Chinese Pharmacopoeia (current edition), it should be 5.0-7.0.

[0060] 3. Osmotic pressure molar concentration detection: according to the General Rules 0632 in Volume III of the Chinese Pharmacopoeia (current edition), it should be 280±65 mOsmol / kg.

[0061] 4. Antigen adsorption rate: the test product was centrifuged at 6500 g for 5 min to obtain the supernatant, and the antigen content in the supernatant was detected by enzyme-linked immunoassay. The antigen content in the supernatant was substituted into the formula to calculate the unadsorbed antigen ratio P (%) in the test product: P = (C s / C t ) × 100%, wherein C s is the antigen content in the centrifuged supernatant of the test product, μg / ml; and C t is the theoretical protein content, μg / ml.

[0062] 5. CpG adjuvant adsorption rate:

[0063] (1) CpG standard curve establishment: different sequence CpG is accurately diluted to 200 μg / ml with sterile purified water, and 2-fold dilution is carried out respectively, a total of 8 gradients (100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 1.563 μg / ml, 0.781 μg / ml), take purified water (1-2 μl), drop on the sample table as background deduction, click "blank" to zero twice; wipe the mirror paper to clean the sample table and the metal part on the measuring arm, respectively add 1-2 μl of different gradient concentration of CpG adjuvant, cover the measuring arm, select "ss-DNA", click "Measure" to measure, the screen displays the sample test result, wipe the sample table and the metal part on the measuring arm. According to the detection results of different gradient concentrations of CpG, the standard curve of different sequence CpG is drawn with the theoretical CpG concentration-detection concentration, so as to calculate the actual concentration of CpG in each sample.

[0064] (2) Sample CpG adsorption rate detection: centrifuge the sample at 6500g for 5min, take the supernatant. Take the diluent (1-2 μl) corresponding to the sample as background deduction, drop on the sample table, click "blank" to zero twice; wipe the mirror paper to clean the sample table and the metal part on the measuring arm, then add 1-2 μl of supernatant, cover the measuring arm, select "ss-DNA", click "Measure" to measure, the screen displays the sample test result, wipe the sample table and the metal part on the measuring arm. According to the standard curve drawn according to the corresponding CpG, the actual concentration of each CpG in the supernatant is calculated, and the formula is calculated CpG adjuvant adsorption rate M (%): M=(1-C s / C t )×100%, wherein, C s is the CpG concentration of the supernatant of the test product, μg / ml; C t is the total CpG concentration before adsorption of the test product, μg / ml.

[0065] 6, serum IgG antibody level (GMT value) detection:

[0066] (1) Preparation before experiment: dilute 10×PBS buffer and 10×PBST buffer with purified water by 10 times, mix well, prepare coating solution (1×PBS) and washing solution (1×PBST) for standby; dissolve a proper amount of skimmed milk powder with 1×PBST buffer, mix well, prepare blocking solution (PBST+3% skimmed milk powder), prepare sample dilution and antibody dilution (PBST+1% skimmed milk powder) for standby;

[0067] (2) Coating: Prepare a 96-well enzyme-coated plate, and directly dilute the Omicron BA.4 / 5-Delta strain recombinant novel coronavirus protein vaccine (CHO cell) stock reference product to a final concentration of 1000 YU / ml, i.e. 1 μg / ml, into the 96-well enzyme-coated plate at 100 μl per well, cover the lid, and place it at 2-8°C overnight;

[0068] (3) The next day, remove the enzyme-coated plate, pour out the well liquid, add 250 μl of washing liquid to each well, stand for about 30 s, pour out the well liquid, and dry on qualitative filter paper;

[0069] (4) Blocking: Add 200 μl of blocking liquid to each well of the enzyme-coated plate, cover the lid, and incubate at 37°C for 2 h;

[0070] (5) Remove the incubated enzyme-coated plate, pour out the well liquid, add 250 μl of washing liquid to each well, stand for about 30 s, pour out the well liquid, and dry on qualitative filter paper. Then add 250 μl of washing liquid, place the enzyme-coated plate in the plate washer, and suck dry the liquid, and dry on qualitative filter paper;

[0071] (6) Mix 10 μl of each of the blank control group serum to obtain a blank control group mixed serum, and dilute the blank control group mixed serum with the sample diluent to the same starting dilution as the high and low dose groups;

[0072] (7) Serum dilution: Dilute the serum at a certain dilution for eight gradients in succession. Specifically, add 180 μl of sample diluent to columns 1-10 of row A and 100 μl of sample diluent to columns 1-10 of rows B-H of the enzyme-coated plate. Dilute each group of serum with sample diluent in the 96-well cell culture plate, then take 20 μl of the diluted serum and add it to columns 1-10 of row A, with two replicate wells for each serum. Mix row A with a multichannel pipette, then take 100 μl from row A and add it to row B, mix, and then dilute by two in turn, and finally mix row H, discard 100 μl of liquid, and ensure that each well of the enzyme-coated plate contains 100 μl of diluted serum. Operate all enzyme-coated plates in the same manner. At the same time, add 100 μl of diluted blank control group mixed serum to each column of the 11th column of the enzyme-coated plate;

[0073] (8) Cover the diluted enzyme-coated plate with a lid, incubate at 37°C for 1 h, remove the incubated enzyme-coated plate, pour out the well liquid, add 250 μl of washing liquid to each well, stand for about 30 s, pour out the well liquid, and dry on qualitative filter paper. Then add 250 μl of washing liquid, place the enzyme-coated plate in the plate washer, and suck dry the liquid, and dry on qualitative filter paper; dilute the goat anti-mouse IgG HRP enzyme-labeled secondary antibody with the antibody diluent at a dilution of 1:5000, add 100 μl to each well of the enzyme-coated plate, and incubate at 37°C for 1 h;

[0074] (9) Take out the incubated enzyme-labeled plate, pour out the hole liquid, add 250 μl washing liquid to each hole, stand for about 30 s, pour out the hole liquid, repeat the washing four times and dry. Add 250 μl washing liquid again, put the enzyme-labeled plate into the plate washing machine to absorb the liquid, and dry on qualitative filter paper; add 50 μl TMB single-component color developing liquid to each hole, place in the dark at room temperature for 5-10 min (the specific time is determined according to the reaction condition); add 50 μl termination liquid to each hole to terminate the color developing reaction, and place in the enzyme-labeled instrument to measure the absorbance at 450 nm;

[0075] (10) GMT value calculation: the GMT value is calculated according to the following formula

[0076]

[0077] Wherein, f represents the number of serum, and X represents the reciprocal of the antibody titer in serum;

[0078] (11) Result determination: when the OD450 value of the test serum is greater than the Cut off value, the test serum is determined to be positive. The Cut off value = average OD450 value of the blank control group mixed serum x 2.1, the average OD450 value of the blank control group mixed serum is lower than 0.05, and the actual OD450 value is calculated. The titer of serum antibody is represented by the reciprocal of the last positive serum dilution. If the OD450 values of different dilutions of the test serum are all greater than the Cut off value, the titer is calculated according to the maximum dilution factor; if the OD450 values of different dilutions of the test serum are all not greater than the Cut off value, the titer of the serum of the animal is calculated as 0.

[0079] 7. ELIspot detection:

[0080] (1) Extraction of BALB / c mouse spleen lymphocytes

[0081] 1) The mice (BALB / c) in each group were killed in turn 14 days after the second needle immunization, soaked in 75% ethanol for 3 min, opened the abdominal cavity, removed the connective tissue around the spleen, and ground the extracted spleen in a 200-mesh screen to rinse with 4-5 ml of mouse lymphocyte separation medium;

[0082] 2) The suspension containing spleen lymphocytes was immediately transferred to a 15-ml centrifuge tube, covered with 500-1000 μl RPMI1640;

[0083] 3) Centrifuge at 800 g for 30 min, and set the acceleration and deceleration of the instrument to 3, respectively. After centrifugation, the cells in the middle white layer were spleen lymphocytes;

[0084] 4) Aspirate the lymphocyte layer and put it into 10 ml RPMI 1640, invert to wash. Centrifuge at 250g for 10 min, collect the cells;

[0085] 5) Discard the supernatant, add appropriate amount of RPMI 1640 (containing 10% FBS) (4 factors are detected, generally 1.0 ml), count the cells, and adjust the final concentration of lymphocytes to 1.5 x 10 7 cells / ml for use.

[0086] (2) ELISpot detection factor

[0087] 1) Take out the ELISpot pre-coated plate from the aluminum foil sealed bag, wash it with sterile PBS at 200 μl / well for 4 times, add RPMI 1640 (containing 10% FBS) at 200 μl / well for blocking treatment, and incubate at room temperature for not less than 30 min;

[0088] 2) Prepare antigen stimulant with RPMI 1640 (containing 10% FBS), the concentration is 100 μg / ml (use OD-RBD stock solution and XB-RBD stock solution respectively);

[0089] 3) Take the lymphocytes with the final concentration adjusted to 1.5 x 10 7 cells / ml;

[0090] 4) Discard the blocking solution in the 96-well plate, dry it on the sterilized and dried paper, add the corresponding stimulant at 100 μl / well, and then add the prepared spleen lymphocyte suspension at 100 μl / well. The control wells are set as follows:

[0091] Positive control well: 100 μl concanavalin A (100 μg / ml) + 100 μl spleen lymphocyte suspension;

[0092] Negative control well: 100 μl RPMI 1640 (containing 10% FBS) + 100 μl spleen lymphocyte suspension;

[0093] Background control well: add 200 μl RPMI 1640 (containing 10% FBS)

[0094] 5) Cover the cover, place it in the CO2 incubator (37°C, 5% CO2), and incubate for 12-48 h (generally 48 h, to avoid evaporation, tin foil paper can be wrapped) under humidified conditions;

[0095] 6) Discard the cell suspension, wash it with PBS at 200 μl / well for 5 times;

[0096] 7) Dilute the corresponding detection antibody to 1 μg / ml with PBS (containing 0.5% FBS), add 100 μl per well, and incubate at room temperature for 2 h;

[0097] 8) Discard the detection antibody solution, and wash with PBS at 200 μl / well for 5 times;

[0098] 9) Dilute the alkaline phosphatase (ALP) labeled streptavidin solution to 1:1000 with PBS (containing 0.5% FBS), add 100 μl per well, and incubate at room temperature for 1 h;

[0099] 10) Discard the ALP labeled streptavidin solution, and wash with PBS at 200 μl / well for 5 times;

[0100] 11) Filter the substrate solution (BCIP / NBT-plus) with a 0.45 μm filter, add 100 μl per well, and stand to observe until specific spots appear;

[0101] 12) Discard the above color developing solution, rinse with purified water to terminate the color developing reaction. Remove the 96-well plate base, and invert it to dry at room temperature overnight. Put it into a machine for spot counting analysis, and store it in the dark.

[0102] The detection results are shown in Tables 2-3, Figures 1A-1B , Figures 2-3 .

[0103] Table 2: Physicochemical detection results of the vaccines of each group

[0104]

[0105] Table 3: Animal experiment detection results of the vaccines of each group

[0106]

[0107]

[0108] Note: "N / A" means not applicable or not detected.

[0109] (1) From the IgG binding antibody level (GMT) detection results in Table 3, it can be seen that the GMT results of the CpG sequences of each example after the second immunization have high effects;

[0110] (2) Example 6, Example 8, Comparative Examples 4-8 CPG sequences are based on the adjustment of individual bases of the CPG sequence of Example 7. Compared with the CPG sequence (ZF-CpG7) of Example 7, the CPG sequence (ZF-CpG6) of Example 6 increases the bases AATCG, the CPG sequence (ZF-CpG8) of Example 8 increases the bases GGAG, the CPG sequence (ZF-CpG9) of Comparative Example 4 changes two bases at both ends, the CPG sequence (ZF-CpG10) of Comparative Example 5 changes three bases at both ends, the CPG sequence (ZF-CpG11) of Comparative Example 6 changes three bases at both ends, the CPG sequence (ZF-CpG12) of Comparative Example 7 lacks two bases T and G at both ends, and the CPG sequence (ZF-CpG13) of Comparative Example 8 lacks one C at the right end. In addition, Comparative Example 1 is a blank control, and Comparative Examples 2-3 are positive controls. According to the results of the second immunization GMT of each group, the data of Example 7 is higher than that of each of the above comparative examples, and the data has a significant difference. Figure 1A As can be seen, the second immunization GMT results of Examples 6-8 have no significant difference, and all have a high IgG binding antibody level. This shows that ZF-CpG7 is the basic sequence, and adding a small amount of base on the basis of the basic sequence does not change the original basic structure, and can maintain a high effect, and changing the basic sequence will reduce the IgG binding antibody level of the mouse to varying degrees, and reduce the immunogenicity of the vaccine; Figure 1B

[0111] (3) According to the results of the second immunization GMT of each group, Figure 1B As can be seen, the second immunization GMT results of each example group have no significant difference, and all have a high IgG binding antibody level;

[0112] (4) According to the results of the second immunization GMT of each group, Figure 2 and Figure 3 As can be seen, the vaccine containing the CpG sequence has a high immunogenicity, which may be related to the regulation of Th1 type immune response (IL-2) and Th2 type immune response (IL-5) in the body.

[0113] The above describes the preferred embodiments of the present application, but the present application is not limited to the specific details of the above embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.​

Claims

1. CpG oligodeoxynucleotides, characterized in that, It is selected from any of the nucleotides shown in SEQ ID NO. 1 to 10.

2. The CpG oligodeoxynucleotide according to claim 1, characterized in that, One or more nucleotides of the CpG oligodeoxynucleotide are thiolated.

3. The CpG oligodeoxynucleotide according to claim 1, characterized in that, All nucleotides of the CpG oligodeoxynucleotide were thiolated.

4. An adjuvant, characterized in that, It comprises the CpG oligodeoxynucleotide as described in any one of claims 1 to 3.

5. The adjuvant according to claim 4, characterized in that, The adjuvant also contains aluminum salts.

6. The adjuvant according to claim 5, characterized in that, The aluminum salt is aluminum hydroxide.

7. A vaccine, characterized in that, It comprises an antigen and the adjuvant as described in any one of claims 4 to 6.

8. The vaccine according to claim 7, characterized in that, The antigen is a microbial antigen or an autoantigen.

9. The vaccine according to claim 8, characterized in that, The microbial antigen is selected from any one of bacterial antigens, viral antigens, and parasitic antigens.

10. The vaccine according to claim 9, characterized in that, The antigen is a viral antigen.

11. The vaccine according to claim 10, characterized in that, The antigen is a recombinant protein antigen of the novel coronavirus.

12. The vaccine according to claim 11, characterized in that, The antigen is the recombinant novel coronavirus protein of the OmicronBA.4 / 5-Delta strain.

13. The vaccine according to claim 12, characterized in that, The vaccine contains OmicronBA.4 / 5-Delta strain recombinant novel coronavirus protein, CpG oligodeoxynucleotides, and aluminum adjuvant. The final protein concentration is 45–55 μg / ml, the aluminum concentration is 0.4–0.6 mg / ml, and the CpG oligodeoxynucleotide concentration is 45–55 μg / ml.

14. The vaccine according to claim 13, characterized in that, The final protein concentration was 50 μg / ml, the aluminum concentration was 0.5 mg / ml, and the CpG oligodeoxynucleotide concentration was 50 μg / ml.

15. Use of the CpG oligodeoxynucleotide of any one of claims 1 to 3, or the adjuvant of any one of claims 4 to 6, in the preparation of a vaccine.

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